Paper 1. "Modelling Biological Macromolecules in Solution: the General Triaxial Ellipsoid can now be employed" S.E. Harding, M. Dampier & A.J. Rowe, IRCS Med. Sci., 7, 33 (1979).

In this paper an extension to the classical Simha-Einstein formula for the viscosity increment for a dispersion of macromolecules is presented. Einstein presented the formula for spheres, Simha for ellipsoids of revolution (ellipsoids with the restriction of two equal axes). This paper announces the new formula (derivation to be subsequently published in full –Paper #4) for the more general tri-axial ellipsoid allowing a much greater variety of macromolecules to be represented. This was the first paper published by Harding as a PhD student with Dr. Rowe.

Paper 2. "The combination of the viscosity increment with the harmonic mean relaxation time for determining the conformation of biological macromolecules in solution" S.E. Harding, Biochem. J. 189, 359-361 (1980). & corrigenda to Vol 189.

In this paper the viscosity increment is combined with the harmonic mean rotational relaxation time to yield a new combined macromolecular shape function "Λ" which does not require for its measurement knowledge of the extent of molecular hydration. The new parameter is illustrated by application to bovine fibrinogen.

Paper 3. "Viscosity parameters for myoglobin" S.E. Harding, IRCS Med. Sci., 8, 610 (1980).

In this paper, the newly derived viscosity increment for tri-axial ellipsoids is applied to intrinsic viscosity data for myoglobin. Comparison of the solution viscosity data with the dimensions of the myoglobin molecule yielded an estimate for the molecular hydration of the myoglobin molecule in solution, with a suggestion that the myoglobin molecule may be more asymmetric in solution compared to the crystalline state.

Paper 4. "The viscosity increment of a dispersion of triaxial ellipsoids under dominant Brownian motion" S.E. Harding, M. Dampier & A.J. Rowe, J. Coll. Int. Sci., 79, 7-13 (1981).

The full derivation of the explicit formula for the viscosity increment, ν, for dispersions of tri-axial ellipsoids in solution (cf. Paper #1) is given, and is shown to be in agreement with the numerical procedure of Rallison. The "line solution" nature of the relationship of n with the two axial ratios which characterise a triaxial (or "centrisymmetric") ellipsoid is discussed in terms of combination with other hydrodynamic parameters to obtain a unique solution for the axial ratios.

Paper 5. "A compound hydrodynamic shape function derived from viscosity & molecular covolume measurements" S.E. Harding, Int. J. Biol. Macromol. 3, 340-341 (1981).

In this paper the viscosity increment is combined with the molecular covolume to yield another new combined macromolecular shape function "Π" which, like the Λ function (Paper #2) does not require for its measurement knowledge of the extent of molecular hydration. The new parameter is illustrated by application to hemoglobin.

Paper 6. "Could egg albumin be egg shaped?" S.E. Harding, Int. J. Biol. Macromol. 3, 398-399 (1981).

In this paper, the new shape function P is used together with another hydration-independent function produced earlier to show that the ovalbumin has a gross conformation axial ratio of ~(1.5+0.3). Intriguingly, the axial ratio of an average standard UK class A egg is the same.

Paper 7. "The viscosity increment for ellipsoids of revolution: Some observations on the Simha formula" S.E. Harding, M. Dampier & A.J. Rowe, Biophys. Chem. 15, 205-208 (1982).

Outstanding uncertainties are resolved concerning the derivation by Simha of his widely used viscosity increment for ellipsoids of revolution, published in 1940. The uncertainties had been raised by Saito in 1951, who had suggested Simha had arrived at the correct result by a fortuitous cancellation of a wrong assumption and a mistake in calculation. This paper identifies the wrong assumption and the calculation error made by Simha.

Paper 8. "Self association, polydispersity & non-ideality in a Cystic Fibrotic glycoprotein" S.E. Harding & J.M. Creeth, IRCS Med. Sci. 10, 474-475 (1982).

This short paper, based on a presentation by Harding at a British Biophysical Society meeting, highlights the problems of interpreting sedimentation equilibrium profiles for mucus glycoproteins compared with protein solutions. The problem is considered in terms of data for a mucus glycoprotein purified from the bronchial secretion of a cystic fibrosis patient. Profiles are a function of polydispersity, non-ideality and possible associative phenomena, and the paper indicated the steps that are in progress to unravel this information. This was the first paper of Harding as a postdoctoral worker with J.M. Creeth.

Paper 9. "A simple test for macromolecular heterogeneity in the analytical ultracentrifuge" J.M. Creeth & S.E. Harding, Biochem. J. 205, 639-641 (1982).

Heterogeneity of macromolecular systems containing a quasi-continuous distribution of molecular weights can be very difficult to detect using the analytical ultracentrifuge. A simple check for heterogeneity is provided by comparing Rayleigh sedimentation equilibrium patters for two solutions of the same concentration expressed in Rayleigh fringe numbers but different absolute concentrations (compensated by being in different optical path length cells). The method is illustrated by application to a bronchial mucus glycoprotein.

Paper 10. "A computer program for evaluating the hydrodynamic parameters of a macromolecule for any given value of its axial dimensions"S.E. Harding, Comput. Biol. Med., 12, 75-80 (1982).

In this paper, a FORTRAN algorithm is presented, employing packages for the stable solution of elliptic integrals, for evaluating hydrodynamic parameters for macromolecule from specification of its axial dimensions or two axial ratios

Paper 11. "The conformation of C-protein in solution: use of new hydrodynamic shape functions" A.J. Rowe & S.E. Harding, J. Muscle Res. & Cell Mot., 3, 488 (1982).

In this paper, new hydration independent hydrodynamic shape functions are employed to obtain an estimate for the overall conformation of C-protein in solution. A prolate shape is confirmed with an axial ratio of ~10, in agreement with an earlier model postulated by G. Offer.

Paper 12. "Some observations on a new type of point average molecular weight", J.M. Creeth & S.E. Harding, J. Biochem. Biophys. Methods, 7, 25-34 (1982).

Measurement of molecular weights and related parameters of difficult heterogeneous systems by sedimentation equilibrium, is facilitated by the derivation and testing of a new type of operational point average molecular weight, M*, which is shown to have some intriguing and useful properties, including faciltating the evaluation of the meniscus concentration and the weight average molecular weight.

Paper 13. "Modelling Biological Macromolecules in solution I: The ellipsoid of revolution" S.E. Harding & A.J. Rowe, Int. J. Biol. Macromol., 4, 160-164 (1982).

The comparative suitability of the complete range of hydration and hydration independent ellipsoid of revolution shape functions (including several new functions) are compared in terms of (i) their relative sensitivity (ii) experimental measurability (iii) their dependence or independence of knowledge of the extent of molecular hydration. The R function and the Harding Λ and Π functions are shown to be particularly useful.

Paper 14. "Modelling Biological Macromolecules in Solution III: The Λ - R Intersection Method for Triaxial ellipsoids" S.E. Harding & A.J. Rowe, Int. J. Biol. Macromol., 4, 357-361 (1982).

This paper (which appeared in print just before Paper II in the series) establishes a new method based on measurement of the intrinsic viscosity, concentration dependence of the sedimentation coefficient and the harmonic mean rotational relaxation time from flourescence spectroscopy for evaluating uniquely the hydrodynamic shape of a macromolecule in terms of the two axial ratios specifying a general ellipsoid. The new method is illustrated by application to neurophysin monomers and dimers, and shows that dimerisation takes place as a side-by-side rather than end-to-end process.

Paper 15. "Modelling Biological Macromolecules in Solution II: The general tri-axial ellipsoid" S.E. Harding & A.J. Rowe, Biopolymers, 22, 1813-1829 (1983).

Hydration independent shape functions are presented for modelling the conformation of macromolecules in solution in terms of the two axial ratios (a/b, b/c) characterising a general ellipsoid. Combinations of these functions are presented for providing a unique solution for (a/b, b/c) for a given macromolecule

Paper 16. "Triaxial ellipsoids as models for macromolecules in solution; procedures for numerical inversion of the shape functions leading to a stable unique solution" S.E. Harding, Comput. Biol. Med. 13, 89-97 (1983).

A FORTRAN computer algorithm is presented for providing a unique solution to the hydrodynamic shape of a macromolecule (in terms of the two axial ratios a/b, b/c) using a graphical inversion procedure for the hydration independent Λ and R shape functions.

Paper 17. "Further evidence for a flexible and highly expanded spheroidal model for mucus glycoproteins in solution" S.E. Harding, A.J. Rowe & J.M. Creeth, Biochem. J. 209, 893-896 (1983).

Hydrodynamic and electron microscopic evidence is presented for a large, highly hydrated, flexible model for the conformation of mucus glycoproteins, based on measurements for a glycoprotein from the bronchial secretion of a cystic fibrosis patient.

Paper 18. "Mathematical models for determining intestinal permeability using polyethylene glycol" (Correspondence) S.E. Harding & S.O. Ukabam, Gut, 24, 456 (1983).

This short paper analyses an algorithm recently published by Sundqvist and co-workers for assessing the relative absorption of polyethylene glycol through the intestinal wall: this algorithm, if workable, would represent a convenient procedure for quantifying the passage of low and high molecular weight molecules in diseased patients. The algorithm as presented by Sundqvist et al involving the extraction of seven parameters from nine data points was shown to be unstable in its present form.

Paper 19. "Modelling the conformation of mucus glycoproteins in solution" S.E. Harding, J.M. Creeth & A.J. Rowe, Proc. 7th. Int. Glycoconjugates Conf., Lund-Ronneby, Sweden (A. Chester, D. Heinegard, A. Lundblad, S. Svensson eds.) pp 558-559, Olsson-Reklambyra (1983).

In this short paper, general principles are established representing the assembly of mucin glycoprotein basic units into large multi-unit linear assemblies. The model, based on hydrodynamic and electron microscopy data, is illustrated with a mucin from the bronchial secretion of a cystic fibrosis patient

Paper 20. "Polyelectrolyte behaviour in mucus glycoproteins" S.E. Harding & J.M. Creeth, Biochim et Biophys. Acta 746, 114-119 (1983).

Polyelectrolyte mediated conformation contraction of mucin glycoproteins, induced by increasing the ionic strength, is revealed from viscosity experiments for mucin glycoproteins from an ovarian cyst and from the bronchial secretion form a cystic fibrosis patient. Sedimentation equilibrium experiments reveal no change in molecular weight. The consquences for this in terms of a recently presented paper on the effect of ionic strength on the viscosity of mucus during the oestral cycle are considered.

Paper 21. "Photon correlation spectroscopy as a probe for bacterial spore germination" S.E. Harding & P. Johnson, IRCS Med. Sci. 12, 200-201 (1984).

In this paper a novel application of Photon correlation spectroscopy is used in an attempt to delineate between theories concerning the role of the cortex in comtrolling the dormancy of bacterial spores. The change in hydrodynamic radius of a dispersion of spores in aqueous solution is monitored as a function of time after additon of germinant. No significant change in the hydrodynamic radius is observable in the initial stages, consistent with the "Ellar theory" of dormancy. This is the first paper published by Harding as a postDoc with Dr. Paley Johnson.

Paper 22. "An analysis of the heterogeneity of mucins. No evidence for a self association" S.E. Harding, Biochem. J. 219, 1061-1064 (1984).

Distinguishing polydispersity (non-interacting heterogeneity) from molecular interaction phenomena using sedimentation equilibrium profiles is not easy. This paper describes how non-superposability of plots at different loading concentration together with the lack of effect of potential dissociative agents is evidence for strong polydispersity as opposed to self-association of the mucin glycoprotein systems studied.

Paper 23. "Quasi-elastic light scattering studies on dormant & germinating Bacillus subtilis spores", S.E. Harding & P. Johnson, Biochem. J., 220, 117-123 (1984).

This paper shows how quasi-elastic light scattering can be used as a dynamic method for following volume changes in germinating bacterial spore systems, germination confirmed by conventional turbidity measurements. The method is illustrated by application to spores of Bacillus subtilis. Lack of "scaling", i.e. superposability of autocorrelation plots above an angle of 35^o was interpreted to be a manifestation of the asymmetry of the spores.

Paper 24. "Erratum: Modelling biological macromolecules in solution. II. The General Tri-axial Ellipsoid". Biopolymers 23, 843 (1984).

A correction to a printer error in Paper #15 which erroneously gave the formula for the translational frictional ratio

Paper 25. "A highly expanded spheroidal model for a mucus glycoprotein from a cystic fibrosis patient: new evidence from electron microscopy" P. Hallett, A.J. Rowe & S.E. Harding, Biochem. Soc. Trans. 12, 878-879 (1984).

This paper provides further supportive evidence for the overall domain of a linear mucin molecule as being highly swollen or expanded though hydration. Earlier evidence came from hydrodynamics. This new evidence comes from electron microscopy where the mucin had been critical point dried to minimise distortion of the macromolecular shape during sample preparation.

Paper 26. "Excluded volumes for pairs of triaxial ellipsoids at dominant Brownian motion" J.M. Rallison & S.E. Harding, J. Coll. Int. Sci. (1985) 103, 284-289.

In this paper an extension to the Ogston-Winzor and Isihara formulae for the exclusion volume for a dispersion of macromolecules is presented, to the case for general tri-axial (or "centrisymmetric") ellipsoids. The potential significance of this finding for dealing with the non-ideality problem in characterising interacting systems is discussed.

Paper 27. "The representation of equilibrium solute distributions for non-ideal polydisperse systems in the analytical ultracentrifuge. Application to mucus glycoproteins". S.E. Harding, Biophys. J. (1985) 47, 247-250.

This method provides a new method for modelling sedimentation equilibrium profiles for a polydisperse non-ideal system, for moderate degrees of polydispersity. The method is successfully applied to a solution of a mucin glycoprotein from a cystic fibrosis patient: the solution data is shown to be consistent with observations from electron microscopy.

Paper 28. "The concentration dependence of macromolecular parameters" S.E. Harding & P. Johnson, Biochem. J. (1985) 231, 543-547.

Thiis paper examines in detail the various theoretical work describing the concentration deoendence of solutions of macromolecules. A new equation for the concentration dependence of the diffusion coefficient is also presented.

Paper 29. "Physicochemical Studies on Turnip Yellow Mosaic Virus: Homogeneity, molecular weights, hydrodynamic radii & concentration dependence of parameters" S.E. Harding & P. Johnson, Biochem. J. (1985) 231, 549-555.

This paper describes a thorough study on the ultracentrifuge and dynamic light scattering properties of a quasi-spherical virus (TYMV) to probe concentration dependence hydroynamic theories. It also provides for accurate determination of the thermodynamic second virial coefficient using independent methods.

Paper 30. "The Brownian Diffusion of Dormant and Germinating spores of Bacillus megaterium" S.E. Harding & P. Johnson, J. Appl. Bact. (1986) 60, 227-232. 70%.

This paper builds on Paper #23 and uses dynamic light scattering to probe the germination characteristics of Bacillus megaterium spores, confirming the observations on B. subtilis of no significant changes in volume of the spores during the initial stages of germination. Dormant and germinating spores give quite different scaling characteristics.

Paper 31. "A combined transient electric birefringence and excluded volume approach to macromolecular shape" S.E. Harding Biochem. Soc. Trans. (1986) 14, 857-858.

This paper describes a combination of the electric birefringence decays constants with intrinsic viscosity and the thermodynamic second virial coefficient to uniquely describe the macromoleculat shape of a macromolecule in solution (in terms of the two general ellipsoid axial ratios) without the requirement of knowledge of the extent of molecular hydration.

Paper 32. "Optimisation of products and processes" J. Mitchell, H. Back, K. Gregson, S.E. Harding & S. Mather, Royal Society of Chemistry Special Publication No. 56 (1986) 236-250.

This paper reviews various considerations and criteria for optimization of a set of parameters with particular reference to food processin parameters, and uses a FORTRAN algorithm with numerical minimization or maximization of a non-linear function involving several variables, similar to that used for Harding’s work on macromolecules in Paper #27.

Paper 33. "A comparison between the hot and cold water soluble fractions of two locust bean gum samples" S.E. Gainsford, S.E. Harding, J.R. Mitchell & T.D. Bradley, Carbohyd. Polym. (1986) 6, 423-442.

The hydrodynamic and rheological properties of two galactomannan polysaccharides solubulisable by different routes are compared with a conventional guar sample. No effects of self association was observed for any of the samples.

Paper 34. "General ellipsoid modelling of macromolecular shape using light scattering & viscometry" S.E. Harding, Macromolecular Preprints (1986), 145.

This short paper describes a combination of the radius of gyration from with intrinsic viscosity and the thermodynamic second virial coefficient to uniquely describe the macromolecular shape of a macromolecule in solution (in terms of the two general ellipsoid axial ratios) without the requirement of knowledge of the extent of molecular hydration.

Paper 35. "The molecular weight of one of the largest known polypeptides: titin" S.E. Harding & R.G. Bardsley, Macromolecular Preprints (1986), 146.

Sedimentation equilibrium in the analytical ultracentrifuge using the M* procedure introduced earlier in Paper #12, is used to demonstrate the largest known single chain polypeptide, titin from muscle: this is shown to have a monomer moleculat weight of ~950,000Da and to self associate.

Paper 36. "Applications of light scattering in microbiology" S.E. Harding, Biotechnology & Appl. Biochem. (1986) 8, 489-509.

This paper reviews the contributions made by simple turbidity, total intensity and dynamic light scattering to our understanding of the structure and function of microbial systems, focussing on viral and bacterial systems

Paper 37. "A General Method for Modelling Macromolecular Shape in Solution. A Graphical (Π - G) Intersection Procedure for Triaxial Ellipsoids" S.E. Harding, Biophys. J. (1987), 51, 673-680.

This paper descrines how the general triaxial shape of a macromolecule in solution can be obtained uniquely by a combination of two hydration independent shape functions: the Π function (from viscosity and 2^nd thermodynamic virial coefficient measurements) and the G function (from light/x-ray or neutron solution scattering measurements of the radius of gyration). The method is illustrated by application to myosin.

Paper 38. "Polyuronide solubilization during ripening of normal and mutant tomato fruit" G.B. Seymour, S.E. Harding, A.J. Taylor, G.E. Hobson & G.A. Tucker, Phytochemistry (1987) 1871-1875.

Solubility of pectin type polyuronides were found to show an increased solubility in ripening, corresponding to a loss of molecular integrity (molecular weights obtained by sedimentation equilibrium in the analytical ultracentrifugation were shown to fall). The decrease in molecular weight, although significant, was not as dramatic as those claimed earlier using non-absolute methods for molecular weight calculation.

Paper 39. "Analysis of the molecular size of tomato (Lycopersicon esculentum Mill) fruit polyuronides by gel filtration and low-speed sedimentation equilibrium" G.B. Seymour & S.E. Harding, Biochem. J. (1987) 245, 463-466.

A combination of sedimentation equilibrium in the analytical ultracentrifuge with gel filtration was used to establish the effect of ripening (with its associated enzymatic degradation processes) on the moelcular weight and molecular weight distribution of cell wall polyuronides from tomato fruit.

Paper 40. "The molecular mass and trimeric nature of chloramphenicol transacetylase" S.E. Harding, A.J. Rowe & W.V. Shaw, Biochem. Soc. Trans. (1987) 15, 513.

This paper describes a molecular weight and oligomeric composition study of the bacterial enzyme chloramphenicol transacetylase. Comparison with the result for the monomeric species from SDS-PAGE reveals that in solution this protein is a stable trimer, believed to be the first demonstration of such for proteins in solution.

Paper 41. "Rayleigh Interferometric fringe patterns from sedimentation equilibrium experiments: Use of an LKB Ultroscan for data capture and of a simple Fourier deconvolution algorithm for data analysis" S.E. Harding & A.J. Rowe Proc. Automatic Fringe Analysis Symp. (J. Tyrer ed.) pp189-200 Open Tech. Press, Loughborough (1987).

The first of a series of papers describing the "off-line" capture (via photographic film) of optical data from the analytical ultracentrifuge. This paper describes an adaptation of a commercial scanner for gel electrophoresis patterns to capture Rayleigh interference optical patterns from sedimentation equilibrium experiments. A PASCAL Fourier cosine series algorithm is described which accurately transforms the captured interference image into a data set of relative concentration versus radial displacement. Paper presented at the Automatic Fringe Analysis Symposium, Loughborough UK.

Paper 42. "A comparative study on the Physico-chemical Properties of Tomato Bushy Stunt Viruses" A. Molina-Garcia, S.E. Harding R.S.S. Fraser, Proc. 7th Int. Biophysics Conference, Jerusalem (1987) 94.

Abstract of a poster presented by Harding’s PhD student, Molina-Garcia, describing a detailed hydrodynamic charactetisation of TBSV.

Paper 43. "The relative molecular mass, heterogeneity and subunit composition of the 12S globulin from oil seed rape" S.E. Harding, P. Kyles, G. West & G. Norton, Biochem. Soc. Trans. (1987) 15, 684.

