Interaction of H+-ions with α-crystallin:: solvent accessibility of ionizable side chains and surface charge

Interaction of H+-ions with alpha-crystallin from goat lens has been studied at three different ionic strengths using the potentiometric titration method. Titrations have also been carried out in the presence of 1.5 M and 6 M GuHCl (guanidine hydrochloride). The isoionic pH of the protein in water and the effect of KCl on it have been determined. Titration 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 have been determined in this work. For alpha-crystallin aggregate, 130 +/- 2 histidine side chains out of a total of 300 and about 134 +/- 4 lysine 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, however, the nontitratable histidine side chains are found to be available for titration as are the nontitratable lysine and tyrosine side chains in 6 M GuHCl. The theoretical titration curve computed on the basis of Linderstrom-Lang model is found to fit quite comfortably with the 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 somewhat higher than usual indicating that the carboxyl groups in the protein are probably in some constrained condition which is released in the presence of a denaturant. Below pH 4.6, there begins a conformational change in the alpha-crystallin aggregate as is corroborated from the circular dichroism studies. The value of electrostatic interaction factor w which remains more or less constant between pH 4.6 and 9.25 is also found to gradually fall off below pH 4.6. (C) 1998 Elsevier Science B.V.