Abstract
The ionization behavior of the tyrosyl groups of bovine pancreatic carboxypeptidase A has been determined by spectrophotometric titration in M sodium chloride solution at 25°C. Of 16 to 17 tyrosyl groups present, eight ionize freely and reversibly in the pH range of 8 to 10.5. Further ionization of eight to nine groups above pH 10.5 is time-dependent. These groups are almost instantaneously ionized at pH 13. After exposure of the enzyme solution to pH 13 for 16 hours, all the tyrosyl groups are normalized and reversibly titrated, with the apparent pK value of 9.9.
The tyrosine content of carboxypeptidase A was determined spectrophotometrically as 16 residues per mole of the enzyme and in good agreement with that obtained by amino acid analysis, although somewhat lower than that reported by other workers.
The effect of a perturbing solvent, 20% sucrose, on the absorption spectrum of carboxypeptidase A was investigated by the method of difference spectrum. The result suggests the presence of approximately three moles of “exposed” tryptophan in the native enzyme.
The authors wish to express their thanks to the Teikoku Hormone Manufacturing Co., Ltd. for kindly supplying the frozen bovine pancreas.
