Characterisation of insulin analogues therapeutically available to patients.

The structure and function of clinical dosage insulin and its analogues were assessed. This included 'native insulins' (human recombinant, bovine, porcine), 'fast-acting analogues' (aspart, glulisine, lispro) and 'slow-acting analogues' (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein.

Application of novel analytical ultracentrifuge analysis to solutions of fungal mannans.

Polysaccharides, the most abundant biopolymers, are required for a host of activities in lower organisms, animals, and plants. Their solution characterization is challenging due to their complex shape, heterogeneity, and size. Here, recently developed data analysis approaches were applied for traditional sedimentation equilibrium and velocity methods in order to investigate the molar mass distribution(s) of a subtype of polysaccharide, namely, mannans from four Candida spp. The molecular weight distributions of these mannans were studied using two recently developed equilibrium approaches: SEDFIT-MSTAR and MULTISIG, resulting in corroboratory distribution profiles. Additionally, sedimentation velocity data for all four mannans, analyzed using ls-g*(s) and Extended Fujita approaches, suggest that two of the fungal mannans (FM-1 and FM-3) have a unimodal distribution of molecular species whereas two others (FM-2 and FM-4) displayed bi-modal and broad distributions, respectively: this demonstrates considerable molecular heterogeneity in these polysaccharides, consistent with previous observations of mannans and polysaccharides in general. These methods not only have applications for the characterization of mannans but for other biopolymers such as polysaccharides, DNA, and proteins (including intrinsically disordered proteins).