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    Unravelling the molecular contributions to collagen higher order structure : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Visser, Danielle Renee
    Abnormal levels of cross-linking in fibrillar collagen strands have been shown to cause a number of human and animal diseases. Cross-linking is a vital step in fibrillogenesis and contributes greatly to the structural integrity of collagenous tissues. Conversely, defects in cross-link formation can significantly alter fibrillar organisation and lead to pathogenesis. Because collagen cross-links form on collagen-specific hydroxylated lysine residues, an understanding of the link between hydroxylysine and cross-link concentrations is needed to determine whether the level of hydroxylysine, the stereochemistry of these hydroxylysine residues, or other post-translational modifications such as glycosylation affect the level of cross-linking in tissue. While some research has been done to elucidate the connection between the two in different tissue types from the same animal, little has been undertaken to relate hydroxylation and glycosylation of lysine and hydroxylysine to the concentration and types of cross-links in different species. Furthermore, no research has been done to compare the relative distribution of diastereomers of hydroxylysine even within the same species. In order to make a valid comparison, collagen needs to be purified from skin to a high degree and separated into different collagen types and sub- structures as much as possible. To achieve this, the extraction and purification of collagen from the skins of four different mammalian species displaying different skin tensile strengths has been optimised. Different extraction methods were used to prevent the loss of specific features of the collagens that were characterised that may otherwise be lost. Amino acid analysis revealed that while the ratios of the two hydroxylysine diastereomers differed between different animals and extraction methods, the differences were not significant. Mass spectral analysis of cross-links showed that goat skin differed from the other three animals in its cross-link profile. Amino acid analysis combined with mass spectral analysis revealed that on average 70% of proline residues were hydroxylated, a figure much higher than previously thought. Mass spectral analysis also revealed that there are some differences between the glycosylation pattern of different animals, and the ratios of the different types of collagen which are extracted from each animal. While these findings need to be confirmed, they challenge some long held beliefs about the collagen molecule and provide a firm foundation for future work.
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    Stabilization of enzymes by chemical modifications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, New Zealand
    (Massey University, 2011) Pattamawadee, Tananchai; Tananchai, Pattamawadee
    This study focused on thermostabilization of enzymes in solution by intramolecular crosslinking of the specific functional groups within an enzyme molecule. Three model enzymes were used: α-amylase of Aspergillus oryzae (EC 3.2.1.1), β-galactosidase of Aspergillus oryzae (EC 3.2.1.23) and extracellular invertase (EC 3.2.1.26) of Saccharomyces cerevisiae. Crosslinking was examined using the following homobifunctional reagents: diisocyanates (O=C=N(CH2)nN=C=O, n = 4, 6, 8), diimidoesters (CH3O(=NH)C(CH2)nC(=NH)OCH3, n = 4, 5, 6) and diamines (NH2(CH2)nNH2, n = 0, 2, 4, 6, 8, 10, 12). The concentration of the enzymes was kept low at 0.9 μM in attempts to promote intramolecular crosslinking as opposed to intermolecular crosslinking. Only invertase could be stabilized relative to controls by crosslinking with diisocyanates. Invertase (0.9 μM) crosslinked with 1,4-diisocyanatobutane (n = 4; or butamethylene diisocyanate, BMDC) and 1,6-diisocyanatohexane (n = 6) showed enhanced thermostability. Stability was improved dramatically by crosslinking invertase with 20-30 μM of the reagent. Molecular engineering of invertase by crosslinking reduced its first-order thermal denaturation constant at 60 °C from 1.232 min-1 for the native enzyme to 0.831 min-1 for the stabilized enzyme. Similarly, the best crosslinking treatment increased the activation energy for thermal denaturation from 372 kJ mol-1 for the native invertase to 517 kJ mol-1 for the stabilized enzyme. Values of the Michaelis- Menten parameters (Km and νmax) showed a reduced efficiency of invertase after the crosslinking treatment. The nature of the crosslinking was examined using size exclusion chromatography (SEC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) and multiple angle laser light scattering (MALLS). Depending on the conditions used, both intermolecular and intramolecular crosslinking occurred. The estimated molecular weight of the intermolecularly crosslinked invertase appeared to be much higher compared to the intramolecularly crosslinked invertase and the native invertase. In attempts to simplify certain analyses, attempts were made to remove the carbohydrate moiety from crosslinked invertase (a glycoprotein) molecule. Deglycosylation with PNGase F achieved a significant reduction of carbohydrate for the native invertase but not for the intra- and intermolecularly crosslinked invertase. Circular dichroism (CD) measurements showed that crosslinking with BMDC affected slightly the secondary structure of invertase. The nature of the crosslinking that might be occurring in invertase molecule was further studied using small model oligopeptides, small nonglycosylated enzymes (hen egg white lysozyme and pepsin) and glycoprotein models (ovalbumin). Crosslinking of the model pentapeptide (0.9 μM) suggested that crosslinking with BMDC involved reaction between BMDC and the amino group of lysine or the carboxylate at C-terminal of the pentapeptide. Using a heptapeptide (1 mM) in crosslinking with BMDC showed a changed hydrophobicity of the crosslinked peptide. The crosslinking treatment of lysozyme (3.5 mM) with BMDC clearly produced an intermolecularly crosslinked lysozyme as evidenced by SEC and SDS-PAGE. A changed net charge of lysozyme after the crosslinking treatment was demonstrated using native PAGE. Mass spectrometry was used to then prove the intramolecular crosslinking of lysozyme with BMDC. CD spectra of the intramolecularly crosslinked lysozyme showed it be more resistant to thermal unfolding relative to native lysozyme.