This paper describes a study by sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge on the oligomeric state of the 12S rape seed globulin, a subject of some debate by other researchers. The results clearly point in favour of the hexameric model of Schwenke.

Paper 44. "High precision automated off-line analysis of sedimentation equilibrium data" S.E. Harding & A.J. Rowe, Biochem. Soc. Trans. (1987) 17, 1046-1047.

The new algorithm "ANALYSER" introduced in paper #41 applied to sedimentation equilibrium data for tomato fruit polyuronides, indicating the realistic possibility of not only extracting whole-cell and point weight-average molecular weight data but also z-average and "y2-average" data.

Paper 45. "Automatic data capture and analysis of Rayleigh Interference Displacements in Analytical Ultracentrifugation" S.E. Harding & A.J. Rowe, Optics & Lasers in Engineering (1988) 8, 83-96. 70%.

Description of the ANALYSER method for the capture of Rayleigh Interference sedimentation equilibrium data from the ultracentrifuge via photographic film and an LKB gel scanner. Peer reviewed version of Paper #41.

Paper 46. "Polysaccharide molecular weight determination: which technique?" S.E. Harding Gums and Stabilisers for the Food Industry 4 (1988) 15-23.

Paper, presented at the 4^th Gums and Stabilisers meeting, puts the case forward that, particularly in the light of recent methodological and technical developments (such as M* and ANALYSER) sedimentation equilibrium in the ultracentrifuge is a powerful tool for characterising the molecular weight and molecular weight distribribution of polysaccharides

Paper 47. "Destruction of bacterial spores by hydrogen peroxide and ultraviolet light" W.M. Waites, S.E. Harding, D.E. Fowler, S.H. Jones, D. Shaw & M. Martin, Proc. 10th Int. Spores Conf. (Wood Hole, Mass. 1988) 65.

Abstract of paper presented at the Woods Hole (USA) meeting on bacterial spores. Describes the synergistic effects of hydrogen peroxide and irradiation by ultra violet light on spore destruction. Peer reviewed version appears as Paper #51.

Paper 48. "A combined quasi-elastic light scattering & electron microscopic study on the stability of Bacillus cereus spores" N.A. Jan, S.E. Harding, A. Molina-Garcia & W.M. Waites Proc. 10th Int. Spores Conf. (Woods Hole, Mass. 1988) 69.

Abstract of paper presented at the Woods Hole (USA) meeting on bacterial spores. Describes the use of quasi-elastic light scattering in conjunction with electron microscopy for the study of Bacillus cereus spores. Peer reviewed version appears as Paper #58

Paper 49. "Further evidence for structural homology of pyruvate ferrodoxin oxidoreductases: sedimentation velocity behaviour" S.E. Harding & K.P. Williams, Int. J. Biol. Macromol. (1988) 10, 318-319.

This paper describes a sedimentation velocity characterisation of a PFOR from the protozoan Trichomonas vaginalis, and yields results in excellent agreement with corresponding results for PFOR’s from other species.

Paper 50. "Combined low-speed sedimentation equilibrium/gel permeation chromatography approach to molecular weight distribution analysis. Application to a sodium alginate" A. Ball, S.E. Harding & J.R. Mitchell, Int. J. Biol. Macromol. (1988) 10 259-264.

This paper presents a new method for obtaining molecular weight distributions of polysaccharides by using molecular weights evaluated by sedimentation equilibrium in the ultracentrifuge applied to fractionated material to "self-calibrate" gel permeation columns for a given polysaccharides. The method is illustrated by application to an alginate.

Paper 51. "The destruction of spores of Bacillus subtilis by the combined effects of hydrogen peroxide and ultraviolet light" W.M. Waites, S.E. Harding, D.R. Fowler, H. Jones, D.Shaw & M. Martin, Letters in Appl. Microbiol. 71 (1988), 139-140.

This paper describes the combination of UV light with hydrogen peroxide for the destruction of bacterial spores. Maximum kill is not at the DNA absorption maximum of ~254nm but at ~270nm suggesting the destruction mechanism does not involve direct action on the DNA by UV but via the production of free hydroxyl.

Paper 52. "Protein transport processes in the water-water interface in incompatible two phase systems", M.P. Tombs & S.E. Harding in Advances in Separations using Aqueous Phase Systems in Cell Biology & Biotechnology (Fisher, D. & Sutherland, I.A. eds.) pp 229-232, Plenum, NY (1988).

This paper was presented at the Advances in Separations Meeting in Oxford by Tombs and considers the events occurring at the interface during the diffusion of proteins between non-compatible aqueous phases.

Paper 53. "The macrostructure of mucus glycoproteins in solution " S.E. Harding, Advances in Carbohydrate Chemistry & Biochemistry 47, (1989) 345-381.

This invited review assesses in detail the advances made in our understanding of the structure and assembly of mucus glycoproteins "mucins" from a variety of sources, and in relation to their biological function in health and disease. The structural hierarchy is built up from simple basic units (molecular weight ~ 500,000) to "subunits" (~2.5 x 10⁶ Da) through to large macrostructures (up to 50 x 10⁶ Da): the review considers the hydrodynamic and electron microscopic evidence for this assembly. The overall conformation, polydispersity and enormous hydration of these substances is also described, and the consequences of these properties are considered.

Paper 54. *"Protein diffusion through interfaces" S.E. Harding & M.P. Tombs, in Interactions in Protein Systems (Schwenke, K.D. & Raab, B. eds) pp 65-72, Abhandlungen der Akademie der Wissenschaften der DDR, Akademie-Verlag, Berlin (1989).

This invited paper presented at Reinhardsbrunn, German Democratic Republic, describes the novel use of the optical system on the analytical ultracentrifuge for monitoring diffusion of proteins through matrices and towards and through interfaces separating incompatible two- phase systems. Diffusion kinetics, density inversion and fingering and interfacial accumulation phenomena are all considered in the light of the theories of Ogston, Albertsson and Wells.

Paper 55. "The Thermal Degradation of Guar Gum" T.D. Bradley, A. Ball, S.E. Harding & J.R. Mitchell, Carbohydrate Polymers, 10 (1989) 205-214.

This paper includes the application of sedimentation equilibrium in the analytical ultracentrifuge (for molecular weight) and a slit viscometer (for intrinsic viscosity) to describe the effect of thermal treatment on the degradation of guar. The set of data also permits an examination of the dependence of intrinsic viscosity on molecular weight: no simple Mark-Houwink (-Kuhn-Sakurada) type of relation could satisfactorily describe the data set, either because of conformation change or through self-association phenomena.

Paper 56. "Sedimentation analysis of the Pf1 gene 5 protein" P.J. Morgan, S.E. Harding, S.E. Plyte & G.G. Kneale, Biochem. Soc. Trans. 17 (1989) 234-235.

This paper describes the application of a combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge to study the purity/heterogenity and oligomeric state of the gene 5 protein from filamentous bacteriophage Pf1. Sedimentation velocity showed a significant proportion of high molecular weight aggregate. The macromolecular component was a dimer with moderate degree of asymmetry (axial ratio between 2 and 3). The aggregate was estimated to contain ~ 50 dimer units on average.

Paper 57. "Subunit interactions in Propionibacterium shermanii methylmalonyl-CoA mutase studied by analytical ultracentrifugation" E.N. Marsh, S.E. Harding & P.F. Leadley, Biochem. J. 260 (1989), 353-358.

This paper describes the effect of increasing ionic strength on the dissociation between the dimeric subunits of P. shermanii methylmalonyl-CoA mutase, an important enzyme in biosynthesis. Sedimentation velocity and equilibrium demonstrate a rapid equilibrium between the subunits and the dimer. Progressive dissociation corresponds to a loss of activity.

Paper 58. "Dynamic Light Scattering studies on the effect of heat and disinfectants on spores of Bacillus cereus", A.D. Molina-Garcia, S.E. Harding, L. de Pieri, N. Jan & W.M. Waites, Biochem. J. 263 (1989) 883-888.

This paper describes the novel combination of dynamic light scattering with scanning electron microscopy to examine the overall morphology of B. cereus spores and the comparative effect of heating or adding the disinfectants sodium hypochlorite and peracetic acid The thermal data shows a good correlation with independent measurements of heat-resistance coefficients.

Paper 59. "Characterisation of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH", T.K. Chin, S.E. Harding & P.A.M. Eagles, Biochem. J. 264 (1989), 53-60.

This paper describes a study of the NFM and NFH proteins (which project as "side arms" from mammalian neurofilaments) in terms of their molecular weights and oligomeric state (the latter by comparison with results for the proteins in potentially dissociating solvents). Both proteins were monomeric and showed no tendency to interact with each other. Dephosphorylation only resulted in a slight change in sedimentation behaviour. The results provided evidence against a proposed function of these side arms in cross linking neurofilaments together.

Paper 60. "The Calibration of GPC for Pectins" G. Berth, H. Dautzenberg & S.E. Harding, Proc. 5th European Symposium on Carbohydrates, (1989) Prague.

Abstract of a paper presented at the 5^th European Carbohydrate Symposium describing the use of three different methods for obtaining molecular weights of fractions of pectin polysaccharides (light scattering, sedimentation equilibrium and osmotic pressure), in conjunction with gel filtration. Interesting correlations involving molecular weight distribution and universal calibration with intrinsic viscosity data can be made for these substances.

Paper 61. "On the interaction in solution of a candidate mucoadhesive polymer, diethylaminoethyl-dextran, with pig gastric mucus glycoprotein" M.T. Anderson, S.E. Harding & S.S. Davis, Biochem. Soc. Trans., 17, 1101-1102 (1989).

The first of a number of papers appearing over the next decade by Harding, Davis and colleagues on biopolymer mucoadhesives. This one looks at the interaction in solution between the candidate cationic mucoadhesive polymer DEAE-dextran with a pig gastric mucin. Change in sedimentation coefficient of the mucin was used as the key criterion for interaction. A definite, but modest interaction was observed over a range of mixing conditions and ionic strengths.

Paper 62. "Analysis of solute concentration and concentration derivative distribution by means of frameshift Fourier and other algorithms applied to Rayleigh interferometric and Fresnel fringe patterns", A.J. Rowe, S. Wynne-Jones, D. Thomas & S.E. Harding, SPIE Vol. 1163 Fringe Pattern Analysis (1989) 138-148.

This extends the earlier papers describing the Fourier algorithm ANALYSER for capturing Rayleigh Interfence optical records of sedimentation equilibrium concentration distributions to the case for Schlieren records, based on capture and analysis of the zero’th order Fresnel fringe. The new method provides for accurate measurement of the z-average molecular weight of polymers, complementing the Rayleigh data for the weight average.

Paper 63. "Modelling the gross conformation of assemblies using hydrodynamics: The Whole Body approach" S.E. Harding in Dynamic properties of Biomolecular Assemblies (Harding S.E. & Rowe, A.J. eds), pp 32-56, Royal Society of Chemistry, Cambridge (1989).

Paper presented at the Techniques Group meeting of the UK Biochemical Society, the first international meeting to be organized by Harding. This describes an overview of methods for representing the gross conformation of quasi-rigid macromolecules in solution using "whole body" i.e. ellipsoid (axisymmetric or general "centrally symmetric") models. It brought together the work of Harding throughout the previous decade on this topic.

Paper 64. "Predicting the Viscosity of mixtures of polysaccharide thickening agents in aqueous solutions" R.O. Mannion, C.D. Melia, J.R. Mitchell, S.E. Harding, A.P. Green & M.C. Davies, J. Pharm. Pharmacol. 41 (1989), 15P.

This short paper based on a presentation at a British Pharmaceutical Society meeting by Harding’s PhD student Mannion describes the use of viscosity based methods to probe the interactions in mixed solutions of polysaccharide. A modification to an equation previously presented by Morris was found to represent the data well for a mixed dispersion of hydroxypropylcellulose and hydroxypropylmethylcellulose.

Paper 65. "Low-speed flotation equilibrium of macromolecules in the analytical ultracentrifuge" S.E. Harding, P.J. Morgan & K. Petrak, J. Phys. Chem. 94 (1990), 978-980.

The application of the M* procedure for sedimentation equilibrium in the analytical ultracentrifuge is applied for the first time to flotation equilibrium: flotation of a dispersion of a block poly[(isoprene)-b-(ethylene oxide)] block copolymer dissolved in chloroform. In water this copolymer forms micelles (and conventional sedimentation equilibrium profiles). Comparison of molecular weights obtained for the monomer (flotation equilibrium in the dissociating solvent chloroform) and the micelle (sedimentation equlibrium in water) yields an estimate of (430+80) for the number of unimer units in a micelle.

Paper 66. "Hydrodynamic evidence for an extended conformation for citrus pectins in dilute solution" S.E. Harding, G. Berth, A. Ball & J.R. Mitchell, Gums and Stabilisers for the Food Industry, 5 (1990), 267-271.

This paper provides two sources of evidence for abn extended rather than coiled conformation of citrus pectin in solution. Wales van Holde ratios for all (five) bar one pectin fraction are all consistent with a rigid rod conformation. A sedimentation Mark-Houwink (-Kuhn-Sakurada) plot also gives a parameter consistent only with a rigid rod, conistent with the polyannionic nature of this substance.

Paper 67. "On the Molecular Weight Distribution of dextran T-500" A. Ball, S.E. Harding & N.J. Simpkin, Gums and Stabilisers for the Food Industry, 5 (1990), 447-450.

This paper applies the new method, introduced in Paper#50 for obtaining molecular weight distributions of polysaccharides by combing sedimentation equilibrium with gel filtration, to the case of dextran T-500. Results are in excellent agreement with another new method introduced by Wyatt technology (USA) of combining gel filtration with multi-angle laser light scattering.

Paper 68. "A rheological investigation of some polysaccharide interactions" R.O. Mannion, J.R. Mitchell, S.E. Harding, N.E. Lee & C.D. Melia, Gums and Stabilisers for the Food Industry, 5 (1990), 451-457.

This paper examines mixtures in solution of the ionic sodium carboxymethylcellulose and non-ionic hydroxypropyl methylcellulose in terms of ultracentrifuge viscosity and rheological properties. Enhanced viscosities observed on mixing support a model of coil expansion without hydrogen bonding or other specific association, a model consistent with the sedimentation velocity results. By contrast mixtures of xanthan and locust bean gum show a clear synergistic interaction.

Paper 69. "Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes", C.J. Smith, C.F. Watson, P.C. Morris, C.R. Bird, G.B. Seymour, J.E. Gray, C. Arnold, G.A. Tucker, W. Schuch, S. Harding & D. Grierson, Plant Mol. Biol. 14 (1990) 369-379.

One of the first papers describing the use of genetic modification of foods: antisense technology to arrest the rotting of tomatoes on ripening. In this pioneering work led by Grierson, the Harding team compared the effect on pectin molecular weight by compromising the polygalacturonase using sedimentation equilibrium in the ultracentrifuge and the M* function. The degradation of pectin on ripening was indeed arrested.

Paper 70. "Physicochemical studies on Tomato Bushy Stunt Virus variants" A.D. Molina-Garcia, S.E. Harding & R.S.S. Fraser, Biopolymers 29 (1990) 1443-155.

A range of TBSV strains were compared with regard their hydrodynamic properties (by analogy with Paper #29 for TYMV. All four strains appear very similar. The hydrodyamic data is consistent with observations using transmisson electron microscopy.

Paper 71. "Hydrodynamic Properties of a polyisoprene/ polyoxyethylene block copolymer", P.J. Morgan, S.E. Harding & K. Petrak, Macromolecules 23 (1990), 4461-4464.

This paper follows on from paper #65 and involves a thorough hydrodynamic characterisation of polyisoprene/poly(oxyethylene) block copolymer systems which form micelles in aqueous solution. Unimers (in chloroform) have a Wales van-Holde ratio of ~1.1 consistent with an extended conformation. In water, where the association number of unimers into a micelle is as previously reported ~430 (based on a comparison of flotation/sedimentation equilibrium results in the two solvents): a hydrodynamic radius of (42+3)nm is estimated. The micelles are shown to encapsulate a model drug compound (sudan black) by the principle of co-sedimentation

Paper 72. "Self-interaction of dynein from Tetrahyamena cilia", C. Wells, A.D. Molina-Garcia, S.E. Harding & A.J. Rowe., J. Mus. Res & Cell Motility 11 (1990), 344-350.

This paper describes the characterisation of the solution properties of the motility protein dynein. Molecular weights were obtained by either sedimentation velocity combined with the dynamic light scattering diffusion coefficient (Svedberg equation) or by sedimentation equilibrium. Dynein forms a dimer under low ionic strength conditions and this dissociates as the ionic strength is increased. This corresponds to an increase in ATP-ase activity.

Paper 73. "Physical & biological properties of water soluble polyelectrolyte complexes" C.J. Davison, K.E. Smith, L.E.F. Hutchinson, J.E. O'Mullane, S.E. Harding, L. Brookman & K. Petrak J. Bioactive and Compatible Polymers 5 (1990) 267-283.

This paper describes an analysis of a candidate polyelectrolyte complex for use in drug delivery involving the polycation QPVI and dextran rendered polyanionic by partial sulfonation. Sedimentation velocity and size exclusion chromatography showed that complexation was present under all the conditions studied.

Paper 74. "A model for the solution conformation of rat IgE" K.G. Davis, M. Glennie, S.E. Harding & D.R. Burton, Biochem. Soc. Trans. 18 (1990) 935-936.

The first demonstration that IgE is cusp shaped in solution. Mutli-sphere or bead modelling was applied to the sedimentation coefficient data. An average hydration was assumed based on comparison of the crystallographic with hydrodynamic data for the hingeless mutant IgG. This model was later shown to be correct by end-to-end distance measurements using NMR and by binding studies of the Fc region to the IgE receptor.

Paper 75. "Interactions of a model block copolymer drug delivery system with two serum proteins and myoglobin" P. J. Morgan, S.E. Harding & K. Petrak, Biochem. Soc. Trans. 18 (1990) 1021-1022.

The model block copolymer drug delivery system described in papers #65 and 71 is scrutinised in this current paper with regards to its potential intearctions with two plasma proteins (human serum albumin and hemoglobin) and myoglobin, using the principle of co-sedimentation in the ultracentrifuge. This drug delivery system was shown to be clear of any such interaction.

Paper 76. "A preliminary investigation of the hydrodynamic properties of two novel monoclonal antibodies", O. Byron, S.E. Harding & S.K. Rhind, Biochem. Soc. Trans. 18 (1990) 1030-1031.

The hydrodynamic characteristics of a chimeric IgG antibody molecule B72.3c produced from the murin monoclonal IgG B72.3 to negate the human anti-mouse antibody (HAMA) response are compared with the native B72.3 using sedimentation velocity and intrinsic viscosity. s^o_20,w values of (6.5+0.3)Sand (6.9+0.3)S are obtained formB72.3 and B72.3c respectively. These compare with a value of (6.7+0.3)S for human IgG4. More significantly, from the concentration dependence sedimentation data, formation of the chimera appears to stop self-association phenomena clearly observed in B72.3.

Paper 77. "Hydrodynamic characterisation of Chromobacter viscosum lipase, N.J. Simpkin, S.E. Harding & M.P. Tombs, Biochem. Soc. Trans. 18 (1990) 1031.

The molecular weight, oligomeric state and sedimentation velocity characteristics of a microbial lipase are described. Molecular weight characteristics are entirely consistent with a monomeric molecule in solution (comparison of sedimentation equlibrium, SDS-PAGE and sedimentation velocity results). Sedimentation velocity is possibly suggestive of some interaction although if present is not of hydrophobic in nature.

Paper 78. "An explanation of the rheological behaviour of mixed aqueous solutions of anionic and non-ionic cellulose ethers" R.O. Mannion, C.D. Melia, J.R. Mitchell, S.E. Harding & A.P. Green, J. Pharm. Pharmac. 42 (1990) 23P.

A comparison of zero-shear viscosities is given as a function of mixing ratio of the ionic cellulose derivative sodium carboxymethylcellulose and the non-ionic hydrodypropylmethylcellulose. Experimental values were compared witn those predicted by a further modification to an equation presented in Paper #64.

Paper 79. "Use of Bioluminescence to study heat resistance of spores of Bacillus megaterium KM" L.R. Hall, S.E. Harding & W.M. Waites, J.Appl. Bact. 69 (1990) xxiv.

Abstract of paper presented at the Society of Applied Bacteriology. B. megaterium spores have been produced containing the bioluminescent lux gene, which is expressed only during germination. Loss of bioluminescence (or lack thereof) in the presence of germination conditions is shown as a useful and rapid monitor of wet heat sterilization.

Paper 80. "A comparison of the hydrodynamic properties of two novel monoclonal antibodies: B72.3 and chimeric B72.3" O. Byron, S. Harding & S. Rhind, 10th International Biophysics Congress P2.2.20 (Vancouver, 1990).

Abstract of paper presented by Harding’s PhD student Byron. The propeties of a chimeric antibody molecule "B72.3c" formed from the constant regions of IgG4 and the variable regions of a monoclonal murine IgG1 called B72.3 are compared with native B72.3 and IgG4.

