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This method was adapted to conditions of chro-rnatographical separation of a mixture of methyl esters of DNS derivatives of amino acids of the BR hydrolysate. Optimization of eluant ratios, the gradient type, and the rate of elution from the column were per­formed. The maximum separation was observed after gradient elution with a mixture of solvents containing acetonitrile and trifluoroacetic acid (at a volume ratio of 100 : 0.1-0.5). In this case, tryptophan and a hardly degraded pare of phenylalanine/tyrosine were success­fully separated. Degrees of chromatographical purities of isolated methyl esters of N-DNS [2,3,4,5,6-2H5]phe-nylalanine, N-DNS [3,5-2H2]tyrosine, and N-DNS [2,4,5,6,7-2H5]tryptophan were 97%, 96%, and 98%, respectively. The yield was 97-85%. Figure 5b con­firms the result obtained. This figure shows the El mass spectrum of methyl ester of N-DNS [2,3,4,5,6-2H5]phe-nylalanine isolated by reverse-phase HPLC (scanning at m/z 70-600; the base peak at m/z 170; 100%). The mass spectrum is represented in relation to unlabeled methyl ester of//-DNS phenylalanine (scanning at m/z 150-700; the base peak at m/z 250; 100%) (Fig. 5a). The peak of a heavy molecular ion of methyl ester of N-DNS phenylalanine ([M]+, 59% at m/z 417; instead of [M]+, 44% at m/z 412 for unlabeled derivative of phe­nylalanine) and the additional peak of the benzyl frag­ment of phenylalanine, C7H7 (61% at mlz 96; instead of 55% at mlz 91 for control; data not shown), confirm the presence of deuterium in phenylalanine. The peaks of secondary fragments of various intensities with m/z 249, 234, and 170 correspond to products of secondary degradation of the dansyl residue to N-dimethylaminon-aphthalene. The low-intensity peak of [M+-COOCH3] (7%) at m/z 358 (m/z 353, 10%, control) represents the detachment of the carboxymethyl group from methyl ester of N-DNS phenylalanine. The peak of [M + CH3]+ (15%) at m/z 430 (m/z 426, 8%, control) represents the additional methylation at a-amino group of phenylala­nine. The difference between molecular weights of

light and heavy peaks of [M]+of methyl ester of N-DNS phenylalanine is five units. This is in agreement with the earlier obtained result and the data on the level of deutera-tion of initial [2,3,4,5,6-2H5]phenylalanine added into the growth medium.

Thus, these data indicate a high efficiency of incor­poration of 2H-labeled aromatic amino acids into the BR molecule. Completely deuterated protein prepara­tions for reconstruction (into 2H2O) of functionally active systems of membrane proteins with purified 2H-labeled lipids and other deuterated biologically active compounds are proposed to be obtained using the method elaborated. In the future, these studies will pro­vide the means for solving the problem of functioning of 2H-Iabeled BR in the composition of artificially con­structed membranes under conditions of deuterium-sat­urated medium.

ACKNOWLEDGMENTS

This work was supported by grant no. 1B-22-866 ("High chemical technologies"). We are grateful to Dr. B.M. Polanuer (GNU GENETICA) for careful attention and helpful remarks in discussions of the results.

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