Contamination of Commercial L-leucine Preparations with Methionine and Cystine.

methods to remove methionine from L-leucine (Fox, Science 84:163, 1936; Bergmann and Stein, J. Biol. Chem. 129:609, 1939), I was surprised to find that certain current preparations of commercial L-leucine, supposedly of high quality, are heavily contaminated with methionine and also with cystine. In an examination of the requirements of a series of Bacillus subtilis mutants, the four types shown in Table 1 were of particular interest, because I was aware of no close relationship between the biosynthesis of leucine and that of the sulfur-containing amino acids. As a representative of group A, strain MB-1794 was examined in more detail and was found to be truly blocked in methionine biosynthesis. It responded to ketomethionine, homocysteine, and cystathionine; cyanocobalamine was inactive. The requirements of the four types of mutants led to the suspicion that the L-leucine was contaminated with methionine and cystine. The grade of L-leucine used was listed as chemically pure. A more expensive grade produced by the same manufacturer (Mann Research Laboratories, Inc., New York, N.Y.) was L-leucine (methioninefree), Mann assayed. This preparation was claimed to be of highest purity and chromatographically pure in two solvent systems. Its analysis was given in the catalog as C, 54.94%; H, 9.99%; N, 10.67%; specific rotation, +15.6° in 5 N HCl; C= 2. Upon testing a sample of "methionine-free" L-leucine, we found that, like the chemically pure material, it allowed good growth of all four groups of mutants. When it was observed that only cultures of group D could grow on DL-leucine, it became apparent that even the "methioninefree" L-leucine was contaminated with methionine (and cystine). As expected, DL-methionine supported growth of groups A and C, and DLcystine was active for groups B and C. Paper chromatographic analysis confirmed the above suspicions. Both the chemically pure and "methionine-free" samples of L-leucine, when chromatographed in four solvent systems, showed ninhydrin spots corresponding to leucine, methionine, and cystine. As final proof, chromatograms which were not sprayed with ninhydrin were placed on agar seeded with strain MB-1796 of group A. After overnight incubation, chromatograms of chemically pure and "methionine-free" L-leucine showed only a single spot of growth, which corresponded to methionine. Chromatograms of L-methionine (chemically pure) from the same manufacturer showed only one ninhydrin spot which was biologically active. A sample of L-leucine was obtained from a second manufacturer (Nutritional Biochemicals Corp., Cleveland, Ohio; no grade listed). It did not support the growth of strain MB-1796. When examined by paper chromatography, it showed only one ninhydrin spot which was inactive in the bioautographic test. It is obvious that the true requirements of groups A, B, and C are methionine, cystine, and methionine/cystine, respectively. These experiments point out the danger in using commercial L-leucine without first checking for contamination by methionine and cystine.