Paper 81. "A comparitive physical study of particles of some tombus viruses in swollen and compact states" A. Molina Garcia, R.S.S. Fraser and S.E. Harding, 10th International Biophysics Congress P2.9.1 (Vancouver, 1990).

Abstract of paper presented by Harding’s PhD student Molina-Garcia. The hydrodynamic properties of tomato bushy stunt virus and three other types of Tombusvirus are compared under native and conditions of pH and divalent cation which promote swelling.

Paper 82. "Molecular Weight Determination of Polysaccharides" S.E. Harding, K.M. Vårum, B.T. Stokke & O. Smidsrød in Advances in Carbohydrate Analysis (C.A. White ed.) 1, (1991), 63-144.

This invited review gives a detailed objective review of all the methods used to characterize molecular weight and molecular weight distribution of polysaccharides, highlighting how the difficulties caused by the polydispersity and thermodynamic non-ideality of these substances can be overcome.

Paper 83. "Laser Light Scattering in Biochemistry: Introductory Remarks" S.E. Harding, D.B. Sattelle and V.A. Bloomfield, Biochem. Soc. Trans., 19 (1991), 477.

The opening introductory paper based on a meeting organized for the Techniques Group of the Biochemical Society at Queens College, Cambridge, in which Harding, Sattelle and Bloomfield were the organizers.

Paper 84. "Total intensity and quasi-elastic light scattering applications in Microbiology", S.E. Harding, Biochem. Soc. Trans., 19 (1991), 499.

Short paper based on a presentation at the Biochemical Society Techniques Group meeting (cf. Paper #83) highlighting the advances in both total intensity and quasi-elastic light scattering of viral and bacterial systems, particularly with regard to the potential of the latter to assay the dynamics of processes involving possible volume changes.

Paper 85. "Gel permeation chromatography - multi angle laser light scattering characterisation of the molecular mass distribution of ‘Pro-nova’ sodium alginate", J.C. Horton, S.E. Harding & J.R. Mitchell, Biochem. Soc. Trans. 19 (1991), 510-511.

This paper based on a presentation at the Biochemical Society Techniques Group meeting of Paper #83 describes the charactetisation of an alginate by GPC-MALLs and examines the consequences of ignoring a correction for the second thermodynamic virial coefficient. Errors of only 10-15% accrue for a loading concentration of 1mg/ml (diluted by the elution process of GPC).

Paper 86. "The Molecular Weight Distribution and Conformation of Citrus Pectins in solution studied by Hydrodynamics" S.E. Harding, G. Berth, A. Ball, J.R. Mitchell & J. Garcia de la Torre, Carbohyd. Polym. 16 (1991), 1-15.

This paper describes a hydrodynamic study on fractions of citrus pectin from which conclusions about the molecular weight distribution and conformation are drawn. The method introduced in paper #50 of combining GPC with sedimentation equilibrium for obtaining an absolute molecular weight distribution is employed and gives a result in excellent agreement with a similat procedure involving GPC with light scattering. Mark-Houwink treatments of the viscosity/molecular weight and sedimentation coefficient/molecular weight data all point to a rigid rod conformation, as does the Wales-van Holde ratio.

Paper 87. "Thermodynamic non-ideality of dilute solutions of sodium alginate studied by sedimentation equilibrium ultracentrifugation", J.C. Horton, S.E. Harding, J.R. Mitchell & D.F. Morton-Holmes, Food Hydrocolloids, 5 (1991), 125-127.

The concentration dependence of the apparent molecular weight of alginate is followed up to a concentration of 2.5mg/ml. The incorporation of a third virial coefficient is found to be necessary to adequately represent the data. The dangers for not correcting for non-ideality are clearly illustrated, particularly at low ionic strength (0.1) where charge suppression is clearly inadequate.

Paper 88. "Books in Brief: An Introduction to Centrifugation", by T.C. Ford & J.N. Graham" (Book Review), S.E. Harding, Trends. Biochem. Sci. (1991), 392.

Paper 89. "Solution behaviour of Chromobacter viscosum and Pseudomonas sp. lipases: no evidence of self-association" N.J. Simpkin, S.E. Harding & M.P. Tombs. Biochem. J. 273 (1991) 611-613.

A hydrodynamic study using a combination of sedimemtation velocity, sedimentation equilibrium and SDS PAGE gives clear evidence against associative phenomena or the existence of any significant surface hydrophobic patches on two microbial lipases. The active form of at least these lipases is the monomer.

Paper 90. "Effect of xanthan/ locust bean gum synergy on ibuprofen release from hydrophilic matrix tablets" R.O. Mannion, C.D. Melia, J.R. Mitchell, S.E. Harding & A.P. Green, J. Pharm. Pharmacol., 43 (1991), 79P.

This paper demonstrates the favourable sustained release characteristics of hydrophilic matrix tablets containing both xanthan and locust bean gum. Comparison of release with the rheological G’ and G’’ profiles as a function of mixing ratio shows that release is modulated by the gel strength rather than the viscosity of the surface layer, and is highly depenedent on the mixing ratio.

Paper 91. "Total intensity and quasi-elastic light scattering applications in Microbiology", S.E. Harding in Laser Light Scattering in Biochemistry (Harding, S.E., Sattelle, D.B. & Bloomfield, V.A. eds.), Royal Society of Chemistry, Cambridge, pp 365-386 (1992).

This article reviews the applications of turbdity, total intensity (or "classical") and quasi-elastic (or "dynamic") light scattering procedures for the study of viral and bacterial systems. It highlights the differences between the treatments for microbes compared to macromolecules with regard to the validity of the Rayleigh, Rayleigh Gans Debye and Mie scattering theories, and the differences between application to viruses and bacterial systems, highlighting the special case of bacterial spores. The virtue of combined approaches is clearly indicated.

Paper 92. "Use of scanning absorption optics for sedimentation equilibrium analysis of labelled polysaccharides: molecular weight of blue dextran", N.C. Errington, S.E. Harding & A.J. Rowe, Carbohyd. Polym. 17 (1992) 151-154.

The new XL-A ultracentrifuge from Beckman instruments possessed only uv/visible absorption optics, potentially excluding its application to macromolecules lacking a chromophore, such as polysaccharides. Using commercially available "blue" dextran as an example, it is shown that labelling with a chromophore permits the reliable measurement of the sedimentation coefficient (so long as incorporation of the label does not significantly alter the conformation or molecular weight) and the molecular weight (so long as the label does not alter the molecular weight, and, after dialysis, negligible amounts of free chromophore are present in solution).

Paper 93. "News and Views: First Advanced Course on alginates and their applications", S.E. Harding, Carbohyd. Polym. 17 (1992) 155.

Conference report on a meeting held at Trondheim, Norway.

Paper 94. "Methods for off-line analysis of sedimentation velocity and sedimentation equilibrium patterns" A.J. Rowe, S. Wynne-Jones, D.G. Thomas & S.E. Harding in (S.E. Harding, A.J. Rowe & J.C. Horton eds.) Analytical Ultracentrifugation in Biochemistry & Polymer Science, Royal Society of Chemistry, pp49-62 (1992).

This paper introduces the algorithm ANALYSE2, upgrading the Rayleigh interference data capture algorithm ANALYSER introduced and described in Papers #41, 44&45. The upgrade provides for the capture and analysis via a two dimensional data acquisition system as opposed to a series of individual one-dimensional scans. Zero’th order Fresnel fringe recording from Schlieren optical records is also developed.

Paper 95. "MSTAR: A FORTRAN Program for the model independent molecular weight analysis of macromolecules using Low Speed or High Speed sedimentation equilibrium" S.E. Harding, J.C. Horton and P.J. Morgan in (S.E. Harding, A.J. Rowe & J.C. Horton eds.) Analytical Ultracentrifugation in Biochemistry & Polymer Science, Royal Society of Chemistry, pp275-294 (1992).

Two new versions of the FORTRAN algorithm MSTAR produced in 1982 for the manual capture of optical data from sedimentation equilibrium records are launched for the capture of the large data sets being generated by the new on-line data capture system for the absorption optical records of sedimentation equilibrium profiles from the XL-A ultracentrifuge and the new off-line data capture system ANALYSE2 for the Rayleigh Interference optical systems from the Model E ultracentrifuge. They are called respectively MSTARA and MSTARI. These algorithms are particular useful for the characterisation of heterogeneous systems, and, unlike other algorithms, do not require an assumed model.

Paper 96. "Sedimentation analysis of polysaccharides" S.E. Harding in (S.E. Harding, A.J. Rowe & J.C. Horton eds.) Analytical Ultracentrifugation in Biochemistry & Polymer Science, Royal Society of Chemistry, pp495-516 (1992).

A detailed review is given of the application of the analytical ultracentrifuge to the study of the purity, molecular weight, polydispersity, conformation of polysaccharides in solution in the light of recent developents of instrumentation and theory.

Paper 97. "The use of anti-oxidants to control viscosity and gel strength loss on heating of galactomannan systems", J.R. Mitchell, S.E. Hill, K. Jumel & S.E. Harding, Gums and Stabilisers for the Food Industry 6 (1992) 303-309.

Viscosity measurements are used to demonstrate the effectiveness of various mixing ratios of the anti-oxidants sodium sulphite and propyl gallate in arresting the degradation of galactomannan polysaccharides during heating regimes as experienced in food processing.

Paper 98. "Xanthan/ locust bean gum interactions at room temperature" R.O. Mannion, C.D. Melia, B. Launay, G. Cuvelier, S.E. Hill, S.E. Harding & J.R. Mitchell, Carbohyd. Polym. 19 (1992), 91-97 .

A combined rheological and analytical ultracentrifuge study is used to probe the interactions between xanthan and galactomannans possessing different mannose:galactose ratios. Two mechanisms for interaction are proposed.

Paper 99. "Ultracentrifugation: The solution conformation of novel antibody fragments studied using analytical ultracentrifugation" P.J. Morgan, O.D. Byron & S.E. Harding, Technical Information (Beckman Instruments, Palo Alto USA) (1992) DS-83.4.

This paper represents part of an evaluation study of the new XL-A analytical ultracentrifuge. It describes a characterisation of the Fab’ and (Fab’)2 fragments of IgG using sedimentation equilibrium (data analysed by MSTARA introduced in paper #95) and sedimentation velocity, followed by the generation of a hydrodynamic bead model for Fab’.

Paper 100. "Physico-chemical studies on di-iodotyrosine dextran" N. Errington, S.E. Harding, L. Illum & E. H. Schacht, Carbohyd. Polym. 18 (1992) 289-294.

A detailed hydrodynamic characterisation is given of a dextran labelled for medical imaging studies, to see if the effect of labelling has any deleterious effect on the dextran’s properties. The study involved sedimentation velocity, sedimentation equilibrium (analysed using ANALYSE 2 and MSTARI – see paper #95) and intrinsic viscosity measurements. Labelling appears to produce a more compact conformation from the original near randomly coiled state. Possible consequences of this for drug delivery are considered.

Paper 101. "A comparison of the enzymological and biophysical properties of two distinct classes of dehydroquinase enzymes" C. Kleanthous, R. Deka, K. Davis, S.M. Kelly, A. Cooper, S.E. Harding, N.C. Price, A.R. Hawkins and J.R. Coggins, Biochem. J. 282 (1992) 687-695.

Two dehydroquinase enzymes are shown to be quite different in their solution structural properties. Sedimentation analysis shows that the enzyme from E. coli is predominatly a dimer, whereas from Aspergillus nidulans it is a dodecamer. This data showing strong structural differences of these enzymes correlates with the observation that they operate though different catalytic mechanisms.

Paper 102. Chemistry in Britian Feature on P.J. Lillford and S.E. Harding, Chemistry in Britain 28 (1992). 370.

A description of the award of RSC Food Chemistry Group medals, the Junior medal going to Harding.

Paper 103. "The solution conformation of novel antibody fragments studied using analytical ultracentrifugation" P.J. Morgan, O.D. Byron & S.E. Harding, Discovery (Beckman Instruments, Palo Alto, USA) (1992) 1-4.

A second paper (cf Paper #99) sponsored by Beckman Instruments expounding the virtues of the XL-A ultracentrifuge for characerising antibodies and antibody fragments.

Harding’s NCMH laboratory had been provided with one of the four original prototype XL-A ultracentrifuges for evaluation, the others going to the laboratories of Schachman, Laue and Shire in the USA.

Paper 104. "Effect of coat protein mutations in bacteriophage fd studied by sedimentation analysis" A. Molina-Garcia, S.E. Harding, G. Diaz, D. Rowitz & R.N. Perham Biophys. J. 63 (1992) 1293-1298.

Sedimentation equilibrium and velocity in the ultracentrifuge are used as monitors for the effect of incorporation of two different point mutations K48Q and K48A in the coat protein on the overall assembly of the filamentous bacteriophage virus fd. The effects are dramatic with a significant increase in molecular weight/ viral length, and change in flexibility.

Paper 105. "Methylmalonyl mutase from P. shermanii: characterisation of the cobalamin-inhibited form and subunit-cofactor interactions studied by analytical ultracentrifugation" N. Marsh & S.E. Harding, Biochem. J. 290 (1993) 551-555.

This paper describes a study on the interactions between the enzyme methylmalonyl mutase and its co-factor, and also on interactions between its subunits. It introduces the principal of co-sedimentation in the analytical ultracentrifuge as a means for assaying for interactions between reactants possessing differing chromophores. A delicate interplay between denaturation and dissociation was revealed. Even 5M GuHCl did not dissociate the cofactor from the enzyme – the addition of dithiothreitol was necessary.

Paper 106. "Expression, purification and characterisation of B72.3 Fv Fragments" D.J. King, O.D. Byron, A. Mountain, N. Weir, A. Harvey, A.D.G. Lawson, K.A. Proudfoot, D. Baldock, S.E. Harding, G.T. Yarranton & R.J. Owens, Biochem. J. 290 (1993) 723-729.

This first paper of Harding’s PhD student Byron describes the production and analysis of the antibody fragment Fv. Using sedimentation equilibrium and velocity in the analytical ultracentrifuge, together with small angle x-ray scattering, the study investigated the association/dissociation of the two domains that make up Fv, and showed that the intact fragment was necessary for the binding of antigen.

Paper 107. "News and Views: Report on "Symposium on Conformational Studies of Oligosaccharides, Polysaccharides and Glycoconjugates" S.E. Harding & G. Berth Carbohyd. Polym. 20 (1993) 63-64.

Conference report on behalf of Carbohydrate Polymers of a major polysaccharide meeting at Le Croissic, France.

Paper 108. "Hydrodynamic characterisation of chitosans of differing degrees of acetylation" N. Errington, S. Harding, K. Vårum & L. Illum, Int. J. Biol. Macromol. 15 (1993) 113-117.

This paper describes a thorough hydrodynamic characterization of five chitosans (chitin derivatives solubilised by treatment with alkali) all of differing sizes and two with differing degrees of acetylation. The Wales-van Holde property and the relationship between molecular weight and hydrodynamic parameters are considered and coil structures of varying stiffnesses is revealed.

Paper 109. "Cinderella will go to the ball: Analytical ultracentrifugation has revived as a tool for protein characterisation" S.E. Harding, Laboratory News, March (1993) 20.

Short invited article expounding the virtues of the new analytical ultracentrifuge produced by Beckman instruments for protein characterisation. Illustrates the versatility of the technique and also its power when used in conjunction with other techniques.

Paper 110. "Examining Cinderella" S.E. Harding, The Biochemist Jun/Jul (1993) 30-31.

Report on the 7^th Harden Discussion Meeting (of the U.K. Biochemical Society) on glycopolymers in which Harding was the principal organizer.

Paper 111. "Evidence for protein-polysaccharide complex formation as a result of dry-heating of mixtures" K. Jumel, S.E. Harding, J.R. Mitchell and E. Dickinson, in Food Colloids and Polymers: Stability and Mechanical Properties (Dickinson, E. and Walstra, P.eds.), Royal Society of Chemistry, Cambridge, pp156-160 (1993).

Paper on dry heating mixtures of dextran polysaccharides with bovine serum albumin, prior to dissolution. Clear complexation is observed over and above aggregation phenomena observed in treating bovine serum albumin in isolation. Good agreement is found between the techniques of sedimentation equilibrium in the analytical ultracentrifuge and GPC/MALLS (gel permeation chromatography coupled on-line to multi-angle laser light scattering).

Paper 112. "The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity", H. Djaballah, A.J. Rowe, S.E. Harding and A.J. Rivett, Biochem. J. 292 (1993) 857-862.

This paper is another demonstration of how the physical state of an enzyme – in this case a proteasome – has a great affect on activity. Sedimentation velocity/equilibrium and dynamic light scattering, together with electron microscopy show a clear correlation between change in shape of the complex and the effect on activity. Clear evidence for intermediates during dissociation of the complex is shown using sedimentation velocity.

Paper 113. "The Structure and Nature of Protein-Polysaccharide Complexes" S.E. Harding, K. Jumel, R. Kelly, E. Gudo, J.C. Horton and J.R. Mitchell in Food Proteins: Structure and Functionality" (Schwenke, K.D. and Mothes, R. eds.) VCH Verlagsgesellschaft, Weinheim, Germany, pp216-226 (1993).

The behaviour of mixed systems of protein and polysacharide are compared: unheated and heated solutions of bovine serum albumin (BSA) with an alginate or a pectin and dry heated solutions of bovine serum albumin with dextran. Clear interactions are observed except for the case of BSA and pectin, where the latter appears thermally unstable.

Paper 114. "Analytical Ultracentrifugation and the Genetic Engineering of Macromolecules" S.E. Harding, Biotechnology & Genetic Engineering Reviews 11 (1993) 317-356.

This invited review considers the contribution sedimentation velocity, sedimentation equilibrium and density gradient analytical ultracentrifugatiin have made to our understanding of not only the genetically engineered molecules themselves, but also those macromolecules asociated with the engineering process: nucleic acids and nucleic acid binding proteins

Paper 115. "Determination of macromolecular heterogeneity, shape and interactions using sedimentation velocity analytical ultracentrifugation" S.E. Harding, Methods in Molecular Biology (Jones, C., Mulloy, B., & Thomas, A eds., Humana Press, New Jersey) 22, 61-73 (1994).

This invited Chapter describes the principles of sedimentation velocity in the analytical ultracentrifuge, principally for the benefit of the non-expert, and considers what can be measured with this technique, and its limitations.

Paper 116. "Determination of absolute molecular weights using sedimentation equilibrium analytical ultracentrifugation" S.E. Harding , Methods in Molecular Biology (Jones, C., Mulloy, B., & Thomas, A eds., Humana Press, New Jersey) 22 , 75-84 (1994).

This invited Chapter describes the principles of sedimentation equilibrium in the analytical ultracentrifuge, principally for the benefit of the non-expert, and considers what can be measured with this technique, and its limitations. .

Paper 117. "Classical Light Scattering for the determination of molecular weight and gross conformation of biological macromolecules" S.E. Harding, Methods in Molecular Biology (Jones, C., Mulloy, B., & Thomas, A eds., Humana Press, New Jersey), 22, 85-95 (1994).

This invited Chapter describes the principles of "Classical" or "Total Intensity" light scattering, principally for the benefit of the non-expert, and considers what can be measured with this technique, and its limitations.

Paper 118. "Determination of diffusion coefficients of biological macromolecules by Dynamic Light Scattering (DLS)" S.E. Harding, Methods in Molecular Biology (Jones, C., Mulloy, B., & Thomas, A eds., Humana Press, New Jersey) 22, 97-108 (1994).

This invited Chapter describes the principles of Dynamic light scattering (also known as "Quasi-elastic light scattering" or "Photon correlation spectroscopy"), principally for the benefit of the non-expert, and considers what can be measured with this technique, and its limitations.

Paper 119. "Some observations on the nature of heated mixtures of bovine serum albumin with an alginate and a pectin" R. Kelly, E.S. Gudo, J.R. Mitchell and S.E. Harding, Carbohydrate Polymers 23 (1994) 115-120.

This paper demonstrates quite different behaviour of heat treated solutions of an alginate, in the presence of bovine serum albumin and a highly esterified pectin in the presence of bovine serum albumin. A combination of dynamic light scattering, relative viscosity, and sedimentation velocity in the analytical ultracentrifuge was employed.

Paper 120. "Physical Techniques for the Study of Food Biopolymers" (Book review) S.E. Harding, Trends in Food Science and Technology 5 (1994) 126.

Paper 121. "Shapes and sizes of food polysaccharides by sedimentation analysis - recent developments" S.E. Harding Gums and Stabilisers for the Food Industry, 7 (1994), 55-65.

This short review highlights the application of ultracentrifuge methods to the characterisation of food-grade polysaccharides: Sedimentation velocity (analysis of sample homogeneity, shape and molecular interaction phenomena); sedimentation equilibrium (size and polydispersity); density gradient methods (sample purity) and as a diffusion tool (movement of molecules through a matrix and interface transport phenomena).

Paper 122. "Sedimentation equilibrium analysis of glycopolymers" S.E. Harding Prog. Colloid Polym. Sci. 94 (1994) 54-65.

