Methionine is present in small amounts in proteins, and methods for its determination usually involve acid hydrolysis of the protein and subsequent amino acid analysis (1) although there are a number of colorimetric methods available (2). However, methionine sulfur is easily oxidized, and it is possible in a hydrolyzate to have a mixture of methionine, methionine sulfoxide, and the sulfone. Oxidation of the sulfur to the VI oxidation state prior to analysis overcomes this difficulty, but this method (3) is not readily applicable to heterogeneous samples which contain carbohydrate. Acid hydrolysis of proteins in the presence of carbohydrate also leads to hydrolytic losses of methionine. Since methionine is the limiting essential amino acid in many legumes (4), seed methionine screening studies would be much enhanced if the desired amino acid were determinable exclusively and the problems associated with acid hydrolysis were avoided.
Proteins react at methionyl residues with cyanogen bromide yielding homoserine and methyl thiocyanate (5). The recoveries of methyl thiocyanate have been determined for two proteins ( 6) and it appears that the method, with modifications, should be generally applicable to methionine quantitation in proteins. This report gives the results of the reaction of cyanogen bromide with a number of proteins and seed meal samples, and presents an alternative method for the determination of methionine in seeds that avoids acid hydrolysis.
Reagents. Cyanogen bromide, reagent grade, Eastman Kodak Co. Formic acid, reagent grade, distilled, mp 7ยฐC. Bovine Ribonuclease, Worthington Biochemicals. Methyl thiocyanate, reagent grade, Eastman Kodak Co. Glucagon was obtained from J. G. Manns, Western College of Veterinary Medicine. The globulin from P. sativum was prepared by the method of Danielsson (7). The seed meals (5% moisture) were from our own supplies (8).
Procedure. A stock solution of cyanogen bromide in formic acid (mp 7ยฐC) was prepared at about a 10% (w/v) concentration (6). It is stable for 2 NRCC No. 15084.