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Mass spectra. Mass spectra of methyl esters of N-DNS derivatives of amino acids were obtained by the method of electron impact on an MB-80 A instrument (Hitachi, Japan) at the energy of ionizing electrons of 70 eV, accelerating potential of 8 kV, and a temperature of the cathode source of 180-200°C. Scanning of the samples analyzed was performed at a resolution of 7500 conditional units and a 10% image definition.

Incorporation of [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan into the molecule of BR. The method of incorporation of 2H-labeled amino acids into the molecule of BR was selected because of the fact that this work was designed to reveal the possibility for obtaining 2H-labeled prepa­rations of the membrane protein (in semipreparative amounts) for the reconstruction of artificial membranes. [2,3,4,5,6-2H5]PhenyIalanine, [3,5-2H2]ryrosine, and [2,4,5,6,7-2H5;]tryptophan play important roles in hydrophobic interaction of the BR molecule with the lipid bilayer of the cell membrane. They are stable to the 'H-2H exchange in water medium under growth conditions. Moreover, high-sensitivity El mass spec-trometry can be used for the analysis of their incorpo­ration, which was performed microbio logically by growing the strain of halophilic bacteria Halobacte-rium halobium on a synthetic medium containing 2H-labeled aromatic amino acids. Thus, these compounds were selected as sources of deuterium. Under the opti­mum growth conditions (exponential growth on a syn­thetic medium with 4.3 M NaCl at 35-37°C and illumi­nation), the cells synthesized a purple pigment whose spectral characteristics were identical to those of native BR. Figure 1 shows the dynamics of (2) bacterial growth on the medium containing -H-labeled aromatic amino acids in relation to (1) growth under control con-

Fig. 1. The dynamics of Che growth of Che strain//, halobium under various experimental conditions: (/) protonated synthetic medium and (2) synthetic medium with [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2Jtyrosine, and [2,4,5,6,7-2H5]tryptophan.

ditions. The growth of this strain on the medium con­taining 2H-Iabeled aromatic amino acids was only slightly inhibited. This is important for producing the raw 2H-labeled biomass for further isolation of BR.

The main stages of isolating 2H-labeled BR (Fig, 2) were the following: production of 1 g of 2H-labeled bio-mass; isolation of the fraction of PMs; removal of low-molecular-weight and high-molecular-weight admix­tures, cellular RNA, carotenoids, and lipids; fraction-ation of solubilized (in 0.05% SDS) protein by metha-nol; and purification on Sephadex G-200. Low-molec­ular-weight admixtures and the intracellular contents were eliminated by osmotic shock induced by distilled water (after removing 4,3 M NaCl) followed by destruction of cell membranes by ultrasound. The cel­lular homogenate was then treated with RNase I (two-three units of activity) to induce the maximum destruc­tion of cellular RNA. The PM fraction obtained con­tained the complex of the desired protein with Hpids and polysaccharides, as well as admixtures of fixed car­otenoids and foreign proteins. Therefore, it was neces­sary to use special methods of protein fracdonation, which would not damage the native structure of the pro­tein native structure or cause its dissociation. This made the isolation of pure individual BR performed by the use of special fine methods for removing carotenoids and lipids, purification, and column chromatography more difficult. Decarotenoidation was conducted by a repeated treatment of PMs with 50% ethanol at -5°C. Although it was a routine procedure, this stage was neces­sary (despite of considerable chromoprotein losses). The treatment was repeated no less than five times to obtain the absorption band of the PM suspension freed of caro­tenoids. Figure 3 shows (curves b, c) these bands at vari­ous stages of treatment in relation to (curve a) the band of

Growth of Halobacterium halobium on synthetic medium containing [2,3,4,5,6-2H5]phenyIalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan

Disintegration by ultrasound Water-soluble products of cellular content, inorganic salts, and other low-molecular-weight compounds

DNS chloride, 2 MNaHCO3, and ethyl acetate jV-Nitroso-N- methyl-

urea, 40% KOH diethyl ester, and diazomethane Purified BR ± Mixture of free amino acids I Modification into methyl esters of /V-DNS derivatives of amino acids Reverse-phase HPLC BaSO4 after neutralization with 2 M 2 M H2SO4 Individual methyl esters of/V-DNS[2,3,4,5,6-2H5]phenylalanine N-DNS-[3,5-2H2]tyrosine, and N-DNS [2,4,5,6,7-2H5]tryptophan El mass spectrometry Fig. 2. Experimentally designed method for isolating H-labeled BR. native BR. In this case, an 80-85% efficiency of remov­ing carotenoids was

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