THERMAL STABILIZATION OF MULTIMERIC PROTEINS - A CASE-STUDY WITH ALPHA-GLOBULIN

Preferential interaction parameters of multisubunit protein, alpha-glo bulin and monomeric protein human serum albumin (HSA) were determined in different cosolvents using precision densitymetry. The apparent par tial specific volumes were determined under both isomolal and isopoten tial conditions for alpha-globulin in 0.02 M glycine-NaOH buffer at pH 10 and the values were 0.692+/-0.002 and 0.688+/-0.001, ml/g, respect ively, at 20.00+/-0.01 degrees C. From the partial specific volume dat a with cosolvents the preferential interaction parameter (xi(3)) and o ther thermodynamic parameters were calculated at different solvent con centrations. The (xi(3)) values increased with an increase in the solv ent concentration up to 30% and reached a maximum with the values of - 0.111+/-0.018 g/g and -0.076+/-0.012 g/g in sucrose and sorbitol, resp ectively. In glycerol the (xi(3)) values decreased with an increase in solvent concentration. The above data is further supported by thermal denaturation profiles in which the apparent thermal denaturation temp erature (apparent T-m) of alpha-globulin shows an increase om 63 degre es C to higher temperatures in the order of sucrose, sorbitol and glyc erol. alpha-globulin showed coagulation due to protein interaction at temperatures above 50 degrees C. The apparent T-m of 63 degrees C for control protein was increased significantly up to 75 degrees C in 40% sorbitol with two fold increase in the Delta S values showing the incr eased structural stability of alpha-globulin. At high solvent concentr ations the protein gets dissociated and the resultant monomers are hyd rated which was evident by fluorescence data and the difference spectr al results with a 6 nm red shift in the emission maximum and 2 nm blue shift in UV-absorption maximum arising out of perturbation of aromati c chromophores. The studies were performed both at native pH of 7.9 wh ere the protein is in its oligomeric form and at pH of 10 where it is in dissociated form and the results compared. The data showed that the solvent is excluded more from the protein vicinity in the dissociated state.