Purification, activite enzymatique potentielle et composition en acides amines du chymotrypsinogene et des trypsinogenes du pancreas de rat

Abstract

Purification, enzymatic activity and amino acid composition of the chymotrypsinogen and the trypsinogens of the rat pancreas 1. 1.|Pancreatic hydrolases, except for phospholipase A, are almost quantitatively solubilized when rat pancreatic tissue is homogenized in a 0.2 M Tris-HCl buffer (pH 8.5). The hydrolases are stable in solution at pH 4.75 in the presence of 75 mM ε-aminocaproate. Their filtration on a Sephadex G-100 column gives three main peaks corresponding, respectively, to lipase, chymotrypsinogen and trypsinogens 1 and 2 together, and α-amylase. 2. 2.|500 mg of proenzymes are successfully fractionated by column chromatography on phosphocellulose (85 cm × 1.2 cm). Trypsinogen 1 and chymotrypsinogen are separated with a 75 mM ε-aminocaproate, 50 mM acetate, 35 mM NaCl buffer (pH 4.75). Trypsinogen 2 is eluted at a higher ionic strength. 3. 3.|The purification of each proenzyme is achieved by a second chromatography on phosphocellulose at pH 5.25. Trypsinogen 1 and chymotrypsinogen can only be separated at this pH, which is close to their isoelectric point. Each of the three purified proenzymes shows a single band on polyacrylamide gel disc electrophoresis. 4. 4.|The amino acid composition of rat chymotrypsinogen resembles those of beef chymotrypsinogens A and B. The E 1cm 1% at 280 mμ is 16. The molecular weight is near 25 000 and the specific activity on acetyl- l-tyrosine ethyl ester is 480 units/mg at 25°. 5. 5.|The E 1cm 1% values at 280 mμ of trypsinogens 1 and 2 are 13 and 15, respectively, the molecular weights are the same (24 000), and the specific activities at 25° are, respectively, 40 and 50 in terms of N-α- benzoyl- l-arginine ethyl ester units/mg and 200 and 100 in terms of N-α- tosyl- l-arginine methyl ester units/mg.