molecular ions [M]+ of methyl esters of N-DNS derivatives of aromatic amino acids had low intensities and were polymorphously split. Therefore,
their molecular enrichment ranges were considerably
widened. Moreover, mass spectra of the mixture components were additive. Therefore, these mixtures can be analyzed only in the case of the presence of spectra of various components recorded under the same conditions. These calculations involve solution of the system of n equations in n unknowns for the mixture containing n components. For the components, whose concentrations are more than 10 mol %, the validity and repro-ducibility of the analysis results can be 0.5 mol % at a confidence probability of 90%. Therefore, chromato-graphical isolation of individual derivatives of 2H-labeled amino acids from the protein hydrolysate is necessary for a obtaining a reproducible result. Reverse-phase HPLC on octadecylsilane silica gel, Separon C18 (whose potency was confirmed by separation of methyl esters of //-DNS derivatives of 2H-labeled amino acids of another microbial objects, e.g., methylotrophic bacteria and microalgae ), was used. 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.
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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