SPECTROPHOTOMETRIC TITRATION OF TYROSYL RESIDUES IN ALKALINE MESENTERICOPEPTIDASE

Abstract

At pH 7.0 the alkaline mesentericopeptidase has ultraviolet absorption spectrum with a minimum at 251 nm and a maximum at 280 nm and no visible absorption. From the tyrosine to tryptophan ratio a value of 3 tryptophyl residues per mole of protein is obtained. The molar extinction coefficient at 280 nm is 3.55 times 10⁴M^-1cm^-1. Spectrophotometric titration studies show that the molecule of mesentericopeptidase contains seven phenolic groups with a pK_app = 9.92 and four to five groups with a pK_app = 11.96. Denaturing agents, such as 5 M guanidine hydrochloride or alkali, normalize the ionization of the tyrosyl residues. There is a good correlation between the spectrophotometric titration data and the results for the reactivities of the tyrosines in mesentericopeptidase towards tetranitromethane. The correlation is explained by the mechanism of nitration. Conclusions about the state of the tyrosyl residues and the three-dimensional structure of mesentericopeptidase are made.