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    Explorations into the nature of insulin binding to oxidized dextran : this thesis was presented in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University
    (Massey University, 1998) Li, Yuming
    The results reported in this thesis comprise an investigation into the conjugation of insulin to oxidized dextran, various release studies from the conjugates, and an attempt to interpret the binding nature of the conjugates. A model system involving the sustained release from insulin-dextran conjugates has been employed in this study. For insulin, up to 3 potential sites only (A1-Gly. B1-Phe and B29-Lys) were expected to bind to oxidized dextran. The rate of release and the maintenance of activity of the released protein are vital to such systems. Success in the interpretation of the binding nature of the conjugate will allow us to investigate its relationship to the rate of release. The desired rate of release for the sustained release of protein could then be achieved, once the projected binding could be obtained. Activation of dextran was achieved by periodate oxidation to give levels of 8%, 16% and 27% oxidized dextran. Insulin was chosen for its relatively 'uncomplicated' structure and few possible sites available for binding with activated dextran. Insulin was bound to the dextran through imine bonds. Complex formation was examined under a wide range of conditions. Initial studies were begun with the determination of a desirable basic molar ratio. A molar ratio of insulin to 8% activated dextran of 10 : 1 arose from this set of experiments. Insulin was bound to 27% activated dextran at pH 7.4, pH 9 and pH 10. In the cases of pH 9 and pH 10, many more lower MW complexes were formed than at pH 7.4. It seemed that the higher the pH of formation, the more crosslinks occurred between an insulin molecule and dextran molecules in the lower MW range. Approximate physiological pHs (pH 7.1-7.8) were used for complex formation in all subsequent experiments. Release studies were carried out under approximate physiological conditions (pH 7.4, 37°C). Immediate release was observed upon isolation by size exclusion chromatography. The greatest release occurred in the first 24 hours for all three activation levels. The higher the activation level of dextran, the lower the level of release. An equilibrium was established after several days' release and studies at 37°C produced the expected result: greater release relative to ambient. A number of studies were carried out with complex after sodium cyanoborohydride had been used to reduce the imine bonds. The first set of experiments on the reduced complexes was enzymatic cleavage studies, which employed trypsin and α-chvmotrypsin. The results for trypsin digestion of the reduced insulin-27% oxidized dextran complex indicated partial binding had occurred at B29-Lys, in combination with full binding at B1 and/or Al. Amino acid analysis results of the isolated complex after trypsin digestion indicated about 90% binding occurred at B29-Lys for the complex, which formed at pH 7.1. The results of α-chymotrypsin digestion study were shown questionable due to its incomplete cleavage. The reduced complexes were analyzed by amino acid analysis. The insulin-27% activated dextran complexes formed at pH 7.4, pH 9 and pH 10 showed similar extents of binding at B1-Phe, indicating B1 might be the prime binding site. There was more binding at B29 and A1 for the pH 9 than at pH 7.4 case. At pH 10 abnormal values arose. The studies for the complexes of insulin with 16% and 27% activated dextran indicated the more highly activated the dextran, the greater the binding at B29 and A1. Trials with the 2, 4-dinitrophenyl-derivativatization method proved to be a useful way to examine the degree of B1 and B29 binding from the amino acid analysis results of complex. The insulin-16% activated dextran complex formed at pH 7.1 was found to be about 100% binding at B1, 60% at A1 and 50% at B29. Oxidative and reductive cleavage studies of A and B chains of insulin and the complex were carried out to investigate the level of A1 binding. After chemical cleavage of the three disulfide bonds in insulin and subsequent chromatography, the amino acid analysis results for the treated complexes indicated a significant proportion of A chain had bound to dextran, i.e. at A1. An estimation of 60-70% of A1 binding was achieved for this study. This exploratory study has shown that varied complex formation conditions such as the level of activation of dextran, pH, and temperature could alter the extent of binding between insulin and dextran molecules. Amino acid analysis of the reduced complex was a useful method to interpret the binding.
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    Dextran enzyme imine complexes : a preliminary study : this thesis was presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1997) Fisher, Louisa Jane
    A model system involving the formation of protein-dextran complexes has been investigated with a view to improving existing methods of drug administration. Activation of the dextran was achieved by periodate oxidation to give levels of 7%, 21% and 56% activated glucose moieties. The protein-dextran complexes were investigated with the prospect of obtaining sustained release of proteins from the dextran in an unmodified form. Covalent conjugation of proteins to carbohydrate polymers is known to confer stability on the protein. The proteins in this study were bound to the dextran through imine bonds. The proteins investigated were lysozyme, trypsin, amylase, alcohol dehydrogenase and catalase. The selection covered a range of molecular weights and varying enzymatic activities. As might be predicted, the speed of complex formation was shown to be greater at the 21% level of activation compared to the 7% activation of dextran in all cases studied. Lysozyme, the smallest protein, readily formed complexes at all three levels of activation. At the 56% level the resulting complex had an extremely high MW, greater than 1MDa. The extensive binding between the dextran and lysozyme molecules resulted in a complex that was inactive and showed no signs of releasing any lysozyme, active or inactive. At the lower levels of activation, complex was formed with relative ease. Upon conjugation lysozyme exhibited only minimal activity. Release of a lysozyme-like species with normal lytic activity was observed. Precautions were taken to minimise possible autolysis in the trypsin study. Once complexed it was postulated that autolysis would be prevented or minimised. Similarly the 56% level of activation appeared to be too high to obtain a viable complex for facile trypsin release. Sustained release of a trypsin-like protein was observed with complexes at the 7% and 21% levels. SEC and SDS-PAGE, in conjunction with a positive BAPNA assay gave support to the released species being trypsin-like. While complexed to the dextran trypsin showed no signs of activity. Released trypsin-like species and unreacted trypsin showed similar tryptic maps from a synthetic peptide, the peptide was designed to show distinctive fragments. α-Amylase, twice the MW of trypsin and over three times the MW of lysozyme, formed complexes with ease at both 7% and 21% levels of activation. Conjugation to dextran did not effect the activity of α-amylase. Over time the release of an α-amylase-like species from the complex was observed. Alcohol dehydrogenase and catalase are both high MW proteins. Complex formation was observed for each protein. Subsequent experiments showed that upon release the proteins appeared to dissociate, most probably into their subunits. It is also possible that the dimers and monomers bound to the dextran. The main advantage of conjugation in this case appeared to be to confer stability on the proteins. The ADH-complex exhibited enzymatic activity. At 7% and 21% activation levels the lower MW proteins formed complexes with dextran that exhibited release of a protein species. The higher MW proteins were possibly stabilised when conjugated to dextran, but dissociated upon release. Investigations have shown that the level of activation chosen affects the extent of binding and therefore the functions of the resultant complex. Thus activation levels can be manipulated depending on the desired result. While lower dextran activation levels appeared to be more suited for smaller MW proteins, there were indications that the larger MW proteins could form beneficial complexes at higher activation levels. Results indicated that conjugation to periodate activated dextran could be extended to further proteins with the possibility of therapeutic or commercial applications.