This review describes the sedimentation equilibrium technique, highlighting the recent advances in methodology (such as MSTAR and ANALYSER) relevant to glycopolymer analysis, and then describes its application to the study of molecular weights, molecular weight distribution, and interaction phenomena, with particular reference to polysaccharides and mucin glycoproteins. The special problem of thermodynamic non-ideality for these substances is illustrated and addressed.

Paper 123. "Sedimentation analysis of potential interactions between mucins and a putative bioadhesive polymer" I. Fiebrig, S.E. Harding and S.S. Davis, Prog. Colloid Polym. Sci. 94 (1994) 66-73.

The mucoadhesive potential of the polycationic polysaccharide chitosan is assessed by sedimentation velocity in the analytical ultracentrifuge, after the mucin glycoprotein substrate had been thoroughly characterised by analytical isopycnic density gradient and GPC-MALLS. Clear interaction was evident with the formation of large complexes.

Paper 124. "Simultaneous determinations of the molecular weight distributions of amyloses and the fine structures of amylopectins of native starches" M.H. Ong, K. Jumel, P.F. Tokarczuk, J.M.V. Blanshard and S.E. Harding, Carbohydrate Research 260 (1994) 99-117.

The technique of SEC MALLs was used to characterise the molecular weight distributions of debranched starch amyloses from a variety of sources: wheat, waxy rice, potato, cassava and "sweet potato" and all found to be in the range 1-9 x 10⁶ Da. Chain length distributions of amylopectin are also compared.

Paper 125. "The analytical ultracentrifuge spins again" S.E. Harding, Trends in Analytical Chemistry, 13 (1994) 439-446.

This article draws attention to the chemistry/physical chemistry community of recent developments in analytical ultracentrifuge, explaining why it has a leading role to play in the characterisation of, and understanding of, polymers, macromolecules and macromolecular assemblies – whether they be biological or not. The interesting case of micelle forming synthetic copolymers, considered for use in drug delivery is highlighted.

Paper 126. "The Sedimentation Equilibrium analysis of polysaccharides and mucins: A guided tour of problem solving for difficult heterogeneous systems" S.E. Harding in (T.M. Schuster and T.M. Laue eds) Modern Analytical Ultracentrifugation, (1994) 315-341, Birkhäuser, Boston USA.

This Chapter, in a book where all the other chapters are devoted to protein systems, highlights the special problems concerning the analysis of sedimentation velocity and equilibrium of polysaccharides and mucus glycoproteins, using the analytical ultracentrifuge. It describes how these difficult non-ideal heterogeneous systems can be properly studied.

Paper 127. "Functional properties of Lupinus luteus proteins" I.M.N. Sousa, M.L. Beirao da Costa, S.E. Hill, J.R. Mitchell and S.E. Harding" in (T.Yano, R. Matsuno and K. Nakamura eds) Developments in Food Engineering: Proceedings of the 6th International Congress on Engineering and Food, (1994) 206-208 Blackie Academic and Professional, London, UK.

Lupin protein isolates were studied by sedimentation velocity in the analytical ultracentrifuge with regards their heterogeneous composition and the sedimentation coefficients of the various globulin components. Results were correlated with differential scanning calorimetry data.

Paper 128. "The potential of chitosan as a mucoadhesive drug carrier: studies on its interaction with pig gastric mucin on a molecular level" I. Fiebrig, S.E. Harding, B.T. Stokke, K.M. Vårum, D. Jordan and S.S. Davis, Eur. J. Pharmaceut. Sci. 2 (1994) 185.

Abstract of a presentation at a European Phramaceutical Sciences meeting. Sedimentation velocity analysis shows large complex formation between chitosan and mucin glycoprotein in solution. Particle sizes of the order ~1mm are consistent with observations under the electron microscope, with the more chitosan appearing towards the centre rather than the outside of the complex.

Paper 129. "The structure of secreted mucins isolated from the adherent mucus gel: comparison with the gene products" F.J.J. Fogg, A. Allen, S.E. Harding and J.P. Pearson, Biochem. Soc. Trans. 22 (1994) 229S.

Short paper based on presentation at a Biochemical Society meeting. Hydrodynamic properties of pig colonic mucin are presented, in the light of current developments in the molecular biology of mucins and structures obtained for mucins from different sources.

Paper 130. "Characterization of pullulans produced from agro-industrial waste" C.Israelides B. Scanlon, A. Smith, S.E. Harding and K. Jumel, Carbohyd. Polym. 25 (1994) 203-209.

SEC-MALLs is used to characterize the molecular weights and heterogeneity of a wide variety of pullulan fermentation products as a function of fermentation substrate.

Paper 131. "Product induced stabilisation of tertiary and quaternary structure in E. coli dehydroquinase" A. Reilly, P. Morgan, K. Davis, S.M. Kelly, J. Greene, A.J. Rowe, S.E. Harding, N.C. Price, J.R. Coggins and C. Kleanthous, J. Biol. Chem. 269 (1994) 5523-5526.

A follow up to Paper #101, specifically on the E. coli dehydroquinase. The effect of increasing concentrations of guanidine hydrochloride in the absence or presence of borohydride were studied with regards sedimentation coefficient, molecular weight, fluorescence emission at 320nm and (CD) ellipticity. All assay procedures show excellent correlation.

Paper 132. "On the hydrodynamic analysis of macromolecular conformation" S.E. Harding, Biophys. Chem. 55, (1995) 69-93 {anniversary volume special edition}.

A review based on an invited talk at a special Biophysical Chemistry anniversary meeting held in Holland, May 1994. The various approaches for obtaining conformation information using hydrodynamic parameters – axisymmetric ellipsoids, centrally symmetric ellipsoids, bead assemblies and general conformation/flexibility analysis are considered and compared.

Paper 133. "Ultracentrifugation of food biopolymers" S.E. Harding in (E. Dickinson ed.) New physico-techniques for the characterisation of complex food systems (1995) 117-146, Blackie Academic and Professional, London UK.

A comprehensive review of the use of the ultracentrifuge for characterising food poteins and polysaccharides, based on a presentation given at a meeting in Leeds, September 1994.

Paper 134. "Some recent developments in the analytical ultracentrifugation of food proteins". S.E. Harding, Nahrung 39 (1995) 375-395.

Another invited review, specifically on food protein systems.

Paper 135. "Inversion formulae for ellipsoid of revolution macromolecular shape functions" S.E. Harding and H. Cölfen, Analyt. Biochem. 228 (1995) 131-142.

This paper describes a new PC algorithm ELLIPS1 (written in BASIC) for evaluating the ellipsoid of revolution axial ratio (representing the overall shape of macromolecules than can be reasonably represented by an axi-symmetric shape) from a given value of a hydrodynamic shape parameter. Based on numerical inversion of the full hydrodynamic equations (that cannot be inverted analytically) using polynomial fits.

Paper 136. "Intrinsic viscosity and Mark-Houwink parameter of lupin proteins in aqueous solutions" I.M.N. de Sousa, J.R. Mitchell, S.E. Hill and S.E. Harding, Les Cahiers de Rheologie 14 (1995) 139-148.

This paper describes the analysis of the intrinsic viscosity and associated viscosity Mark-Houwink (-Kuhn-Sakurada) parameters for a lupin protein isolate.

Paper 137. "Investigations of the oligomeric state of the 42kDa repressor isoform from the Streptomyces temperate bacteriophage fC3", K. Jumel, S. Wilson, M. Smith and S.E. Harding, Prog. Coll. Polym. Sci. 99 (1995) 11-16.

This paper demonstrates using sedimemtation equilibrium in the analytical ultracentrifuge, that the 42kDa isoform (one of three) of this repressor isoform from Streptomyces phage fC31 exists as a dimer in solution: a His-tagged variant was shown to be capable of forming higher order association products, such as tetramers, depending on the conditions.

Paper 138. "A study on Schlieren patterns derived with the Beckman Optima XL-A absorption optics" H. Cölfen and S.E. Harding, Prog. Coll. Polym. Sci. 99 (1995), 168-187.

Artifactual Schlieren images can be produced from the absorption optical system of the XL-A ultracentrifuge for concentrated solutions. Higher concenrations refract light stronger, and sometimes to an extent where the path of the refracted light is so distorted it catches part of the optical system which acts as an artificial "knife edge" producing a reproducible Schlieren pattern. The consequences of this for commercial development are indicated.

Paper 139. "A comparitive "Schlieren" study of the sedimentation behaviour of three polysaccharides using the Beckman Optima XL-A and Model E analytical ultracentrifuges" R. Dhami, H. Cölfen and S.E. Harding, Prog. Coll. Polym. Sci. 99 (1995), 188-193.

Advantage is taken of the artifactual Schlieren images described in Paper#138 to the evaluation of the sedimentation coefficient for three polysaccharides: xylan, xanthan and locust bean gum: results are comparable to those from the conventional Model E ultracentrifuge, reinforcing the suggestion that the XL-A could be adapted with a proper knife-edge Schlieren system. This opens up the possibilities of applying the XL-A to macromolecular systems not possessing an absorbing chromophore.

Paper 140. "Alternative light sources for the Schlieren optical system of analytical ultracentrifuges" H. Cölfen, P. Husbands and S.E. Harding, Prog. Coll. & Polym. Sci. 99 (1995), 194-199.

High pressure mercury lamps, commonly used for the refractometric optical system of the Model E ultracentrifuge are unreliable and short-lived. The design and installation of a new LED based lamp system with a much greater lifetime is described in this paper.

Paper 141. "Estimation of the dissociation constant of the cell adhesion molecules srCD2 and srCD48 using analytical ultracentrifugation", H. Silkowski, O Byron, S.J. Davis, A.N. Barclay, E.A. Davies, A.J. Rowe and S.E. Harding, Biochem. Soc Trans. 23 (1995), 435S.

Dissociation constants K_d for the interaction between srCD2 and srCD48 are measured in this collaboration with the Barclay team by both sedimentation equilibrium in the ultracentrifuge and sedimentation velocity. Advantage is taken of the fact that the reactants have similar molecular weight, so the interaction can be regarded as a simple dimerisation reaction. The interaction is shown to be a weak one, with K_d ~ 100-200mM.

Paper 142. "Physico-chemical studies on a commercial food-grade xanthan. I. Characterisation by sedimentation velocity, sedimentation equilibrium and viscometry". R. Dhami, S.E. Harding, T. Jones, T. Hughes, J.R. Mitchell and K.-M. To. Carbohyd. Polym. 27 (1995) 93-99.

This paper describes a hydrodynamic charactersiation of the microbial polysaccharide xanthan. Sedimentation equilibrium (after correction for non-ideality) reveals a huge molecule of M = 5.9x10⁶ Da. Sedimentation velocity combined with intrinsic viscosity and the Wales-van Holde ratio reveals a fairly rigid rod shape structure. This ratio and the P function (applicable only to ~rigid particles) introduced in Paper #5 both suggest an axial ratio of ~70.

Paper 143. "Methods used to develop mucoadhesive drug delivery systems: bioadhesion in the gasterointestinal tract" I. Fiebrig, S.S. Davis and S.E. Harding in (S.E. Harding, S.E. Hill and J.R. Mitchell eds.) Biopolymer Mixtures, Nottingham University Press, Chapter 18 (1995).

A review of the development of mucoadheisve systems, based on a presentation at the Biopolymer Mixtures Meeting held in Nottingham, September 1994 The virtue of chitosan based systems is highlighted, in the light of macroscopic and molecular (hydrodynamic and electron microscopic) evidence.

Paper 144. Book review: "Rheology of Industrial Polysaccharides: Theory and Applications" (R. Lapasin and S. Pricl. eds.) S.E. Harding, Carbohydr. Research. 237 (1995) c1-c3.

Paper 145. "Active Enzyme Centrifugation", S.E.Harding and A.J. Rowe in (P.Engel, ed.) Enzymology Labfax Chap. 2F (pages 58-61) Bios, Oxford.

Very low concentration enzymes, or enzymes of low purity can be "fingerprinted" if linked to a spectral change involving the reaction they catalysed. The principle, first introduced in 1963 has since fell into complete dis-use. This paper re-launches that method in the light of modern instrumental developments, and highlights special application to the new XL-A ultracentrifuge.

Paper 146. "Identification of a flavin-NADH oxidoreductase involved in the biosynthesis of actinorhodin: Purification and characterisation of the recombinant enzyme", S.G. Kendrew, S.E. Harding, D.A. Hopwood and E.N. Marsh, J. Biol. Chem 270 (1995) 17339-17343.

The subunit structure of this oxidoreductase was investigated by analytical ultracentrifugation and show to be in an equilibrium beween monometic and dimeric forms. The consequences of this for the activity of the enzyme are considered.

Paper 147. "Mycobacterial turberculosis chaperonin 10 forms stable tetrameric and heptameric structures." G. Fossati, P. Lucietto, P. Giulani, A.R. Coates, S. Harding, H. Cölfen, G. Legname, E. Chan, A. Zaliani and P. Mascagni, J. Biol. Chem 270 (1995), 26159-26167.

The first of a number of papers involving the Harding team in characterizing molecular chaperonins. Thorough hydrodynamic (gel filtration and sedimentation equilibrium) analysis shows stable heptameric and also tetrameric forms., the latter associated with immunogenic properties.

Paper 148. "Hydrodynamic characterisation of the molar mass and gross conformation of corn cob heteroxylan AGX" R. Dhami, S.E. Harding, N.J. Elizabeth and A. Ebringerova, Carbohydrate Polym. 28 (1995), 113-119.

This paper examines an earlier claim that the water soluble fraction of heteroxylans may self associate in solution, and provides a hydrodynamic characterisation of the molecular size and shape. No evidence is seen from the concentration dependence of the molecular weight averages nor from sedimentation velocity (shape of sedimenting boundary and concentration dependence of sedmentation coefficient both do not show any signature of association).. A highly expanded asymmetric coiled structure is indicated.

Paper 149. "Some recent developments in the size and shape analysis of industrial polysaccharides in solution using sedimentation analysis in the analytical ultracentrifuge". S.E. Harding, Carbohyd. Polym. 28 (1995) 227-237.

This short review article attempts to establish the contribution that sedimentation velocity and sedimentation equilibrium are making to our understanding of the sizes, shapes and heterogeneity of industrially important polysaccharides in dilute solution and also indicates recent developments in the analysis of gels, diffusion though matrices and interactions/complex formation involving these substances

Paper 150. "Transmission electron microscopy studies on pig gastric mucin and its interactions with chitosan" I. Fiebrig, S.E. Harding, A.J. Rowe, S.C. Hyman and S.S. Davis, Carbohyd. Polym. 28 (1995) 239-244.

Mucin glycoprotein from pig gastric mucus, with a weight average molecular weight form sedimentation equilibrium of 9x10⁶Da have been prepared for electron microscopy by convenional air drying and critical point drying preparation procedures. Images of structures seen earlier for bronchial mucins (Papers # 17&25) are reproduced. Complexes with the mucoadhesive chitosan were then observed, and gave complex sizes conistent with these observed in solution by sedimentation velocity in the analytical ultracentrifuge.

Paper 151. "Characterisation of gliadin-galactomannan incubation mixtures by analytical ultracentrifugation. Part I: Sedimentation velocity" A. Seifert, L. Heinevetter, H. Cölfen and S.E. Harding, Carbohyd. Polym. 28 (1995) 325-332.

Galactomannan polysaccharides have been considered as possible protectants against coeliac allergies to wheat. This paper describes the an investigation on mixed solutions of locust bean gum galactomannan and gliadin. The concentration dependence of the sedimentation coefficient, supported by UV spectroscopy and gel filtration provides evidence for a weak interaction between these susbstances.

Paper 152. "Alternative Bioseparation Techniques". P.G. Righetti, S.E. Harding, and V. Stocchi in (E. Katz ed.) High Performance Liquid Chromatography: Principles and Methods in Biotechnology (1995) Chapter 10, John Wiley & Sons, New York.

This article critically considers alternative methods to HPLC for bioseparations and considers electrophoresis (including capillary), ultracentrifugation (including rate-zonal and isopycnic density gradient procedures) and ultrafiltration.

Paper 153. "A study of highly purified pig gastric mucin by scanning tunneling microscopy". C.J. Roberts, A. Shivji, M.C. Davies, S.S. Davis, I. Fiebrig, S.E. Harding, S.J.B. Tendler and P.M. Williams, Prot. Pept. Lett. 2 (1995), 409-414.

STM has been proposed as a complementary procedure to electron microscopy for visualising macromolecules. This paper uses STM to investigate the macrostructure of pig gastric mucin. Images appear to confirm the observations for this mucin (Paper 150) and other mucins (Papers #17,19) of swollen regions of high glycosylation linked by less swollen regions. The images appear to support the swollen/ linear random coil models for mucins proposed earlier.

Paper 154. "The effect of gamma-irradiation on the macromolecular integrity of guar gum". K. Jumel, S.E. Harding and J.R. Mitchell, Carbohyd. Research 282 (1996) 223-236.

The sale of food subject to low doses of irradiation has been legal in many countries for several years now. Some concerns have been expressed which include the possible damage to structural polysaccharides in foods. In this study nine Guar samples had been subject to various doses of irradiation (up to 9 kGray) and their properties compared with unirradiated material. Molecular weights (from SEC-MALLs) showed a steady decrease from 2.7 million to 565,000, and there were corresponding decreases in intrinsic viscosity and sedimentation coefficient. The Mark-Houwink intrinsic viscosity versus molecular weight relation suggested a random coil conformation, consistent with other observations on guar.

Paper 155. "Homodimeric and expanded behaviour of trimethylamine dehydrogenase in solution at different temperatures" H. Cölfen, S.E. Harding, E.K. Wilson. L.C Packman and N.S. Scrutton. Eur. Biophys. J. 24 (1996) 159-164.

This paper is the first of a trilogy with the Scrutton group on a study of the electron transfer redox partners TMADH and ETF (see papers #175 & 185). In this paper sedimentation velocity and sedimentation equilibrium measurements both confirm a stable homodimeric molecule. From the change in the sedimentation coefficient at various temperatures (after correction for change in solvent viscosity and density) suggest the enzyme is conformationally flexible, assuming a more expanded structure at higher temperatures.

Paper 156. "Rapid size distribution and purity analysis of gastric mucus glycoproteins by size exclusion chromatography/ multi angle laser light scattering" K. Jumel, I. Fiebrig and S.E. Harding, Int. J. Biol. Macromol. 18 (1996) 133-139.

This paper is the first reported application of SEC-MALLs to mucin glycoproteins. The molecular weight distributions of several commercial and fresh preparations of pig gastric mucin are compared. The study demonstrates that commercial material is very much degraded (by a factor of ~ 10x) compared to freshly prepared mucin. Although the method appears to be the method of choice for mucin moleclar weight work, the limitations are discussed, particularly with regard to the high molecular weight end (problems of column separation and the light scattering Rayleigh-Gans-Debye limit).

Paper 157. "Molar mass and viscometric characterization of hydroxypropylmethyl cellulose". K. Jumel, S.E. Harding, K.M To, I. Hayter, J.E. O'Mullane and S.Ward-Smith Carbohyd. Polym. 25(1996) 105-109.

A combined SEC-MALLs and viscometry approach was applied to a study of three HPMC preparations. A simple Mark-Houwink plot constructed on the basis of just the three materials suggests that water is a poor solvent for this neutral polysacharide. Solvent quality effects may explain the higher than expected viscosity values.

Paper 158. "Static Light Scattering studies on xanthan in aqueous solutions" G. Berth, H. Dautzenberg, B.E. Christensen, S.E. Harding, G. Rother and O. Smidsrød, Macromolecules 29 (1996) 3491-3498.

This paper describes the measurement of molecular weight, conformation and oligomeric state of the bacterial polysaccharide xanthan. The classical Zimm procedure yielded the weight average molecular weight which when combined with the contour length yielded a mass per unit length consistent with a double stranded (dimeric) molecule, and consistent also with earlier claims. The persistence length of 150nm is consistent with a rigid rod conformation.

Paper 159. "Maillard induced complexes of bovine serum albumin - a dilute solution study" A. Mat-Easa, H. Armstrong, S.E. Hill, J.R. Mitchell, S.E. Harding & A.J. Taylor, Int. J. Biol. Macromol. 18 (1996) 297-301.

This paper describes a study on large protein (BSA) – sugar (xylose) Maillard type complexes formed on heating . Molecular weights as a function of heating time are calculated from the diffusion coefficient (dynamic light scattering) with the sedimentation coefficient (sedimentation velocity). Mark-Houwink (-Kuhn-Sakurada) plots of sedimentation coefficient versus molecular weight or diffusion coefficient versus molecular weight are consistent with a linear aggregation mechanism.

Paper 160. "Comparison of quasi-elastic light scattering and laser diffractometry as nondestructive probes into the structure of Bacillus sphaericus spores produced at different temperatures". A. Molina-Garcia, L.A. de Pieri, I. Ludlow, W.M. Waites, J. Garcia de la Torre and S.E. Harding, Appl. Env. Micro . 62 (1996) 1699-1704.

