Termodinâmica da ligação de tripsina bovina com o íon benzamidínio na presença de osmólitos naturais

Abstract

Sucrose, sorbitol, glycerol and betaine are stabilizing solutes known in the literature as osmolytes. In this work, we investigate the action of such solutes on the variation of standard free energy and the variation of standard enthalpy of van't Hoff for the formation of complexes between bovine trypsin and benzamidine son. The free energies of association were determined from the dissociation constants of the trypsin-ion benzamidinium complex (Ki) under different solvent compositions. The standard enthalpy variation of van't Hoff was determined from the dependence of In K, with 1 / T at 10, 20 and 30 ° C. The dissociation constants were determined according to a classic method in the literature, using Benzoyl-D, L-Arginine-p-Nitroanilide as a substrate in two or three different concentrations and six or fourteen different concentrations of the inhibitor. Osmolytes were incorporated into each solution used in the experiments at a concentration of 0.5 mol.L-1. The experimental points obtained by locating the reciprocal of the enzymatic activity against the inhibitor concentration were adjusted by linear regression. We determine each dissociation constant from the abscissa value corresponding to the point of intersection of the regression lines. At 25 ° C, the presence of 0.5 mol.L. sucrose, sorbitol, glycerol or betaine, changed the free energy variation of association between bovine trypsin and benzamidinium son from -6.02 +0.061 kcal / mol to -5.21 + 0.042, -5.22 + 0.026, - 5.23 0.060 and -5.25 + 0.087 kcal / mol, respectively. The osmolites considered, regardless of their structure, produced an average increase in the variation of standard free energy (SAG) of formation of the trypsin-ion benzamidinium complex of +0.79 kcal / mol, which means that those solutes increased the relative population of the free enzyme in equilibrium E + I = EI. Glycerol, sorbitol, sucrose and betaine produced van't Hoff's standard enthalpy variations of -11.4 0.86, -10.3 = 0.52, -12.3 - 1.22 and -13.1 1, 76 kcal / mol, respectively. Those values ​​do not differ significantly from the Aho value of -11.7 - 1.24 kcal / mol obtained in the absence of osmolytes. The action of these solutes on trypsin was also investigated by electrophoresis on polyacrylamide gel in which a gradient transverse to the direction of migration of the protein with 0 to 4 mol.L-? of glycerol. To counteract the increase in gel density determined by the osmolyte gradient, we vary the acrylamide concentration from 11 to 8% in the direction of increasing the solute gradient. Trypsin was applied as a continuous band on top of the gel. No disturbance of protein mobility was observed within the limits considered for glycerol concentration. These results agree with the idea that all these compounds, regardless of their structure, act in the same way, changing the properties of the solvent, in order to stabilize the protein structure.