CORNtLL UNIVERSITY MEDICAL P.~LLEQE, I;)¢PAJlt~NF.gT OF PHYSIOJ.0O¥, DETERMINATION OF CERTAIN AMINO ACIDS IN CHYMOTRYPSINOGEN, AND ITS MOLECULAR WEIGHT BY ERWIN BRAND a~ro

Chymotrypsinogen is a crystalline protein, which has been isolated from acid extracts of fresh cattle pancress by Kunitz and Northrop (1). This protein is transformed by minute amounts of trypsin into an active proteolytic enzyme, originally called chymotrypein (1), but later designated a-chymotrypsin by Kunitz (2). This paper deals with the determination of the sulfur amino acids and of tyrosine and tryptophane in chymotrypsinogen, kindly put at our disposal by Dr. Kunitz. Semi-micro methods for the accurate determination (in protein hydrolysates) of the sulfur amino acids, cystine, cysteine, and methionine, and of sulfate sulfur have been described in recent publications (3-6) from this laboratory. A system of analysis has been developed in which each of these constituents is determined by at least two independent methods. Hydrolysis is carried out in an inert atmosphere with HI in presence of hypophosphite, with HCI or with HC1 in the presence of urea (7). During HI digestion methionine (CHr--S---CH~. CH~. CH(NH~). COOH) is split into methyl iodide and the S-lactone of homocysteine (HS. CH2. CH~. CH(NH~). COOH). Both split products are determined, the former as volatile iodide, the latter iodometrically (5). HI hydrolysis leaves cysteine unchanged, while cystine is reduced to cysteine, so that the sum of these compounds can be determined as cysteine by iodometric oxidation (5). Cysteine and cystine are determined separately by our photometric method in HC1 hydrolysates (3, 4). Acid-insoluble humin in HCI hydrolysates interferes with the cysteine deterruination, since the humin precipitate may contain appreciable amounts of cysteine, as shown first by Lugg (8). With certain proteins (e.g. egg albumin and lactalbumin) the formation of acid-insoluble humin can be almost entirely prevented by carrying out the HC1 hydrolysis in the presence of urea (7); under these conditions satisfactory results for both cysteine and cystine are obtained. The Sullivan method (cf. 3, 6), which is specific for cystine, cannot be inter167