This paper reports for the first time a comparison of quasi-elastic (or "dynamic") light scattering on ensembles of spores in suspension with laser diffractometry (Mie scattering) on single spores, and scanning electron micrsocopy images. The latter two techniques give comparable overall sizes, whereas dynamic light scattering gives considerably larger values, a difference ascribed to the presence of exosporia.

Paper 161. "Polylactide-poly(ethylene glycol) copolymers as drug delivery systems. 1. Characterisation of water dispesrible micelle-forming systems". S.A. Hagan, A.G.A. Coombes, M.C. Garnett, S.E. Dunn, M.C. Davies, L.Illum, S.S. Davis, S.E. Harding, S. Purkiss and P.R. Gellert. Langmuir 12 (1996) 2153-2161.

This paper describes a comparative study which includes the use of dynamic light scattering and sedimentation equilibrium to asssess the size, and sedimentation velocity (using the principle of co-sedimentation) to assess the drug incorporation capacity (tested using testosterone and sudan black B) of micelle forming polylactide-poly(ethylene glycol) systems in aqueous media – an approach successfully applied earlier by the Harding group to differing micelle systems - Paper#71. Large unimer association numbers of ~4000 and ~7000 were found for two different PLA-PEG block copolymer micelle systems, and they were shown to either have partial or complete incorporation of the "test" drug systems.

Paper 162. "Characterization of pig colonic mucins". F.J.J. Fogg, D.A. Hutton, K. Jumel, J.P. Pearson, S.E. Harding and A. Allen, Biochem. J. 316 (1996) 937-942.

This paper describes a characterisation of mucin glycoprotein isolated from pig colonic mucus by density gradient and gel filtration procedures in the presence of appropriate protease inhibitors. The native mucin together with reduced (to give subunits) and enzyme digested (to give basic units or "T-domains") were analysed. Sedimentation equilibrium gave excellent agreement with SEC-MALLs for the molecular weights. The intrinsic viscosities corresponded well with these values.

Paper 163. "Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure, backbone dynamics and protein association." A. Pintar, M. Hensmann, K. Jumel, M. Pitkeathly, S.E. Harding and I.D. Campbell, Eur. Biophys. J. 24 (1996) 371-380.

Although the main thrust of this paper was NMR, this paper with the Campbell team includes a supportive investigation of the oligomeric state of the SH2 domain, using a combination of dynamic light scattering and sedimentation equilibrium in the ultracentrifuge. Using the molecular weight of the monmetric protein from mass spectrometry, dimerization behaviour was clearly evident across the range of concentrations investigated, with a dissociation constant of ~25 mM, low enough to have some in vivo significance, and was the dominant form in the conditions of the NMR experiments.

Paper 164. "A physico-chemical investigation of the self-association of the DNA binding domain of the yeast transcriptional activator GAL4" P. Gadhavi, P.J. Morgan, P. Alefounder and S.E. Harding , Eur. Biophys. J. 24 (1996) 405-412.

Fragments of different chain length of the yeasr transcriptional activator GAL4 were investigated to see which residues were critical for the dimerisation process. Sedimentation equilibrium studies confirmed that the ‘hydrophobic region’ of the protein (residues 54-97 – which contains a larger proportion of a-helix), is essential for dimerisation. A K_d,app ~ 50mM for a 1-94 residue peptide and a stronger association, K_d,app ~20mM for a 1-147 residue peptide.

Paper 165. "Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, Band 3". H. Cölfen, S.E. Harding, J.M. Boulter and A. Watts, Biophys. J. 71 (1996) 1611-1615.

The first in a trilogy of papers with the Watts group on the Band 3 system. This first paper focusses on the aqueous soluble cytoplasmic domain. Sedimentation equilibrium sedimentation velocity and gel filtration show it to be a clear dimer in solution (K_d ~ 3mM), and hydrodynamic calculations of shape suggest an extended molecule of axial ratio ~10 in solution, consistent with images from electron microscopy.

Paper 166. "Physicochemical studies on Xylinan (Acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering". S.E. Harding, G. Berth, J. Hartmann, K. Jumel, H. Cölfen and B.E. Christensen. Biopolymers 39 (1996) 729-736.

This paper describes the solution physical properties of the polysaccharide Xylinan (or acetan). Light scattdering (classical), sedimentation equilibrium, and the sedimentation coefficient combined with the dynamic light scattering diffusion coefficient both suggest a large (weight average) molecular weight (all three procedures independently give ~2.5 x 10⁶Da). The Wales-van Holde ratio and the r-parameter (from dynamic and total intensity light scattering) both indicate an extended conformaton in solution, consistent with its polyelectrolyte nature.

Paper 167. "A study by analytical ultracentrifugation on the interaction between lysozyme and extensively deacetylated chitin (chitosan)". H. Cölfen, S.E. Harding, K.M. Vårum and D.J. Winzor. Carbohyd. Polym. 30 (1996) 45-53.

This paper describes the first study investigating interactions between lysozyme and chitosan using the analytical ultracentrifuge. MSTAR analysis of the sedimentation equilibrium data confirms a strong interaction between lysozyme and a 99.9% deacetylated chitosan, and a weaker interaction with a 99% deacetylated sample. Further analysis by means of the "omega" function to determine the fraction of free lysozyme in mixtures with defined total concentration indicated that esentially no free lysozyme is present in the mixtures, irrespective of whether the concentration is monitored reractometrically or whether only the lysozyme constituent is monitored by means of the absorption optics.

Paper 168. "Investigation, using analytical ultracentrifugation, of the effect of the incorporation of the fluorophore 9-anthraldehyde on two chitosans of differing degrees of acetylation". H. Cölfen, S.E. Harding and K.M. Vårum. Carbohyd. Polym. 30 (1996), 55-60.

Incorporation of a fluorophore onto chitosans assists with their visualisation during biomedical application. As with Paper #100 for di-iodo dextran, it is important to establish that incorporation of the label does not have a deleterious effect on the chitosan. This paper describes an analysis of the effect of increasing the degree of substitution of the fluorophore 9-anthraldehyde on 2 chitosans of differing degrees of acetylation. Based on sedimentaion coefficient and sedimentation equilibrium measurements, no deleterious effect on molecular weight results from the substitution, but there is possibly a slight change in conformation. The study also accidentally generated "Toepler" Schlieren images using the new XL-A ultracentrifuge equipped with only UV/Visible absorption optics (see papers #138 and 139).

Paper 169. "Hydrodynamic study of carboxymethyl chitin". E.V. Korneeva, G.A. Vichoreva, S.E. Harding and G.M. Pavlov. Abstr. Am. Chem. Soc. 212 (part 1) (1996) 75-cell.

Abstract of paper given at a meeting of the American Chemical Society describing the hydrodynamic properties of the polyannionic soluble derivative of chitin, carbxymethylchitin.

Paper 170. "Construction of a Dimeric Form of Glutamate Dehydrogenase from Clostridium symbiosum by site-directed mutagenesis". A. Pasquo, K.L. Britton, T.J. Stillman, D.W. Rice, H. Cölfen, S.E. Harding, R. Scandurra and P.C. Engel. Biochim. Biophys. Acta. 1297 (1996) 149-158.

In this paper alteration of the hydrophobic Phe-187 residue to the hydrophilic aspartic acid residue is shown to have a drastic affect on the oligomeric state of glutamate dehydrogenase. The wild type protein is shown by sedimentation equilibrium to be a stable hexameric structure (280,000 Da), and a sedimentation coefficient of 11.1S. The F187D mutant showed a concentration dependence of its molecular weight with a value extrapolated to zero concentration of 108000, close to the dimer value.

Paper 171. "Hydrodynamic characterization of lupin proteins: solubility, intrinsic viscosity and molar mass". I.M.N. Sousa, P.J. Morgan, J.R. Mitchell, S.E. Harding and S.E. Hill. J. Agric. Food Chem. 44 (1996) 3018-3021.

Lupin protein globulins were studied with regards their sedimentation coefficients (13S, 7S and 2S). The molar mass of the larger globulin (390,000) is larger than that for the soya bean 11S, but the lupin 7S (105000) was surprisingly much smaller to that of the soya bean 7S.

Paper 172. "Adenosylcobalamin-dependent glutamate mutase: properties of a fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit". D.E. Holloway, S.E. Harding and E.N. Marsh. Biochem. J. 320 (1996) 825-830.

Papers 57 & 105 considered the oligomeric state and subunit interactions of the methylmalonyl mutase system. This paper on the glutamate mutase system includes a study on the interaction between its two protein components MutS and MutE. Clear interaction is seen but with a K_d of ~13mM suggesting a somewhat weaker interaction than observed from kinetic experiments, probably because the former experiments, using the absorption optical system in the XL-A centrifuge had to be performed in the absence of the uv-absorbing adenosylcobalamin.

Paper 173. "Nitrite reductase from Achromobacter xylosoxidans forms stable trimers in dilute solution" P.J. Morgan and S.E. Harding, Biochem. Soc. Trans. 25 (1997) 40S.

This short paper describes another rare instance of a stable trimeric protein in solution (cf Paper #40). Combination of the monomeric molecularv weight from protein sequence and mass spectrometry, with the measured solution molecular weight from sedimentation equilibrium and MSTARA analysis shows clear trimers that are very stable (dissociation constant must be lower than 1mM). This paper resolves an earlier dispute concerning dimer versus trimer.

Paper 174. "Hydrodynamic properties of proteins" S.E. Harding in Protein structure: A Practical Approach". (ed. T.E. Creighton) (1997) Chapter 9, Oxford University Press.

This invited Chapter in the Practical Approach series examines the impact of modern hydrodynamics on our understanding of protein conformation and interaction in solution, and considers gel filtration, dynamic light scattering, sedimentation velocity and equilibrium, and total intensity (or "classical") light scattering coupled to size exclusion chromatography. Several experimental protocols are provided.

Paper 175. "An ultracentrifugal approach to quantitative characterization of the molecular assembly of a physiological electron-transfer complex. The interaction of electron-transferring flavoprotein with trimethylamine dehydrogenase." E.K. Wilson, N.S. Scrutton, H. Colfen, S.E. Harding, M.P. Jacobsen and D.J. Winzor. Eur. J. Biochem. 243 (1997) 393-399.

This second paper of a trilogy with the Scrutton team describes application of sedimentation in the analytical ultracentrifuge to a study of the interaction between the electron transfer proteins ETF and TMADH. The new "psi" function approach is applied to the equilibrium concentration profiles and yields a dissociation constant of ~5mM for the interaction between the two redox partners, with two equivalent and independent sites on the homodimeric ETF.

Paper 176. "Further observations on the size, shape and hydration of kappa-carrageenan in dilute solution" S.E. Harding, K. Day, R. Dhami and P.M. Lowe. Carbohyd. Polym. 32 (1997) 81-87.

Sedimentation equilibrium in the analytical ultracentrifuge yields a value for the weught average molecular weight consistent with light scattering analysis. Large thermodynamic non-ideality is demonstrated. The Wales-van Holde ratio from sedimentation velocity and viscometry is consistent with an extended conformation, and the data is also consistent with a high degree of water binding (a volume expansion through hydration of ~100x).

Paper 177. "Microbial Laser Light Scattering" S.E. Harding. Biotech. and Gen. Eng. Reviews 14 (1997) 145-164.

Update of a review presented earlier (Paper #91) which includes work on the dynamic light scattering of Bacillus sphaericus spores, tobacco mosaic virus and the effect of single amino acid mutations on the conformation of filamentous fd bacteriophage virus particles.

Paper 178. "Monomeric behaviour of Mytilus edulis (mussel) glue protein in dilute solution". M.P. Deacon, S.S. Davis, R. White, J. H. Waite and S.E. Harding. Biochem. Soc. Trans. 25 (1997) 422S.

Short paper based on a presentation by Harding’s PhD student Deacon at a Biochemical Society meeting, describes the hydrodynamic evidence for the monomeric solution state of the mussel "glue" protein: the sedimentation equilibrium "solution" molecular weight is in exact agreement with the value obtained from mass spectroscopy.

Paper 179. "The solution molecular weight and shape of the bacterial exopolysaccharides amylovoran and stewartan" K. Jumel , K. Geider and S.E. Harding. Int. J. Biol. Macromol. 20 (1997) 251-258.

Two bacterial exopolysaccharies are analysed with regard their solution physical properties, using a combination of SEC-MALLS and sedimentation equilibrium in the analytical ultracentrifuge, which gave excellent agreement. The large sedimentation coefficients suggest a more compact rather than extended conformation compared to other polysaccharides of similar molecular weight.

Paper 180. "New developments in analytical ultracentrifugation and related macromolecular modelling techniques" O. Byron and S.E. Harding, Eur. Biophys. J. 25 (1997) 305-306.

Introductory paper for a special edition of European Biophysical Journal dedicated to the 4^th UK Analytical Ultracentrifuge Users Group meeting at Leicester, U.K. on the analytical ultracentrifuge.

Paper 181. "MSTARA and MSTARI: interactive PC algorithms for simple, model independent evaluation of sedimentation equilibrium data" H. Colfen and S.E. Harding, Eur. Biophys. J. 25, (1997) 333-346.

The MSTARA and MSTARI algorithms,. originally presented in 1992 (Paper #95) have now been completely rewritten to facilitate their use on PC’s. The use of these model independent algorithms is illustrated for a range of data-set types (ideal, non-ideal, polydisperse and self-associating), and are seen to be particularly useful for the representation of difficult heterogeneous systems.

Paper 182. "The ELLIPS suite of macromolecular conformation algorithms" S.E. Harding, J.C. Horton and H. Colfen, Eur. Biophys. J. 25 (1997) 347-359.

Harding was extensively involved with developing ellipsoid strategies (axisymmetric and general, hydration dependent and hydration independent) in the 1980’s (see Papers #1-7, 10,11, 13-17, 24, 26, 31, 34, 37, 63 and 132). This paper brings together all the procedures for representing macromolecules in solution in this way conveniently into a suite of four programmes for PC: ELLIPS1, 2,3 and 4.

Paper 183. "SOLPRO: theory and computer program for the prediction of SOLution PROperties of rigid macromolecules and bioparticles" J. Garcia de la Torre, B. Carrasco and S.E. Harding, Eur. Biophys. J. 25 (1997) 361-372.

Many macromolecules cannot be represented by either axisymmetric or general centrisymmetric ellipsoids: antibodies are the classical example. This paper complements Paper 182 for ellipspods by bringing together bead and bead shell modelling, algorithms largely developed by Garcia de la Torre, into a convenient PC programme called "SOLPRO", a development of an earlier programme called HYDRO, but facilitating, like with Paper #182, the use of "universal" size independent shape parameters.

Paper 184. "A trimeric, alpha-helical coiled coil peptide: association stoichiometry and interaction strength by analytical ultracentrifugation" R.M. Thomas, A. Zampieri, K. Jumel and S.E. Harding, Eur. Biophys. J. 25 (1997) 405-410.

The oligomeric state in solution of a 35 residue alpha-helical coiled peptide "FZ" was studied. Originally designed as a dimer, sedimentation equilibrium in the analytical ultracentrifuge proves it exists as a stable trimer (cf other stable trimers – Papers #40 & 173), a result confirmed by calibrated HPLC. Studies using the denaturant GuHCl shows that the unfolding and dissociation process cannot be adequately described by a simple, two-state monomer-trimer equilibrium.

Paper 185. "Low temperature solution behaviour of Methylophilus methylotrophus electron transferring flavoprotein: a study by analytical ultracentrifugation" H. Cölfen, S.E. Harding, E.K. Wilson, N.S. Scrutton and D.J. Winzor, Eur. Biophys. J. 25 (1997) 411-416.

The oligomeric state and solution conformation of the electron transfer protein ETF, one member of the redox pair with TMADH (cf. Paper #175) was studied using sedimentation equilibrium and sedimentation velocity. A heterodimeric state is confirmed, and since the subunits are similar in size, the association can be treated as a simple monomer-dimer equilibrium, yielding from "psi analysis" a dissociation constant of ~1.5mM, viz. a strong interaction. The sedimentation coefficient suggests a globular structure for this heterodimer.

Paper 186. "Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces" J.L.E. Dean, H. Cölfen, S.E. Harding, D.W. Rice and P.C. Engel, Eur. Biophys. J. 25 (1997) 417-422.

Paper #170 described the effect of a single point mutation of a subunit surface hydrophobic residue on the oligomeric structure of C. symbiosum glutamate dehydrogenase. In this paper here, a single point amino acid substitution distant from the subunit interface is shown by ultracentrifugation and gel filtration to be also sufficient to cause disruption of the normal homohexameric oligomeric state (paper #170) into a dimeric form. A possible structural route is indicated for communication between the active sites and subunit interfaces which may be important for relaying allosteric signals between the subunits.

Paper 187. "Characterisation of the low affinity interaction between rat cell adhesion molecules CD2 and CD48 by analytical ultracentrifugation" H. Silkowski, S.J. Davis, A.N. Barclay, A.J. Rowe, S.E. Harding, O. Byron, Eur. Biophys. J. 25 (1997), 455-462.

This paper with the Barclay team describes the use of the analytical ultracentrifuge to characterise the interaction between the cell adhesion molecule CD2 and its structurally related counter-receptor CD48. Both sedimentation velocity and sedimentation equilibrium suggest a weak interaction, with dissociation constant in the range 20-120 mM, consistent with immobilisation studies using surface plasmon resonance. No significant self-association of the reactants was observed.

Paper 188. "A polydisperse linear random coil model for the quaternary structure of pig colonic mucin" K. Jumel, F.J.J. Fogg, D.A. Hutton, J.P. Pearson, A. Allen and S.E. Harding, Eur. Biophys. J. 25 (1997) 477-480. 25%.

A model is presented for the solution structure of colonic mucin glycoprotein on the basis of SEC-MALLS, sedimentation equilibrium, intrinsic viscosity and associated Mark-Houwink-Kuhn-Sakurada relationship. This highly expanded & polydisperse "linear random coil model" or "swollen coil array model" is consistent with earlier models for other mucins (Papers #17, 19, 22, 27).

Paper 189. "Conformation zoning of large molecules using the analytical ultracentrifuge" G.M. Pavlov, A.J. Rowe and S.E. Harding. Trends. Analyt. Chem. 16 (1997) 401-405.

Conformations of quasi-rigid macromolecules in terms of ellipsoid or bead models were considered earlier (cf Papers #182, 183). The current paper addresses general particle conformations. Conformation zones are introduced (A: extra rigid rod; B: rigid rod; C: semi-flexible coil; D: random coil; C: globular or branched) which cover the entire range of partucle conformaions and flexibilities. A simple empirical procedure (with a theoretical basis) is provided for assigning the zone for a given macromolecules, from a combination of sedimentation velocity and mass per unit length information.

Paper 190. "Colloidal gold and colloidal gold labelled wheat germ agglutinin as molecular probes for identification in mucin/chitosan complexes". I. Fiebrig, K.M. Vårum, S.E. Harding, S.S. Davis and B.T. Stokke, Carbohydrate Polymers 33 (1997) 91-99.

A development from Paper #150 which described a thorough electron microscopic study of gastriv mucin and mucoadheshive complex with chitosan. The current paper describes the use of colloid gold labelling of the chitosan to indicate its distribution in the mucoadhesive complex using transmission electron microcsopy. It appears that the chitosan appears to be concentrated more towards the central regions of the complex, surrounded by a possibly more hydrophilic layer of mucin.

Paper 191. "Characterisation of chitosan-mucin complexes by sedimentation velocity analytical ultracentrifugation" in (R.A.A. Muzzarelli and M.G. Peter eds), S.E. Harding, Chitin Handbook, pp457-466, European Chitin Society (1997).

An invited paper at a Europan chitin Society meeting focussing on the use of sedimentation velocity in the analytical ultracentrifuge as a molecular probe for mucoadhesive interactions for drug delivery.

Paper 192. "The intrinsic viscosity of biological macromolecules. Progress in measurement, interpretation and application to structure in dilute solution" S.E. Harding Prog. Biophys. Mol. Biol (T.L. Blundell ed.) 68 (1997) 207-262.

A comprehensive invited review describing the use of high precision viscometry for providing information about conformations of macromolecules in solution, focussing in particular on proteins and polysaccharides. A full set of published data is provided.

Paper 193. "Ultracentrifugation studies on the transmembrane domain of the human erythrocyte anion transporter Band 3 in the detergent C12E8". H. Cölfen, J.M. Boulter, S.E. Harding and A. Watts. Eur. Biophys. J. 27 (1998), 651-655.

This paper with the Watts team reports a sedimentation velocity and equilibrium study on the oligomeric state and conformation of the detergent solubilized transmembrane domain of the Band 3 protein, complementing Paper #165 on the cytoplasmic domain. A strong dimer is observed with no sign of dissociation at low concentration (at least to 0.1mg/ml). It appears hydrodynamically as an asymmetric particle of modest axial ratio (~3.5)

Paper 194. "Thermodynamic stability and folding of GroEL minichaperones" R. Golbik, R. Zahn, S.E. Harding and A.R. Fersht. J. Mol. Biol. 276 (1998) 505-515.

