Abstract
Arginine, histidine, lysine, cystine, methionine, tyrosine, tryptophane, phenylalanine, glycine, alanine, and threonine can all be determined by available micro methods 1 , 2 in only 2 to 4 g of protein, but until recently methods were not available for the estimation of 3 of the essential amino acids : leucine, isoleucine, and valine in small quantities of protein. Last year Fromageot and Heitz 3 described a procedure for the determination of leucine and valine, involving deamination to the corresponding hydroxy acids with nitrous acid, oxidation to acetone with chromate, and measurement of the acetone colorimetrically after reaction with salicylaldehyde. Leucine and valine were estimated in the presence of each other by carrying out the oxidation with chromate on 2 aliquots under conditions such that the proportionate yield of acetone from the 2 amino acids differed considerably. We were unsuccessful in obtaining consistent results with this procedure. In the present study we have made extensive changes in and simplified the procedure of Fromageot and Heitz and in addition have found conditions which permit the determination of isoleucine by measuring the ethylmethylketone formed in the oxidation of the corresponding hydroxy acid. All 3 amino acids can be determined in as little as 100 mg of protein for no one of the other amino acids recognized as occurring in proteins yields acetone or ethylmethylketone under these conditions.
Hydrolysis and Deamination. The protein is hydrolyzed with 10 volumes of 8 N sulfuric acid and the excess acid is removed with barium hydroxide. The precipitate of barium sulfate is centrifuged and thoroughly washed with hot water. The filtrate and washings are reduced in vacua to 50 cc. Amino acid solutions prepared by other methods of hydrolysis are suitable provided the hydrolyzing agent does not yield compounds which would interfere in later steps.
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