✍ Scribed by Bernhardt W. Langer Jr.
No coin nor oath required. For personal study only.
The study of the biological interconversion of methionine, z-keto-4-methylthiobutyric acid '(methioninc keto ,analogue, MKA), and z-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue, MHA) required a method for their chromatographic separation and qualitative detection. The method described herein fulfills' the need for such a system. Sfandard solutions of methionine, MKA, MHA, and a mixture of the compotinds were spotted on thin-layer chromatograms (silica gel; Eastman Chromagram Sheet No. 6061) and dried for IO min in an oven at 85". The chromatogram was developed, ascendingly, using I-butanol-acetic acid (glacial)-water (170 :zs :5) uritil the solvent frrqnt had migrated 15 cm past the origin (about 2 h) after which the chromatogram was air dried. Methionine was detected by spraying with a 1% acetone sqlution of ninhydrin followed by heating at 85". After circling the methionine spot, MKA and MHA were detected by a modification of the method of MCCARTHY AND SULLIVANI. The chromatogram was sprayed with a I y. aqueous solution of glycine immediately followed by spraying with a 14.3 N NaOH soJution.' After 30 set the excess fluid was removed by patting with filter paper followed by spraying with a IO*/* aqueous solution of sodium nitroprusside.'The MKA immediately appeared as a deep purple spot. The chromatogram was again patted dry and the location of the 'MKA was noted. The chromatogram was then heated for 5 min at 85" during which the MKA spot disappeared. Spraying with an acid mixture (12 N HCl-85% H,PO,,, g :I) resulted in the MHA appearing as a light violet-red spot and after about 5 min. the MKA area reappeared as a light blue, area, with all spots fading within 30 min. To establish that the MKA spot was actually the keto analogue, a separate chromatogram was sprayed with 0.4% 2,4_dinitrophenylhydrazine (in 2 N HCl) and IO*/* NaOH according to the method of NEWCOMBE AND REID 2. The yellow spot that developed coincid.ed exactly with the deep purple and light blue spots noted as belonging to the MKA in the former method.
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