The 191-376 apical domain, and 191-345 C-terminally truncated fragment of the chaperonin GroEL, expressed in E. coli to give functional minichaperones were studied with regards their oligomeric state, stability and folding characteristics. As part of this study with the Fersht team, sedimentation velocity and equilibrium demonstrate that the minchaperones remain as functional monomers, and, in agreement with crystal structure information, show that the apical domain of GroEL is not responsible for association of the native subunits in native GroEL.

Paper 195. "Dilute solution properties of carboxymethylchitins in high ionic-strength solvent". G.M. Pavlov, E.V. Korneeva, S.E. Harding and G.A. Vichoreva. Polymer, 39 (1998) 6951-6961.

Molecular weight and conformation characteristics of the soluble chitin derivatives carboxymethylchitins, by sedimentation and viscometric analyses. The equilibrium rigidity is described in terms of the equivalent Kuhn segment length (or the simply related persistence length), and compared with chitosan and methyl cellulose: all are comparable and are semi-flexible polymers.

Paper 196. "Analytical Ultracentrifugation at Nottingham, Leicester and the National Centre for Macromolecular Hydrodynamics". K. Jumel, S.E. Harding and D.J. Winzor. The Biochemist, Oct. (1998) 53-55.

Conference report on a meeting organized for the Biochemical Society in conjunction with the British Biophysical Society hosted by the NCMH at Nottingham. The meeting marked the retirement of Professor Rowe and the 50^th anniversary of analytical ultracentrifugation at Nottingham, highlighting the seminal contribution of D.O. Jordan, J.M. Gulland and J.M. Creeth in those formative years in the work leading to the discovery by Watson and Crick of the double helix.

Paper 197. "Bead modelling using HYDRO and SOLPRO of the conformation of multisubunit proteins: sunflower and rape-seed 11S globulins". B. Carrasco, S.E. Harding and J. Garcia de la Torre. Biophys. Chem. 74 (1998) 127-133.

Application of SOLPRO (introduced in Paper #183) with HYDRO to the study of the subunit arrangements in solution of seed globulins.

Paper 198. "Mucin-biopolymer interactions for mucoadhesion" S.E. Harding. Glycobiology 8 (1998) Abs. 53. Request e-copy

Abstract from the Society for Glycobiology meeting at Baltimore, September 1998, highlighting the molecular evidence underpinning the mucoadhesive potential of chitosan and mussel foot glue protein

Paper 199. "Structure and mucoadhesion of mussel glue protein in dilute solution" M.P. Deacon, S.S. Davis, J.H. Waite and S.E. Harding. Biochemistry 37, 14108-14112.

A model for the solution structure of the mussel foot glue protein mefp1 is presented based on hydrodynamic and circular dichroism measurements, and consists of a non-repetitive globular region with a semi-flexible tail containing alternating flexible and rigid segments. The strong cationic nature of this tail makes it a potential candidate for mucoadhesion: this is clearly demonstrated using the principle of co-sedimentation in the ultracentrifuge.

Paper 200. "New strategies for modelling of antibody structure in dilute solution".S.E. Harding, B. Carrasco and J. Garcia de la Torre. Glycobiology 8 (1998) Abs. 114.

Abstract from the Society for Glycobiology meeting at Baltimore, September 1998, highlighting bead-shell modelling strategies for representing the solution conformation of antibodies, and indicating how the hydration problem may be addressed.

Paper 201. "Usefulness of the bead model algorithm SOLPRO for modelling the conformation of seed globulins" B. Carrasco, S.E. Harding and J. Garcia de la Torre. Plant Proteins from European Crops. Food and Non-Food Applications (eds. J. Gueguen and Y. Popineau) pp 153-155. Springer, Berlin (1998).

Paper presented at the Plant Protein from European Crops meeting, supporting the trigonal antiprism model for assembly of the subunits in 11S seed globulins, based on solution x-ray scattering and sedimentation data.

Paper 202. "Hydrodynamic properties of mucins secreted by primary cultures of Guinea pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution.". S. Dodd, G.A. Place, R.L. Hall and S.E. Harding. Eur. Biophys. J. (1998) 28, 38-47.

This paper describes a hydrodynamic characterisation of the mucin purified from primary culture secretions of guinea-pig tracheal epithelial cells. Comparable results for these cultured mucins were obtained for native human bronchial mucins. Combination of the weight average molecular weight with the sedimentation coefficient yielded an estimate for the free-soluton translational diffusion coefficient. Comparison with the diffusivity values measured in secretion of mucus gel suggested, after the appropriate corrections for viscosity and temperature were made: this suggested that the dissolution phase of the secretion process is not energetically driven but the hydration phase is.

Paper 203. "Dilute solution viscometry of food biopolymers". S.E. Harding. In (S.E. Hill, D.A. Ledward & J.R. Mitchell eds.) Functional Properties of Food Macromolecules, Chap. 1, Aspen/ Chapman & Hall, Maryland.

Review of the application of modern viscometric techniques to the characterisation of food proteins and polysaccharides.

Paper 204. "Advances in Ultracentrifuge Analysis" K. Jumel, D.J. Winzor and S.E. Harding. Biochem. Soc. Trans. 26 (1998) 715.

Introductory paper for the 5^th U.K. Analytical Ultracentrifuge Users meeting

Paper 205. "Advances in modelling solution properties of macromolecules and particles" J. Garcia de la Torre, S.E. Harding and B. Carrasco. Biochem. Soc. Trans (1998) 26, 716-721.

A thorough description of the HYDRO and SOLPRO bead modelling algorithms.

Paper 206. "Models for the multisubunit conformation of oil-seed globulins" B. Carrasco, J. Garcia de la Torre and S.E. Harding. Biochem. Soc. Trans. 26 (1998) 721-725.

Development of the earlier papers (#197, 201) to include new data from intrinsic viscosity and the Flory parameters F_o and P_o. Ambiguities caused by the hydration problem are discussed.

Paper 207. "COVOL: an answer to your thermodynamic non-ideality problems?" S.E. Harding, J.C. Horton and D.J. Winzor (1998). Biochem. Soc. Trans. 26 (1998) 737-741.

This paper introduces the FORTRAN algorithm COVOL for predicting the thermodynamic non-ideality second virial coefficient from user provided information about the molecular shape (tri-axial ellipsoid axial ratios from e.g. a crystal structure), hydration, valency, solution ionic strength and molecular weight. Its particular usefulness in dealing with the characterisation of interacting systems is indicated.

Paper 208. "Light Scattering" S.E. Harding & K. Jumel in (J.E. Coligan, B. Dunn, H.L. Ploegh, D.W. Speicher and P.T.Wingfield eds.) Current Protocols in Protein Science, J. Wiley & Sons, New York 7.8.1. -7.8.14 (1998).

Description of classical or total intensity light scattering (including SEC-MALLs) and dynamic light scattering for characterising proteins in solution. Provides practical protocols and troubleshooting tips.

Paper 209. "Hydrodynamic and molecular characteristics of carboxymethylchitin in solution" G.M. Pavlov, E.V. Korneeva, G.A. Vikhoreva and S.E. Harding. Polym. Sci. Ser. A. 40 (1998) 1275-1281 and Vysokomolekulyarnye Soed. Ser. A. 40 (1998) 2048-2055.

Paper in Russian (and its English translation) describing a full hydrodynamic characterisation of the polyanionic soluble derivative of chitin, carboxymethylchitin.

Paper 210. "Translational and rotational friction of lactodendrimer molecules in solution" G.M. Pavlov, E.V. Korneeva, S.A. Nepogod'ev, K. Jumel and S.E. Harding. Polym. Sci. Ser. A. 40 (1998) 1282-1289 and Vysokomolekulyarnye Soed. Ser. A. 40 (1998) 2056-2064.

Paper in Russian (and its English translation) comparing the translational frictional and viscometric properties of 5 types of glycodendrimer – dendrimers with lactose attached to the primary amine groups.

Paper 211. "Calculation of NMR relaxation, covolume and scattering - related properties of bead models using the SOLPRO computer program" Garcia de la Torre, J; Harding, S.E. and Carrasco, B. European Biophysical Journal 28 (1999), 119-132.

Further development of the SOLPRO programme introduced in Paper #183 to include the calculation, for arbitrary shaped molecules of rotational relaxation times from NMR, the molecular covolume and scattering related properties, namely the distribution of distances for the bead model, which is directly related to the angular envelope of scattering intensity, and the particle’s longest distance.

Paper 212. "The Polysaccharide Biotechnology Royal Society of Chemistry Meeting, 1997" S.E. Harding. Carbohydr. Polym. 38 (1999), 193-194.

Introductory paper for a Royal Society of Chemistry meeting organised by Harding in September 1997.

Paper 213. "Hydrodynamic properties of carbohydrate-coated dendrimers" G.M. Pavlov, E.V. Korneeva, K. Jumel, S.E. Harding, E.W. Meyer, H.W.I. Peerlings, J.F. Stoddart and S.A. Nepogodiev. Carbohydr. Polym. 38 (1999), 195-202.

Full description of the hydrodynamic properties of glycodendrimers of "generation numbers" 1-5, ranging in molecular weight as established from the sedimentation and diffusion coefficients) from 1900 to 45000Da, and corresponding hydrodynamic radius from 10 to 37 Å. Mark-Houwink types of scaling relations (hydrodynamic parameter vs. molecular weight) suggest a spherical conformation for the series with an inhomogeneous distribution of density.

Paper 214. "Size and shape of inulin in dimethyl sulphoxide solution" B.H. Azis, B. Chin, M.P. Deacon, S.E. Harding and G.M.Pavlov. Carbohydr. Polym. 38 (1999), 231-294.

This paper describes the solution properties in DMSO of two small inulin polysaccharides from different sources: Jerusalem artichoke and Chicory root. Both gave similar properties of molecular weight (from sedimentation equilibrium), intrinsic viscosity and sedimentation coefficient, consistent with a compact but highly hydrated structure.

Paper 215. "Are chitosan-mucin interactions specific to different regions of the stomach? Velocity ultracentrifugation offers a clue". M.P. Deacon, S.S. Davis, R.J. White, H. Nordman, I. Carlstedt, N. Errington, A.J. Rowe and S.E. Harding. Carbohydr. Polym. 38 (1999), 235-238.

This paper introduces the technique of "sedimentation fingerprinting" for detecting the presence of an interaction when one of the components is beyond optical registration in the ultracentrifuge (in this case through low concentration). Mucins were isolated from specific regions of the stomach but in low quantities. However their effect on the sedimentation properties of the mucoadhesive chitosan can be assessed from loss of chitosan through supramolecular complex formation. Using this procedure it is seen that mucins from different regions of the stomach (and differing levels of charged groups) show a difference in reactivity.

Paper 216. "Protein Hydrodynamics", S.E. Harding in (G. Allen ed.) Protein Structure: A Comprehensive Treatise, Vol 2. 271-305, JAI Press, Greenwich, Connecticut (1999).

Invited review describing the range of hydrodynamic techniques for size, oligomeric state and conformation studies on proteins in solution.

Paper 217. "Evidence of noncovalent dimerization of calmodulin." Lafitte, D; Heck, A.J.R; Hill, T.J, Jumel, K, Harding, S.E. and Derrick, P.J. European Journal of Biochemistry 261 (1999), 337-344.

This paper includes a determination by sedimentation equilibrium (in conjunction with the monomeric mass from mass spectroscopy measurements) of the oligomeric state of calmodulin. A monomer-dimer system is revealed, but the large dissociation constant (~500mM) indicates a weak non-covalent interaction, consistent with further observations from mass spectroscopy.

Paper 218. "Hydrodynamic properties of human erythrocyte band 3 solubilised in reduced triton X-100." Taylor, A.M, Boulter, J, Harding, S.E, Cölfen, H. and Watts, A. Biophysical Journal 76 (1999), 2043-2055.

This is the final part of a triology of papers with the Watts group on the oligomeric state of the Band 3 membrane protein. Paper #165 characterized the cytoplasmic domain as a strong dimer (in aqueous solution). Paper #193 did likewise for the transmembrane domain in detergent. In this paper, the preparation procedure had an important bearing on the oligomeric state observed in (detergent) solution. The common form showed a reversible association described by a dimer/tetramer/hexamer equilibrium, each form with a different inhibitor binding affinity.

Paper 219. "COVOL: An interactive program for evaluating second virial coefficients from the triaxial shape or dimensions of rigid macromolecules." Harding, S.E., Horton, J.C., Jones, S., Thornton, J.M. and Winzor, D.J. Biophysical Journal 76 (1999), 2432 - 2438.

This paper describes the derivation of the COVOL algorithm for evaluating thermodynamic second virial coefficients B, from molecular dimensions or shape, based on Rallison-Harding excluded volume theory for triaxial ellipsoids (Paper #26). The algorithm requires input of the triaxial shape (from e.g. a crystal structure), molecular weight, hydration estimate and valency (from titration or calculation from the amino acid composition). It can either be employed to eliminate B as a variable in the analysis of self-associating systems, or, for monodisperse systems, as a valuable route to estimating protein hydration, as illustrated in application to ovalbumin, ribonuclease A and hemoglobin.

Paper 220. "Probing activations of the prokaryotic arginine transcriptional regulator using chimeric proteins" Holtham, C.A.M., Jumel, K. Miller, C.M., Harding, S.E., Baumberg, S. and Stockley, P.G., J. Mol. Biol. 289 (1999), 707-727.

This paper includes the use of sedimentation analysis combined with gel filtration to uncover the mode of assembly of the prokaryotic arginine transcriptional regulator. The two major transcriptional factors, called ArgR and AhrC were studied and compared with two chimeras, where sequences for the N- and C-terminal regions have been swapped. All four proteins form hexamers in the presence of l-arginine at high concentration. In the absence of l-arginine or at lower protein concentration, the hexamers are in rapid equilibrium with smaller subunits, whose dominant species appears to be trimers, as expected from the earlier published crystal structure of the ArgR C-terminal fragment, with the exception of the ArgR-C chimera which apparently dissociates to dimers, suggesting that in the intact protein the DNA-binding domains may have a significant dimeric interaction. The hexamer-trimer dissociation constant, K_d is in the mM range, suggesting that trimers are the principal species at in vivo concentrations.

Paper 221. "Biopolymer Mucoadhesives" S.E. Harding, M.P. Deacon, I. Fiebrig and S.S. Davis. Biotech. Gen. Eng. Rev.16 (1999) 41-86.

This review updates an earlier version (Paper #143) which focussed more on the macroscopic aspects, to cover the more molecular and biophysical aspects of mucoadhesive phenomena. It reviews the principles of mucoadhesion for enhancing the oral (and nasal) administration of drugs, the various absorption enhancement strategies, the nature of the mucin substrate – including our understanding of the molecular biological and biophysical properties, the potential mucoadhesives – focusing on chitosan and a mussel glue protein. The assay methods are then considered focusing on the molecular methods (particularly ultracentrifugation and electron microscopy. The review considers a case study – mucoadhesive interactions with pig gastric mucin, and finally on how potential mucoadhesives could be constructed into a stable delivery system.

Paper 222. "The correct analysis of low speed sedimentation equilibrium distributions recorded by the Rayleigh interference optical system in a Beckman XL-I ultracentrifuge". D.R. Hall, S.E. Harding and D.J. Winzor. Prog. Coll. Polym. Sci. 113 (1999) 62-68.

The proliferation of analytical ultracentrifuges has also resulted in a proliferation of uncertainty, and in some cases faulty methodology, with regard correct procedure for extracting molecular weights form sedimentation equilibrium. This article illustrates the fallacy of the common practice of floating absorbance and especially Rayleigh interference base lines to obtain the meniscus concentration – an essential parameter. A procedure is provided for the appropriate extraction of the meniscus concentration from both cases, and then for the correct extraction of molecular weight.

Paper 223. "Normalized scaling relations as a natural classification of linear macromolecules according to size". G.M. Pavlov, S.E. Harding and A.J. Rowe. Prog. Coll.Polym. Sci. 113 (1999) 76-80.

This article extends the classical Mark-Houwink-Kuhn-Sakurada (MHKS or MKHS) scaling treatments for representing molecular conformation and flexibility of polymers to additional cases. This paper builds essentially on the earlier paper on Conformation zoning (Paper # 189) to provide an additional diagnostic test for zoning a macromolecule, namely a double logarithmic plot of intrinsic viscosity x mass per unit length against mass per unit length x molecular weight. A "Master Curve" plot is also presented giving a line onto which most polymer conformations lie.

Paper 224. "Combining ultracentrifugation with fluorescence to follow the unfolding of modules 16-17 of complement receptor type 1".M.D. Kirkitadze, K. Jumel, S.E. Harding, D.T.F. Dryden, M. Krych, J.P. Atkinson and P.N. Barlow. Prog. Coll. Polym. Sci. 113 (1999) 164-167.

A fragment of complement receptor CR1, comprising modules 16 and 17 has been overxpressed as a noglycosylated protein in Pichia pastoris. This paper investigates the effects of increasing GuHCl concentrations on the unfolding of the protein. Sedimentation velocity analysis, and use of the Perrin P parameter indicates that, under native conditions CR1~16-17 has an axial ratio ~5. Since it is known form structural NMR studies that the individual modules have an axial ratio ~3 one can conclude the2 modules are assembled end-to end. Both sedimentation velocity and fluorescence intensity plots gave similar trends with increasing GuHCl concentration.

Paper 225. "Combining sedimentation velocity with SEC-MALLS to probe the molecular structure of heterogeneous macromolecular systems that cannot be preparatively separated: application to three rice starches." Tongdang, T; Bligh, H.F.J; Jumel, K; and Harding, S.E. Progress in Colloid and Polymer Science 113 (1999) 185-191.

Analytical ultracentrifugation and SEC-MALLs facilitates the analysis of those mixed solute systems where it is difficult to separate preparatively. The amylose/amylopectin mixed system in starch is a classical example. SEC MALLs was employed to successfully characterise the molecular weights of the amylose component of three rice starches in DMSO but not the amylopectin which was too large. Sedimentation velocity in both the Model E ultracentrifuge with Schlieren optics and the Beckman XL-I with interference optics, both have satisfactory separation of amylose from amylopectin, permitting sedimentation coefficient evaluation, although with the latter a correction to zero time is necessary to allow for the steady loss of material during the sedimentation experiment.

Paper 226. "UV tagging leaves the structural integrity of an arabino-(4-O-methylglucurono)xylan polysaccharide unaffected" G.A. Morris, A. Ebringerova, S.E. Harding and Z. Hromadkova. Prog. Coll. Polym. Sci. 113 (1999) 201-204.

Another paper in the series of chromophore tagging of polysaccharides (cf Papers #100 & 168). Substitution of hydroxyl groups on xylan by p-carboxybenzyl (CB) bromide groups is shown to have little effect on the molecular weight (sedimentation equilibrium and little apparent effect on the conformation (as monitored by sedimentation velocity and intrinsic viscosity).

Paper 227. "Elevated temperature analytical ultracentrifugation of a low-methoxy polyuronide" G.A. Morris, S.N.G. Butler, T.J. Foster, K. Jumel and S.E. Harding. Prog. Coll. Polym. Sci. 113 (1999) 205-208.

Many macromolecules are subject to prolonged periods of elevated temperature (e.g. during the thermal processing of foodstuffs). The sedimentation behaviour of pectin as a function of temperature increases from 20^oC to 60^oC was observed in a specially adapted Model E ultracentrifuge. Parallel measurements were made of reduced viscosity. A small decrease in the reduced viscosity – if significant – with increase in temperature, suggests a slightly more flexible structure, although from the molecular weight measurements the structural integrity remains.

Paper 228. "Nonequilibrium self-association of a cpn60 chaperonin induced by tryptophan mutation." Walters, C; Cliff, M; Clarke, A. and Harding, S.E. Progress in Colloid & Polymer Science 113 (1999), 216-220.

A single point mutationY203W (tyrosine to tryptophan) causes subtle changes in the mode of assembly of the GroEL chaperonin. From molecular weight and sedimentation coefficient studies of the mutant compared to the wild-type, this mutation causes significant irreversible self-association, and appears to be another example of how sedimentation analysis can be used to probe the effects of a single point mutation (cf papers #104, #170 and #186.)

Paper 229. "Diffusion" S.E. Harding in (T.E. Creighton ed.) "Encylopedia of Molecular Biology" J. Wiley & Sons, New York, 674-678 (1999).

This article in the Encylopaedia of Molecular Biology describes the processes of translational and rotational diffusion of macromolecules in solution, how these processes can be measured and how useful information can be extracted from these parameters.

Paper 230. "Frictional coefficient, ratio" S.E. Harding in (T.E. Creighton ed.) "Encylopedia of Molecular Biology" J. Wiley & Sons, New York, 942 (1999).

This article in the Encylopaedia of Molecular Biology describes the translational frictional ratio, how it can be measured from either the sedimentation coefficient of translational diffusion coefficient and how it depends on the conformation (via the Perrin function, P) and molecular hydration of a macromolecule

Paper 231. "Dynamic light scattering" S.E. Harding in (T.E. Creighton ed.) "Encylopedia of Molecular Biology" J. Wiley &Sons, New York 1847-1849 (1999).

