MASS-SPECTROMETRIC EVALUATION OF THE INCO RPORATION OF H-2 AND C-13 INTO AMINO-ACIDS OF BACTERIA

A highly sensitive EIMS method was used to study the incorporation of stable isotopes H-2 and C-13 into amino acids secreted by the L-phenyl alanine-producing strain of Brevibacterium methylicum and the L-leucin e-producing strain of Methylobacillus flagellatum, as well as into the amino acid residues of the total biomass proteins upon growing the ba cteria on media containing [H-2]methanol, [C-13]methanol, and (H2O)-H- 2 as sources of the stable isotopes. L-[2,3,4,5,6-H-2]phenylalanine, L -[3,5-H-2]tyrosine, and L-[2,4,5,6,7-H-2]tryp tophan were incorporated into bacteriorhodopsin synthesized by Halobacterium halobium ET 1001. For the mass spectrometric analysis, multicomponent mixtures of amino acids in the culture media and protein hydrolysates (hydrolysis with 6 M (HCl)-H-2 containing 3% of phenol and with 2 M barium hydroxide) w ere used. Barium hydroxide was converted into mixtures of N-benzyloxyc arbonyl derivatives of amino acids and methyl esters of N-dimethylamin onaphthalene-5-sulfonyl derivatives of amino acids, which were prepara tively separated by means of reversed phase HPLC. The obtained [H-2]- and [C-13]amino acids represented mixtures, which differed in the numb er of isotopes incorporated into their molecules. The incorporation of H-2 and C-13 into the secreted amino acids and the amino acid residue s of the total biomass proteins depends on the content of labeled subs trates in the growth media and are different for different amino acids (from 10% for L-leucine/isoleucine to 97.5% for L-alanine).