INTERACTION OF H-IONS WITH ALPHA-CRYSTALLIN - SOLVENT ACCESSIBILITY OF IONIZABLE SIDE-CHAINS AND SURFACE-CHARGE()

Interaction of H+-ions with alpha-crystallin from goat lens has been s tudied at three different ionic strengths using the potentiometric tit ration method. Titrations have also been carried out in the presence o f 1.5 M and 6 M GuHCl (guanidine hydrochloride). The isoionic pH of th e protein in water and the effect of KCl on it have been determined. T itration curves have been found to be reversible between pH 3 to 9.25 at all ionic strengths. To aid in the data analyses, the reactivities of alpha-crystallin lysine residues to trinitrobenzenesulfonic acid ha ve been determined in this work. For alpha-crystallin aggregate, 130 /- 2 histidine side chains out of a total of 300 and about 134 +/- 4 l ysine side chains out of 310 have been found to be inaccessible to the solvent in the native condition. The remaining titratable side chains determine the surface charge of the native protein. In 1.5 M GuHCl, h owever, the nontitratable histidine side chains are found to be availa ble for titration as are the nontitratable lysine and tyrosine side ch ains in 6 M GuHCl. The theoretical titration curve computed on the bas is of Linderstrom-Lang model is found to fit quite comfortably with th e experimental one between pH 4.6 and 9.25. The pK(int) value for beta ,gamma-carboxyl side chains has been found to be 5.18 which is somewha t higher than usual indicating that the carboxyl groups in the protein are probably in some constrained condition which is released in the p resence of a denaturant. Below pH 4.6, there begins a conformational c hange in the alpha-crystallin aggregate as is corroborated from the ci rcular dichroism studies. The value of electrostatic interaction facto r w which remains more or less constant between pH 4.6 and 9.25 is als o found to gradually fall off below pH 4.6. (C) 1998 Elsevier Science B.V.