This article in the Encylopaedia of Molecular Biology describes dynamic light scattering, and the two principle types for molecular biologists – fixed 90^o angle and multi-angle photometers. The principles behind the autocorrelation function are briefly described, as is the usefulness of this technique for following the dynamics of processes, particularly changes in large macromolecular assemblies (e.g. swelling by water uptake by a virus)

Paper 232. "Amylose content of rice starch" M. Ramesh, J.R. Mitchell, K. Jumel and S.E. Harding. Starch/Stärke 51 (1999) 311-313.

SEC MALLs analysis on aqueous solutions of debranched starch provided estimates for the molecular weight of the amyloses (assuming debranching enzymes affected only the amylopectin, not the amylose), but also provide a route for estimating the amylose content of starch, and the amylose/amylopectin ratio, provided that there are no losses, the dn/dc of amylose is known accurately and the total concentration of amylose + amylopectin is known accurately. The method is illustrated by application to four rice starches, and results compared with the conventional iodine test.

Paper 233. "Novel size-independent modeling of the dilute solution conformation of the immunoglobulin IgG Fab' domain using SOLPRO and ELLIPS" B. Carrasco, J. Garcia de la Torre, O. Byron, D.King, C. Walters, S. Jones and S.E. Harding. Biophysical J. 77 (1999), 2902-2910.

As a precursor to representing the conformations of intact immunologically active antibody molecules in solution, it is necessary to characterise the individual domains, and in particular, to predict the hydration for the domains by comparing the shape of the molecule from the crystal structure with the experimentally measured frictional ratio (from combination of the sedimentation coefficient with molecular weight). This study involves fitting a crystal structure for IgG Fab’ to a surface ellipsoid using an algorithm of Janet Thornton and then feeding the ellipsoid axial ratios into the programme ELLIPS2 (Paper #182). A bead shell model is fitted to this using SOLPRO (Paper #183) to check that doing so leads to no significant error (bead calculations of hydrodynamic parameters are excellent but still only an approximation). Establishment of lack of error, + experimental combination with f/f_o yields a "bead model" estimate of the hydration of 0.51 g/g (0.43g/g from the ellipsoid fit) IgGFab’ is beautifully monodisperse with clearly defined hydrodynamic properties.

Paper 234. *"What is the true amylose content of rice starch" M. Ramesh, J.R. Mitchell, K. Jumel and S.E. Harding, Gums and Stabilisers for the Food Industry 10 (2000) 86-81.

Paper, based on #232 presented by Visiting Fellow, Ramesh at the Gums and Stabilisers for the Food Industry Meeting, Wrexham July 1999.

Paper 235. "Atomic force microscopy of gastric mucin and chitosan mucoadhesive systems" M.P. Deacon, S. McGurk, C.J. Roberts, P.M. Williams, S.J.B. Tendler, M.C. Davies, S.S. Davis and S.E. Harding, Biochem. J. 348 (2000), 557-563.

This paper applies the relatively new probe of atomic force microscopy to visualizing a mucus glycoprotein (pig gastric mucin) and its mucoadhesive complex with chitosan. Images of the mucin are consistent with the earlier hydrodynamic, electron microscopic and scanning tunnelling microscopic images of mucin: long linear filamentous structures. The technique offers some advantages compared to earlier imaging procedures with being under a liquid environment, although still not a true solution. The chitosan also adopted a linear conformation but a smaller average length and diameter, and appeared as a stiff-coil form, consistent with the solution measurements. The complex formed after mixing revealed large aggregates of ~ 0.7mm in diameter, again consistent with the earlier measurements. The effect of ionic strength on the complex suggested that the interaction is principally electrostatic in nature.

Paper 236."Qualitative assessment of aromatic indica rice (Oryza sativa L.): Proteins, lipids and starch in grain from somatic embryo- and seed-derived plants" K. Azhakanandam, J.B. Power, K.C. Lowe, E.C. Cocking, T. Tongdang, K. Jumel, H.F.J. Bligh, S.E. Harding and M.R. Davey, J. Plant. Physiol. 156 (2000), 783-789.

This paper includes an analysis by sedimentation velocity in the ultracentrifuge of starches produced from somatic embryogensis derived plants of the aromatic indica rice cultiva Pusa basamati. The physico-chemical properties of the amylopectin and amylose showed similar characteristics between material from the grain of somatic embryo derived plants and from the grain of seed derived plants: part of a larger study showing for the first time that the quality of the grain produced by plants regenerated form embryogenic callus of scutellum origin is not affected in any detrimental way by the culture process.

Paper 237. "The effect of the degree of esterification on the hydrodynamic properties of citrus pectin" G.A. Morris, T.J. Foster and S.E. Harding, Food Hydrocolloids. 14 (2000) 227-235.

This paper reports a hydrodynamic study on five citrus pectins of differing degrees of esterification. Estimates for the conformation dependent Wales-van Holde and frictional ratios clearly indicate increasing chain stiffness with decreasing degree of esterification (i.e. increasing charge).

Paper 238. "Trp203 mutation in GroEL promotes a self-association reaction: a hydrodynamic study" C. Walters, A. Clarke, M.J. Cliff, P.A. Lund and S.E. Harding, Eur. Biophys. J. 29 (2000), 420-428.

Builds on paper #194 on minichaperones from GroEL and #228 and examines in detail the effect of the point mutation Y203W on the assembly of GroEL. Wild type studies on GroEL using sedimentation equilibrium and velocity give excellent agreement with the results of Behlke and co-workers showing a uniform 14-mer complex. The mutant causes a significant self-association reaction beyond the 14-mer: the findings are consistent with the observations of Gibbons and coworkers who showed an increase in hydrophobic exposure due to this mutation.

Paper 239. "Polysaccharides/Centrifugation" S.E. Harding, Encylopædia of Separation Science, III, (2000) 3921-3929, Academic Press, New York.

Invited Chapter describing for the non-expert the application of analytical centrifugation to the study of polysaccharide molecular weight, heterogeneity, shape/conformation and water binding. It outlines the types of analytical ultracentrifuge and their optical systems, and the types of experiment: sedimentation equilibrium, velocity, diffusion analysis, density gradient and gel analysis.

Paper 240. "Hydrodynamic study of the behavior of chondroitin sulphate under nondestructive laser irradiation of cartilage" E.N. Sobol, A.I. Omel'chenko, A.P. Sviridov, S.E. Harding, K. Jumel, N. Jones, Proc. SPIE, 3914 (2000) 88-93.

Paper with the Sobol team describing the effect on the molecular integrity of solutions of chondroitin sulphate by irradiation with pulsed laser radiation (at 0.55 J/cm²) to reach local temperatures of 70^oC or 90^oC. SEC-MALLs showed a moderate but not spectacular degradation in molecular weight (reduction of 25% at the high temperature) with little change in the sedimentation coefficient. Intriguigingly, laser irradiated cartilage tissue was seen to induce diffusion of macromolecules into the medium possessing a similar molecular weight to chondroitin sulphate.

Paper 241. "Laser Reshaping of Cartilage" E. Sobol, A. Sviridov, A. Omeltchenko, V. Bagratashvili, M. Kitai, S.E. Harding, N. Jones, K. Jumel, M. Mertig, W. Pompe, Y. Ovchinnikov, A. Shekhter, Valerii Svistushkin, in Biotechnology & Genetic Engineering Reviews, 17 (2000), 539-564.

Review outlining the advances in the use of lasers to reform deformed cartilage. Hydrodynamic methods are contributing in helping to elucidate the mechanism of the stress relaxation process: the structure and water relations of proteoglycans and their interactions with collagen.

Paper 242. "Optoacoustic monitoring of laser correction of the ear shape" Omel’chenko, A.I., Sobol, E.N., Sviridov, A.P., Harding, S., Jumel, K., Walker, R. and Jones, N. Kvantovaya Elektronika 30 (2000) 1031-1033.

The progress of pulse-laser irradiation – similar to that of paper #240 - for the reshaping of porcine ear cartilage was monitored during the process by an optoacoustic detection system: the detection system can distinguish change in form of the tissue from an elastic state to a plastic state (corresponding to the amplitude decay period).

Paper 243. "Further observations on the size, shape and hydration of casein micelles from novel analytical ultracentrifuge and capillary viscometry approaches". Morris, G.A., Foster, T.J. and Harding, S.E., Biomacromolecules 1 (2000), 764-767.

This paper provides a hydrodynamic characterization of casein micelle systems as a prelude to forthcoming work on casein-polysaccharide interactions. The micelles are too large to characterize by SEC-MALLs or sedimentation equilibrium but the Wales-van Holde ratio from sedimentation velocity and intrinsic viscosity confirms a spherical shape. The sedimentation coefficient data can then be interpreted for spheres in terms of a hydrodynamic radius of ~78nm and a weight average molecular weight of ~ 280 millionDa. Comparison with the partial specific volume yields a hydration value of ~3.4g/g. Results are consistent with dynamic light scattering and electron microscopy.

Paper 244. "Hydrodynamic characterization of the exopolysaccharide from the halophilic cyanobacterium Aphanothece halophytica GR02: a comparison with xanthan" Morris, G.A., Li, P., Puaud, M., Liu, Z., Mitchell, J.R. and Harding, S.E., Carbohydrate Polymers 44 (2001) 261-268. Publication date: March 2001.

Paper in conjunction with Visiting Chinese Fellow P. Li reports the first detailed molecular study on the exopolysaccharide from a halophytic bacterium. It appeared to have xanthan like properties in terms of its size and conformation properties (Papers #139, 142, 158)

Paper 245. "Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b." Chebotareva, N.A., Harding, S.E. and Winzor, D.J., Eur. J. Biochem. 268 (2001) 506-513. Publication Date: January 2001.

This paper, in conjunction with Visiting Fellow from Russia, N. Chebotareva, describes a study on the oligomeric state and subunit interactions of muscle glycogen phosphorylase b, and a slow equilibrium (dimer-tetramer) is evident, sufficiently slow to allow the resolution of dimer and tetramer species by sedimentation velocity. This also provides the opportunity to examine the effect of adding an unrelated "space filling" co-solute (TMAO) as a molecular crowder on this equilibrium and the relation between the inactive (T-state) and active (R-state) forms of the subunits. The R-state is necessary for tetramer formation. It is shown that the effects of thermodynamic non-ideality on the dimer-tetramer equilibrium are being countered by those displacing the T-R isomerization equilibrium for dimer towards the smaller, non-associating T-state. This would explain the effects of high concentrations of glycerol, sucrose and ethylene glycol on inhibiting phosphorylase b activity.

Paper 246. "Molecular and structural characteristics of lactodendrimers based on poly(amidoamine)" Pavlov, G.M., Errington, N., Harding, S.E., Korneeva, E.V. and Roy, R., Polymer Science Ser. A. (Russia) 43 (2001) 118-123. Publication date: January 2001.

A new study by dynamic light scattering, analytical ultracentrifugation and intrinsic viscosity on polyamidoamine lactodendrimers investigated previously (Paper #210), and essentially agree with the earlier findings. Moreover, GPC measurements demonstrate that the polydispersity of the samples is almost insignificant, and from density and volume calculations on the data it appears that the end lactose groups are located at the periphery and do not penetrate the bulk of the molecules.

Paper 247. "Sedimentation velocity analytical ultracentrifugation", Harding, S.E. and Winzor, D.J. in (Harding SE, Chowdhry, BZ eds) Protein-Ligand Interactions: Hydrodynamics and Calorimetry" p75-103, Oxford University Press (2001). Publication date: January 2001.

Chapter in the "Practical Approach" series describes the principles of sedimentation velocity – how to measure a sedimentation coefficient, how to obtain molecular mass (incorporating boundary spreading information), the use of the optical systems and analysis of the optical records. The principles of co-sedimentation and concentration dependence are explained for ligand binding analysis, along with sedimentation coefficient ratios and "fingerprinting", and how to assess the shape of the reaction product. Quantitative analysis of ligand binding is then explained. Several examples are given and a full practical protocol is provided.

Paper 248. "Sedimentation equilibrium in the analytical ultracentrifuge" Winzor, D.J. and Harding, S.E., in (Harding SE, Chowdhry, BZ eds) Protein-Ligand Interactions: Hydrodynamics and Calorimetry" p105-135, Oxford University Press (2001). Publication date: January 2001.

Chapter in the "Practical Approach" series describes the principles of sedimentation equilibrium – the different experimental aspects compared with sedimentation velocity, how to extract molecular weight, meniscus concentration and baseline issues, average molecular weights, the MSTAR procedureand point average molecular weights. Curve fitting procedures are then described for interacting systems, the programmes NONLIN and psi analysis. Quantification of ligand binding is considered in some detail, including ligand perturbation of a self-association. Several examples are given as well as a full experimental protocol and a protocol of software.

Paper 249. "Dilute solution properties of lactosylated polyamidoamine dendrimers and their structural characteristics" Pavlov, G.M., Errington, N., Harding, S.E., Korneeva, E.V. and Roy, R., Polymer 42 (2001) 3671-3678.

Paper 250. "Cyanobacterial exopolysaccharides: their nature and potential biotechnological applications" Li, P., Harding, S.E. and Liu, Z. (2001) Biotech. Bioeng. Rev. 18, 375-404.

Paper 251. "The hydration problem in solution biophysics: an introduction" Harding, S.E. (2001) Biophys. Chem. 93, 87-91.

Paper 252. "Crystallohydrodynamics for solving the hydration problem for multi-domain proteins: open physiological conformations for human IgG1" Carrasco, B., Garcia de la Torre, J., Davis, K.G., Jones, S., Athwal, D., Walters, C., Burton, D.R. and Harding, S.E. (2001) Biophys. Chem. 93, 181-196.

Paper 253. "Analysis of thermodynamic non-ideality in terms of protein solvation" Winzor, D.J., Carrington, L.E. and Harding, S.E. (2001) Biophys. Chem. 93, 231-240.

Paper 254. "A potential role for the analytical ultracentrifuge in the experimental measurement of protein valence" Winzor, D.J., Carrington, L.E. and Harding, S.E. (2001) Biophys. Chem. 299, 235-240.

Paper 255. "Pressure cell assisted solution characterization of polysaccharides. 1. Guar gum." Pacout, D.R., Ross-Murphy, S.B., Errington, N. and Harding, S.E. (2001) Biomacromolecules 2, 1301-1309.

Paper 256. "Aspects of the structural integrity of chondroitin sulphate after laser irradiation" Jumel, K., Harding, S.E., Sobol, E., Omel’chenko, A., Sviridov, A. and Jones, N. (2002) Carbohyd. Polym. 48, 241-245.

Paper 257. "A hydrodynamic study of the depolymerisation of a high methoxy pectin at elevated temperatures" Morris, G.A., Foster,T.J. and Harding, S.E. (2002) Carbohyd. Polym. 48, 361-367.

Paper 258. "Self association of phosphorylase kinase from rabbit skeletal muscle in the presence of natural osmolyte, trimethylamine N-oxide" Chebotareva, N.A., Andreeva, I.E., Makeeva, V.F., Kurganov, B.I., Livanova, N.B. and Harding, S.E. (2002) Progr. Coll. Polym. Sci. 119, 70-76

Paper 259. "The analytical ultracentrifuge as a probe for interface transport phenomena" Harding, S.E. and Tombs, M.P. (2002) Biotech. Genet. Eng. Rev. 19, Chap. 2.

Paper 260. "Hydrolysable ATP is a requirement for the correct interaction of molecular chaperonins cpn60 and cpn10" Walters, C., Errington, N.E., Rowe, A.J. and Harding, S.E. (2002) Biochem. J., 364, 849-855.

Paper 261. "Pressure cell assisted solution characterisation of polysaccharides. 2: LBG and tara gums" Picout, D.R., Ross-Murphy, S.B., Jumel, K. and Harding, S.E. (2002) Biomacromolecules, 3, 761-767.

Paper 262. "Modification of pectin with UV-absorbing substitutents and its effect on the structural and hydrodynamic properties of the water-soluble derivatives" Morris, G.A., Hromádková, Z., Ebringerová, A., Malovíková, A., Alföldi, J. and Harding, S.E. (2002) Carbohydrate Polym. 48, 351-359.

Paper 263. "The effect of acid shock on sporulating Bacillus subtilis cells" Lee, J.K., Mohavedi, S., Harding, S.E. and Waites, W.M. (2003) J. Applied Microbiology, 94, 184-190.

Paper 264. "Hydrodynamic characterisation of chemically degraded hyaluronic acid" Hokputsa, S., Jumel, K., Alexander, C. and Harding, S.E. (2003) Carbohydrate Polym. 52, 111-117.

Paper 265. "Partial fractionation of wheat starch amylose and amylopectin using zonal ultracentrifugation" Majzoobi, M., Rowe, A.J., Connock, M., Hill, S.E., and Harding, S.E. (2003) Carbohydrate Polym. 52, 269-274.

Paper 266. "Identification of oligomerization and drug-binding domains of the membrane fusion protein EmrA" Borges-Walmsley, M.I., Beauchamp, J., Kelly, S.M., Jumel, K., Candlish, D., Harding, S.E., Price, N.C. and Warmsley, A.R. (2003) J. Biol. Chem. 278, 12903-12912.

Paper 267. "A physico-chemical comparative study on extracellular carbohydrate polymers from five desert algae" Hokputsa, S., Hu, C., Smestad Paulsen and Harding, S.E. (2003) Carbohydrate Polym, 54, 27-32.

Paper 268. "Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics" Longman, E., Kreusel, K., Tendler, S.B., Fiebrig, I., King, K., Adair, J., O’Shea, P., Ortega, A., Garcia de la Torre, J., and Harding, S.E. (2003), Eur. Biophys. J. 32, 503-510.

Paper 269. "Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome" Pettigrew, G.W., Pauletta, S.R., Goodhew, C.F., Cooper, A., Nutley, M., Jumel, K., Harding, S.E., Costa, C., Krippahl, L., Moura, I. and Moura, J. (2003) Biochemistry 42, 11968-11981.

Paper 270. "Pressure cell assisted solubilization of xyloglucans: Tamarind seed polysaccharide and detarium gum" Picout, D.R., Ross-Murphy, S.B., Errington, N. and Harding, S.E. (2003) Biomacromolecules, 4, 799-807.

Paper 271. "Biochemical interactions in 2D and 3D". Introductory remarks. Harding, S.E. and O’Shea, P. (2003) Biochem. Soc. Trans. 31, 971-972.

Paper 272. "Mucoadhesive interactions" Harding, S.E. (2003) Biochem. Soc. Trans. 31, 1036-1041.

Paper 273. "A comparison of molecular mass determination of hyaluronic acid using SEC/MALLS and sedimentation equilibrium" Hokputsa, S., Jumel, K., Alexander, C. and Harding, S.E. (2003) Eur. Biophys. J., 32, 450-456.

Paper 274. "Correlation of SEC/MALLS with ultracentrifuge and viscometric data for chitosans" Fee, M., Errington, N., Jumel, K., Illum, L., Smith, A. and Harding, S.E. (2003), Eur. Biophys. J. 32, 457-464.

Paper 275. “Studying antibody conformations by ultracentrifugation and hydrodynamic modelling” Harding, S.E., Longman, E., Carrasco, B., Ortega, A. and Garcia de la Torre, J. (2003) Methods in Molecular Biology, 248, 93-113.

Paper 276. “Ligand-mediated dimerization of a carbohydrate-binding module reveals a novel mechanism for protein-carbohydrate recognition” Flint, J., Nurizzo, D., Harding, S.E., Longman, E., Davies, G.J., Gilbert, H.J. and Bolam, D.N. (2004), J. Mol. Biol. 337, 417-426.

Paper 277. “Bioactive polysaccharides from the stems of the Thai medicinal plant Acanthus erbracteatus: their chemical and physical features” Hokputsa, S., Harding, S.E., Inngjerdingen, K., Jumel, K., Michaelsen, T.E., Heinze, T., Koschella, A. and Paulsen, B. (2004) Carbohyd. Res. 339, 753-762.

Paper 278. “The Mycobacterium tuberculosis Chaperonin 10 Monomer Exhibits Structural Plasticity”. Fossati, G., Cremonesi, P., Izzo, G., Rizzi, E., Sandrone, G., Harding, S.E., Errington, N., Walters, C., Henderson, B., Roberts, M.M., Coates, A.R. and Mascagni, P. (2004), Biopolymers 75, 148-162.

Paper 279. “Use of the sedimentation coefficient for modelling antibodies. Refinements to the crystallohydrodynamics approach”. Harding, S.E., Longman, E., Ortega, A., Kreusel, K., Tendler, S.J.B., King, K. and Garcia De La Torre, J. (2004). Progress in Colloid & Polymer Science, 127, 113-118.

Paper 280. “Revisiting Dingesmere”. Cavill, P., Harding, S.E. and Jesch, J. (2004) J. English Place Name Soc. 36, 25-38.

