An investigation of the thermal stability of polyglycyl bovine serum albumin Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/c534fs309

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  • A heat stable derivative of bovine serum albumin containing 300 residues of glycine added to 30 sites on the surface of the protein has been prepared. This derivative may be heated to 100° C for prolonged periods of time without aggregation. By comparison, native BSA aggregates at 62° C under similar conditions. The spatial arrangement of the added glycine peptides is probably responsible for the observed stabilization of the protein. Two possible arrangements of the added glycyl chains have been suggested. The first considers the chains to be fully extended and projecting into the surrounding solvent. The second envisions the added chains folded back along the protein surface. The experimental evidence greatly favors the second suggestion. Using trinitrobenzene sulfonic acid, it was determined that there were thirty reactive amino groups on the surface of native BSA. After denaturation of the protein with 8 M urea; 57 lysine groups could be reacted. Since the preparation of the polyglycyl derivative is carried out under similar reaction conditions to those employed with trinitrobenzene sulfonic acid, it is most likely that the amino groups on the surface of the molecule react with the N-carboxy glycine anhydride. The conformation characteristic of native BSA appears to be maintained in the derivative as indicated by various experimental measurements. The change in rotation associated with the acid expansion of BSA is paralleled by a similar but smaller change in the derivative. At pH 5.5 the specific rotation of the derivative was -47.7° and the native was -64.6°. The value for the polyglycyl BSA is several degrees lower than would be expected if it possessed the identical molar rotation as the native protein. A similar decrease in levorotation has been reported when ten moles of anionic detergent are bound to one mole of BSA The ultraviolet difference spectra observed by perturbing the native and derivative protein by pH and heat indicated that the main matrix of each protein was undergoing a similar transition. In all cases observed the derivative showed less change in optical density than did the native protein. If the glycine chains were folded back along the protein surface shielding some chromophoric groups from contact with the solvent, this decrease could be explained. The hydrodynamic measurements also support the suggestion that the structure of the protein core of the derivative is retained. The derivative undergoes the same reversible acid expansion as the native protein. Polyglycyl BSA with an S₂₀ value of 3.85 at pH 2.5 regained its native conformation which had a sedimentation coefficient of 5.45 by dialysis against phosphate buffer at pH 6.3. By approximations that may be made from the diffusion coefficient, it was shown that the degree of hydration of the derivative could have a large variation depending upon the spatial arrangement of the added peptides. If the added peptides were folded, the hydration would be less than the native protein and if they were extended the hydration would be greater than the native protein. The hydration was evaluated by the determination of the buoyant density of polyglycyl BSA in a series of salt solutions. The values of the hydration of the protein-salt complex determined are as follows: in CsC1, 0.42 gm H₂O/gm protein; in RbBr, 0.37 gm H₂O/gm protein; and in KBr, 0.33 gm H₂O/gm protein. The corresponding values of the hydration of the protein-salt complex for bovine mercaptalbumin reported by Ifft and Vinograd are as follows: in CsC1, 0.51 gm H₂O/gm protein; in RbBr, 0.46 gm H₂O/gm protein and in KBr, 0.37 gm H₂O/gm protein. The decrease in hydration found in the derivative may be adequately explained by the change in the surface to volume ratio that would be expected if the added peptides were folded back along the protein surface. An approximate calculation of the surface to volume ratio of the derivative in this configuration gives a value of 0.83 that of the native protein. A similar calculation assuming that the chains are extended gives a ratio of 1.5. The decrease found is clearly more consistent with a molecule in which the added peptide chains are folded back along the protein surface. An increase of the activity of water accompanied by an increase in the hydration of the protein has been presented previously by Ifft and Vinograd, and was found to be valid for the hydration of the derivative. Considering the experimental evidence that has been presented, it is felt that the added polyglycyl chains are folded back along the surface of the protein. When they are in this configuration they form hydrogen bonds with groups on the surface of the protein and would cover some of the hydrophobic regions on the surface of the protein. Both of these effects would cause the derivative to be more stable than the native protein.
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