Immo Fiebrig

Transmission electron microscopy studies on pig gastric mucin and its interactions with chitosan

Transmission electron microscopy has been employed to visualize the molecular structure and organization of a highly purified preparation of pig gastric mucin (molar mass M~9 × 106g/mol, as determined by low speed sedimentation equilibrium), prepared for microscopy by two completely independent methods. Samples were prepared for imaging by air drying on mica (in the presence of 50% glycerol) as well as critical point drying in acetone/CO2. The data appear consistent with the accepted linear model for mucins, and are consistent with regions of variable degrees of glycosylation along the polypeptide backbone chain, with the overall conformation that of a loose or random coil. The behaviour of this mucin in a dilute solution mixture with the potential mucoadhesive polymer chitosan (M~1 60,000 g/mol) was then explored, and a clear interaction was demonstrated, consistent with dilute solution measurements using sedimentation velocity analysis in the analytical ultracentrifuge.

Rapid size distribution and purity analysis of gastric mucus glycoproteins by size exclusion chromatography/multi angle laser light scattering

The weight average molar masses and molar mass distributions of two commercial pig gastric mucins and two fresh mucin preparations were determined by the technique of size exclusion chromatography coupled on-line to multi angle laser light scattering (SEC/MALLS). Both commercial samples exhibited much lower molar mass averages than the freshly prepared material and contained more impurities. On the other hand, a fresh mucin preparation examined after being stored frozen for 18 months revealed a slight increase in molar mass.

Colloidal gold and colloidal gold labelled wheat germ agglutinin as molecular probes for identification in mucin/chitosan complexes

Polyelectrolyte complex formation between pig gastric mucin and chitosan and its implications to mucoadhesion have recently been studied using techniques such as analytical ultracentrifugation, static light scattering and turbidimetry (Fiebrig, I., Harding, S.E. and Davis, S.S. (1994) Prog. Coll. Polym. Sci.93, 66–73; Fiebrig, I., Harding, S.E., Rowe, A.J., Hyman, S.C. and Davies, S.S. (1995) Carbohyd. Polym.28, 239-44). The findings suggested the formation of large complexes, which were then visualised using electron microscopy. The present investigation employed electron microscopy combined with colloidal gold conjugates in order to identify and localize chitosan within the mucin/chitosan complex. Images of apparently dispersed chitosan molecules could be obtained using an anionic dye molecule, Orange II, which is known to interact stoichiometrically with the cationic sites on chitosan. Once chitosan was complexed with mucin, the colloidal gold labelled techniques revealed that chitosan is concentrated in the centre of the complex, surrounded by a possibly more hydrophilic layer of mucin.

Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(γ4): cB72.3(γ4A), devoid of inter-chain disulfide bridging, and cB72.3(γ4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(γ4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(γ4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(γ4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.