Paper 281. “A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase”. Pauleta, S.R., Cooper, A., Nutley, M., Errington, N., Harding, S.E., Guerlesquin, F., Goodhew, C.F., Moura, I., Moura, J.J.G. and Pettigrew, G.W. (2004) . Biochemistry 43, 14566-14576.

Paper 282. “Characterization of bovine serum albumin/chlorogenic acid solution mixtures by analytical ultracentrifugation”. Seifert, A., Rawel, H.M., Harding, S.E. and Kroll, J. (2004) Progress in Colloid & Polymer Science 127, 83-88.

Paper 283. “Determination of protein charge by capillary zone electrophoresis”. Winzor, D.J., Jones, S. and Harding, S.E. (2004). Analytical Biochemistry 333, 225-229.

Paper 284. “Extent of charge screening in aqueous polysaccharide solutions”. Winzor, D.J., Carrington, L.E., Deszczynski, M. and Harding, S.E. (2004) Biomacromolecules 5, 2456-2460.

Paper 285. “Limitations of the ultracentrifugal approach for measuring the effective net charge of a macroion”. Winzor, D.J., Carrington, L.E. and Harding, S.E. (2004) Analytical Biochemistry 333, 114-118.

Paper 286. “Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b”. Chebotareva, N.A., Kurganov, B.I., Harding, S.E. and Winzor, D.J. (2005) Biophysical Chemistry 113, 61-66.

Paper 287. “Challenges for the modern analytical ultracentrifuge analysis of polysaccharides”. Harding, S.E. (2005). Carbohydrate Research 340, 811-826.

Paper 288. “Analysis of polysaccharide size, shape and interactions” Harding, S.E. In (Scott, D., Harding, S.E., & Rowe, A.J. eds) Analytical ultracentrifugation. Techniques and Methods. Royal Society of Chemistry, Cambridge, pp231-252 (2005).

Paper 289. “The ELLIPS suite of whole-body protein conformation algorithms for Microsoft Windows”. Harding, S.E., Cölfen, H. and Aziz, Z. In (Scott, D., Harding, S.E., & Rowe, A.J. eds) Analytical ultracentrifugation. Techniques and Methods. Royal Society of Chemistry, Cambridge, pp468-483 (2005).

Paper 290. “Analysis of polysaccharides by ultracentrifugation: size, conformation and interactions in Solution” Harding S.E. (2005) in Advances in Polymer Science (Polysaccharides I: Structure, Characterisation and Use, Ed. T. Heinze) 186, Chap. 5.

Paper 291. "Bioactive pectic polysaccharides from Glinus oppositifolius (L.) Aug. DC., a Malian medicinal plant, isolation and partial characterization". Inngjerdingen, K.T., Debes, S.C., Inngjerdingen, M., Hokputsa, S., Harding, S.E. Rolstad, B., Michaelesn, T.E., Diallo, D. and Smestad Paulsen, B. (2005) Journal of Ethnopharmacology, 101, 204-214.

Paper 292. "Structural and immunological studies of a pectin and a pectic arabinogalactan from Veronia kotschyana Sch. Bip. Ex Walp. (Asteraceae)". Negard, C.S., Matsumoto, T. Inngjerdingen, M., Inngjerdingen, K.., Hokputsa, S., Harding, S.E. Diallo, D., Kiyohara, H., Smestad Paulsen, B. and Yamada, H. (2005) Carbohydrate Research, 340, 115-130.

Paper 293. “Identifying differences in solution conformation of two chimeric IgG3 antibodies through triple detection SEC”. Longman, E., Harding, S.E. and Marheineke, N. (2005) LCGC 18, 662-668.

Paper 294. “Identifying differences in solution conformation of two chimeric IgG3 antibodies through triple detection SEC”. Longman, E., Harding, S.E. and Marheineke, N. (2006) LCGC North America 24, 64-72.

Paper 295. "Trends in mucoadhesive analysis". Harding, S.E. (2006) Trends in Food Science & Technology, 17, 25-262.

Paper 296. “Oligomerization of hydroperoxide lyase a novel P450 enzyme in plants”. Khan, A., Hughes, R.K., Belfield, E.J., casey, R. Rowe, A.J. and Harding, S.E. (2006) Progress Colloid Polymer Sci. 131, 116-120.

Paper 297. "Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer-micelle association". Hughes, R.K., Belfield, E.J., Muthusamay, M., Khan, A., Rowe, A., Harding, S.E., Fairhurst, S.A., Bornemann, S., Ashton, R., Thorneley, R.N.F. and Casey, R. (2006) Biochem. J. 395, 641-652. Supplementary material (255 Kb).

Paper 298. “Glycomics: from glycobiology to diagnostics and therapeutics”. Bedford, C., Cass, A., Francois, I. and Harding, S.E. (2006) Drug News Perspect 19, 163-172.

Paper 299. “Damaged starch characterization by ultracentrifugation”, Tester, R.F., Patel, T. and Harding, S.E. (2006) Carbohydrate Research 341, 130-137.

Paper 300. “Negative second virial coefficients as predictors of protein crystal growth: evidence from sedimentation equilibrium studies that refutes the designation of those light scattering parameters as osmotic virial coefficients". Deszczynski, M., Harding, S.E. and Winzor, D.J. (2006) Biophysical Chemistry 120, 106-113.

Paper 301. “Crystallohydrodynamics of Protein Assemblies: Combining Sedimentation, Viscometry, and X-Ray Scattering” Lu, Y., Longman, E., Davis, K., Ortega, A., Grossmann, J.G., Michaelsen, T.E., García de la Torre, J., and Harding, S.E. (2006) Biophys. J. 91, 1688-1697.

Paper 302. "Water-soluble polysaccharides with pharmaceutical importance from Durian rinds (Durio zibethinus Murr.): isolation, fractionation, characterisation and bioactivity" Hokputsa S., Gerddit W., Pongsamart S., Inngjerdingen K., Heinze Th., Koschella A., Harding S.E., Paulsen B.S. (2004) Carbohydrate Polymers 56 (2004) 471-781.

Paper 303. "Weak self-association in a carbohydrate system". Patel, T.R, Harding, S.E, Ebringerova, A., Deszczynski, M., Hromadkova, Z., Togola, A., Paulsen, B.S., Morris, G.A and Rowe, A.J., (2007). Biophys. J. 93, 741-749.

Paper 304. "Solution conformation of wild type and mutant IgG3 and IgG4 immunoglobulins using Crystallohydrodynamics: possible implications for complement activation". Lu, Y., Harding, S.E., Michaelsen, T.E., Longman, E., Davis, K.G., Ortega, A., Grossmann, J.G. Sandlie, I. and Garcia De La Torre, J, (2007). Biophys. J., 93, 3733-3744.

Paper 305. "The Effect of a Point Mutation on the Stability of IgG4 as Monitored by Analytical Ultracentrifugation". Lu, Y., Harding, S.E., Rowe, A.J., Davis, K.G., Fish, B., Varley, P., Gee, C., Mulot, S. (2008) Journal of Pharmaceutical Science. 97. 948-957.

Paper 306. "Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies". Nobbmann U, Connah M, Fish B, Varley P, Gee C, Mulot S, Chen J, Zhou L, Lu Y, Shen F, Yi J, Harding SE. (2007) Biotechnology and Genetic Engineering Reviews. Vol. 24. 117-128.

Paper 307. "Immunological and Structural Properties of a Pectic Polymer from Glinus Oppositifolius", Inngjerdingen, K.T., Patel, T.R., Chen, X., Kenne, L., Allen, S., Morris, G.A., Harding, S.E., Matsumoto, T., Diallo, D., Yamada, H., Michaelsen, T.E., Inngjerdingen, M., Paulsen, B.S. (2007) Glycobiology 17, 1299-1310.

Paper 308. "Molecular Flexibility of Methylcelluloses of Differing Degree of Substitution by Combined Sedimentation and Viscosity Analysis". Patel TR, Morris GA, Garcia de la Torre J, Ortega A, Mischnick P, Harding SE. (2008). Macromolecular Bioscience, 8, 1108-1115.

Paper 309. "Molar mass and solution conformation of branched α(1→4), α(1→6) glucans. Part I: Glycogens in water". Morris, G.A., Ang, S., Hill, S.E., Lewis, S., Shäfer, B., Nobbmann, U. and Harding, S.E. (2008) Carbohydrate Polym. 71, 101-108.

Paper 310. "Pressure cell assisted solution characterisation of galactomannans 3". Application of analytical ultracentrifugation techniques. Patel, T. R.,  Picout, D. R., Harding, S. E. and Ross-Murphy, S. B. (2006) Biomacromolecules. 7, 3513-3520.

Paper 313. "Effect of small, acid-soluble proteins on spore resistance and germination under a combination pressure and heat treatment". Lee, J.K., Movahedi, S., Harding, S.E., Mackey, B.M. and Waites, W.M. (2007) J. Food. Protect. 70, 2168-2171.

Paper 314. "Global hydrodynamic analysis of the molecular flexibility of galactomannans". Morris, G.A., Patel, T.R., Picout, S.R., Ross-Murphy, S.B., Ortega, A., Garcia de la Torre, J. and Harding, S.E. (2008) Carbohydrate Polymers, 72, 356-360.

Paper 315. "In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes". Israilides, C., Kletsas, D., Arapoglou, D., Philippoussis, A., Pratsinis, H., Ebringerova, A., Hribalova, V. and Harding, S.E. (2008) Phytomedicine, 15, 512-519.

Paper 316. "Effect of PEGylation on the solution conformation of antibody fragments",  Lu Y., Harding S.E., Turner A., Smith B., Athwal D.S.,Grossmann J.G., Davis K.G. and Rowe A.J. (2008) J. Pharm. Sci. 97, 2062-2079.

Paper 317. "Molecular flexibility of citrus pectins by combined sedimentation and viscosity analysis". Morris, G.A., García de la Torre, J., Ortega, A., Castile, J., Smith, A. and Harding, S.E. (2008) Food Hydrocolloids, 22, 1435-1442.

Paper 318. "Pectic polysaccharides from Biophytum petersianum Klotzsch, and their activation of macrophages and dendritic cells". Inngjerdingen M, Inngjerdingen KT, Patel TR, Allen S, Chen XY, Rolstad B, Morris GA, Harding SE, Michaelsen TE, Diallo D, Paulsen BS. (2008). Glycobiology, 18, 1074-1084.

Paper 319. "Global conformation analysis of irradiated xyloglucans". Patel TR, Morris GA, Ebringerová A, Vodenicarová M, Velebny V, Ortega A, Garcia de la Torre JG, Harding SE. (2008). Carbohydrate Polymers, 74, 845-851.

Paper 320."Studies on the molecular flexibility of novel dendronized carboxymethyl cellulose derivatives".  Pohl, M, Morris GA, Harding, SE, Heinze T. (2009). European Polymer Journal, 45, 1098-1110.

Paper 321. "Various non-injectable delivery systems for the treatment of Diabetes mellitus". Yadav N, Morris GA, Harding SE, Ang S, Adams GG. (2009). Endocrine, Metabolic & Immune Disorders - Drug Targets, 9, 1-13.

Paper 322. "Polysaccharides, Microbial". Morris GA, Harding SE. (2009). In: Encyclopedia of Microbiology (Third Edition) Schaechter M (Ed.). Elsevier: Amsterdam, pp 482-494.

Paper 323."Looking for Vikings in north-west England". Griffiths D, Harding SE and Jobling MA (2008) British Archaeology 103, 18-25.

Paper 324. "Analysis of the continuous phase of the modified waxy maize starch suspension". DESSE, M., ANG, S., MORRIS, G. A., ABU-HARDAN, M., WOLF, B., HILL, S. E., HARDING, S. E., BUDTOVA, T., and MITCHELL, J. R., (2009). Carbohydrate Polymers, 77, 320-325.

Paper 325."A novel global hydrodynamic analysis of the molecular flexibility of the dietary fibre polysaccharide konjac glucomannan". KöK, M. S., ABDELHAMEED, A. S., ANG, S., MORRIS, G. A., and HARDING, S. E., (2009). Food Hydrocolloids, 23, 1910-1917.

Paper 326. "Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach". MORRIS, G. A., CASTILE, J., SMITH, A., ADAMS, G. G., and HARDING, S. E., (2009). Carbohydrate Polymers, 76, 616-621.

Paper 327. "Combined hydrodynamic approach to size, heterogeneity, conformation and flexbility of bio-based macromolecules", HARDING, S.E., 2009. European Polymer Congress (EPF09) IL1-9, 107.

Paper 328. "The kinetics of chitosan depolymerisation at different temperatures", MORRIS, G.A., CASTILE, J. SMITH, A., ADAMS, G.G. and HARDING, S.E., (2009). Polymer Degradation and Stability, 94, 1344-1348.

Paper 329. "Materials for encapsulation", WANDREY, C., BARTKOWIAK, A. and HARDING, S.E. (2009). In (Zuidam, N.J. and Nedovic, V. eds ) Encapsulation Technologies for Active Food Ingredients and Food Processing, Springer, New York, pages 31-100.

Paper 330. "Structure and heterogeneity of gliadin: a hydrodynamic evaluation". Ang, S., Kogulanathan, J., Morris, G. A., Kök, M. S., Shewry, P. R., Tatham, A. S., Adams, G. G., Rowe A. J. and Harding, S. E. (2010). European Biophysics Journal, 39, 255-261.

Paper 331. "Hydrodynamic and mass spectrometry analysis of nearly-intact human fibrinogen, chicken fibrinogen, and of a substantially monodisperse human fibrinogen fragment X". Cardinali, B., Profumo, A., Aprile, A., Byron, O., Morris, G., Harding, S. E., Stafford, W. F. and Rocco, M. (2010). Archives of Biochemistry and Biophysics, 493, 157-168.

Paper 332. "An analytical ultracentrifuge study on ternary mixtures of konjac glucomannan supplemented with sodium alginate and xanthan gum". Abdelhameed, A. S., Ang, S., Morris, G. A., Smith, I., Lawson, C., Gahler, R., Wood, S. and Harding, S. E. (2010). Carbohydrate Polymers, 81, 145-148.

Paper 333. "Molecular weight distribution evaluation of polysaccharides and glycoconjugates using analytical ultracentrifugation". Harding, S. E., Abdelhameed, A. S. and Morris, G. A. (2010). Macromolecular Bioscience, 10, 714-720.

Paper 334. "Physical characterisation of the rhamnogalacturonan and homogalacturonan fractions of sugar beet (beta vulgaris) pectin". Morris, G. A., Ralet, M-C., Bonnin, E., Thibault, J-F. and Harding, S. E. (2010). Carbohydrate Polymers, 82, 1161-1167.

Paper 335. "Stem cells: the therapeutic role in the treatment of diabetes mellitus". Adams, G. G., Buttery, L., Stolnik, S., Morris, G. A., Harding, S. E. and Wang, N. (2010). Biotechnology and Genetic Engineering Reviews, 27, 285-304.

Paper 336. " Some observations on the effects of bioprocessing on biopolymer stability", Harding, S.E. (2010) J. Drug. Target, 18,732-740.

Paper 337. "Polysaccharide drug delivery systems based on pectin and chitosan". Morris, G. A., Kök, M. S., Harding, S. E. and Adams, G. G. (2010). Biotechnology and Genetic Engineering Reviews, 27, 257-284.

Paper 338. "Insight into protein–protein interactions from analytical ultracentrifugation", Harding, S.E. and Rowe, A.J. (2010)Biochem. Soc. Trans. 38, 901-907.

Paper 339. "Dr. Michael Creeth. Scientist who helped pave the way for Watson and Crick". Harding, S.E. (2010)The Independent, 31st March, page 39.

Paper 340. "James Michael Creeth (1924-2010)". Harding, S.E. and Winzor, D.J. (2010) The Biochemist, 32, April, 44-45.

Paper 341. "James Michael Creeth, 1924-2010". Harding, S.E. and Winzor, D.J. (2010) Macromolecular Bioscience, 10, 696-699.

Paper 342. Subatomic structure of the laminin -1 short arm reveals an extended and curved multidomain assembly. Patel, T. R., Morris, G. A., Zwolanek, D., Koch, M., Harding, S. E., Li, J. and Stetefeld, J. (2010). Matrix Biology,29,565-572.

Paper 343. "The effect of different storage temperatures on the physical properties of pectin solutions and gels". Morris, G. A., Castile, J., Smith, A., Adams, G. G. and Harding S. E. (2010). Polymer Degradation and Stability, 84, 1430-1434.

Paper 344. "On the hydrodynamic analysis of conformation in mixed biopolymer systems". Harding, S. E., Abdelhameed, A. S. and Morris, G. A. (2010). Polymer International, 60, 2-8.

Paper 345. "The hypoglycaemic effect of pumpkins as anti-diabetic and functional medicines". Adams, G.G., Imran, S., Mohammad, A., Kok, S., Gray. D.A., Channell, G.A., Morris, G.A. and Harding, S.E (2011). Food Research International, 44, 862-867.

Paper 346. "On the hydrodynamic analysis of conformation in mixed biopolymer systems". Harding, S.E., Abdelhameed, A.S. and Morris, G.A (2011) . Polymer International, 60, 2-8.

Paper 347. "The effect of prolonged storage at different temperatures on the particle size distribution of tripolyphosphate (TPP) - chitosan nanoparticles". Morris, G.A., Castile, J., Adams, G.G. and Harding, S.E. (2011) Carbohydrate Polymers, 84, 1430-1434.

Paper 348. "Extended Fujita approach to the molecular weight distribution of polysaccharides and other polymeric systems". Harding, S.E., Schuck, P., Abdelhameed, A.S., Adams, G., Kok, M.S. and Morris, G.A. (2011)Methods, 54, 136-144.

Paper 349. "Studies on macromolecular interactions in ternary mixtures of konjac glucomannan, xanthan gum and sodium alginate", Harding, S.E., Smith, I.H., Lawson, C.J., Gahler, R.J. and Wood, S. (2011) Carbohydrate Polymers 83, 329–338

Paper 350. "Protein-like oligomerisation of carbohydrates". Heinze, T., Nikolajski, M., Daus, S., Besong, T.M.D., Michaelis, N., Berlin, P., Morris, G.A., Rowe, A.J. and Harding, S.E (2011) Angewandte Chemie - in press.

Paper 351 "The spin doctors" Associated article by Simon Harvey to paper#196 published by the University of Nottingham Newsletter, December 1998, about the work of Stephen Harding, the NCMH and the Nottingham pioneers of the ultracentrifuge.

Paper 352. "Formation and biochemical characterization of tube/pelle death domain complexes: Critical regulators of postreceptor signaling by the Drosophila Toll receptor". Schiffmann, D.A., White, J.H.M., Cooper, A., Nutley, M.A., Harding, S.E., Jumel, K., Solari, R., Ray, K.P., Gay, N.J., Biochemistry 38, 11722-11733 (1999).

Paper 353. Harding, S.E. (2000) Locations and Legends. In Cavill, P., Harding S.E. and Jesch, J. Wirral and its Viking Heritage. English Place Name Society, UK.

Paper 354. "Protein-ligand interactions and their analysis", Chowdhry, B.Z and Harding, S.E. in (Harding SE, Chowdhry, BZ eds) Protein-Ligand Interactions: Hydrodynamics and Calorimetry" p75-103, Oxford University Press (2001). Publication date: January 2001.

Paper 355. "Genes affecting starch biosynthesis exert pleiotropic effects on the protein content and composition of pea seeds" Hughes, R. K., Desforges, N., Selwood, C., Speirs, C. I., Sinnaeve, G., Gorton, P. G., Wiseman, J., Jumel, K., Harding, S. E., Hill, S. E., Street, V., Wang, T. L. and Hedley, C. L. Journal of the Science of Food and Agriculture 81, 877-882 (2001).

Paper 356. "The electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans". Pettigrew, G.W., Goodhew, C.F., Cooper, A., Nutley, M., Jumel, K. and Harding, S.E. Biochemistry, 42(7), 2046-55.

Paper 357. "The Wirral carrs and holms". Harding, S.E. (2007) Journal of the English Place Name Society, 39, 45-57.

Paper 358. "Paley Johnson, 1917-2011", Harding, S.E. (2011) The Biochemist, 33, December, 66-67.

Paper 359. TIMÓTEO CG, TAVARES P, GOODHEW CF, DUARTE LC, JUMEL K, GÍRIO FMF, HARDING S, PETTIGREW GW and MOURA I, 2003. Ca2+ and the bacterial peroxidases: the cytochrome C peroxidase from Pseudomonas stutzeri. Journal of Biological Inorganic Chemistry, 8, 29-37.

Paper 360. ADAMS, G.G., IMRAN, S., WANG, S., MOHAMMAD, A., KOK, M.S., GRAY, D.A., CHANNELL, G.A. & HARDING, S.E. (2011) The hypoglycaemic effect of pumpkin seeds, trigonelline (TRG), nicotinic acid (NA) and D-chiro-inositol (DCI) in controlling glycaemic levels In Diabetes Mellitus. Critical Reviews in Food Science and Nutrition (in press)