Incorporation of [2,3,4,5,6-2H5]Phenylalanine, [3,5-2H2]Tyrosine, and [2,4,5,6,7-2H5]Tryptophan into...

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nium sul-fate and another traditional salt-eliminating agents is not appropriate. The protein must be transformed into the soluble form by solubilization in 0.5% SDS. The use of this ionic detergent was dictated by the necessity of the most complete solubilization of the protein achieved by combining delipidation and precipitation. In this case, BR solubilized in a low-concentration solution of SDS retained its helical cc-conformation [12]. Therefore, it was not necessary to use organic solvents such as acetone, methanol, and chloroform for removing lipids. Delipidation and precipitation of the protein were combined into the same stage. This noticeably simplified fracdonation. The advantage of this method was that the desired protein (in the complex with molecules of lipids and detergent) was in the supernatant. Another high-molecular-weight admixtures were in the nonreacted precipitate, which was removed by centrifugation. Fractionation of solubilized (in 0.5% SDS) protein and its further isolation in the crystalline form were conducted using a gradual low-temperature (-5C) precipitation by methanol (three stages). The second and the third stages were performed by decreasing the detergent concentration 2.5 and 5 times, respectively. The final stage of BR purification involved the separation of the protein from low-molecular-weight admixtures by gel-permeation chro-matography. The fractions containing BR were passed two times through a column with dextran Sephadex G-200 balanced with 0.09 M Tris-borate buffer (pH 8.35) containing 0.1% SDS and 2.5 mM EDTA. The method designed for fractionation of the protein made it possible to obtain 8-10 mg of pure preparation of 2H-labeled BR from 1 g of bacterial biomass. The homogeneity of BR complied with the requirements on reconstruction of membranes and was confirmed by electrophoresis in 12.5% PAAG with 0.1% SDS, regeneration of apomembranes with trans-retinal, and reverse-phase HPLC of methyl esters of N-DNS derivatives of amino aids. Low yield of BR was no barrier to further studies of isotopic incorporation. However, it must be emphasized that considerable amounts of the raw biomass must be produced in order to provide high yield of the protein.

Hydrolysis of BR. Conditions of hydrolysis of deuterium-containing protein were determined by the necessity of preventing the isotopic (H-2H) hydrogen-deuterium exchange in molecules of aromatic amino acids, as well as retaining tryptophan in the protein hydrolysate. Two alternative variants (acid and alkaline hydrolysis) were considered. Acid hydrolysis of the


400 500 600 700


Fig. 3. Absorption bands (in 50% ethanol) at various stages of treatment: (a) native BR, (b) PMs after intermediate treatment, and (c) P.Ms purified of foreign admixtures. The band (/) corresponds to the spectral form of BR568. The band (2) corresponds to the admixture of the M^ spectral form. The band (J) characterizes the absorption of aromatic amino acids. The bands (4) and (5) correspond to foreign caro-tenoids. Native BR was used as control.

protein performed under standard conditions (6 N HC1 or 8 N H2SO4, 110C, 24 h) is known to induce complete degradation of tryptophan and partial degradation of serine, threonine, and several other amino acids in the protein [13]. These amino acids do not play an important role in this study. The modification of this method involving the addition of phenol [14], thiogly-colic acid [15], and p-mercaptoethanol [16] into the reaction medium allowed retaining tryptophan (to 80-85%). 7-ToIuenesulfonic acid with 0.2% 3-(2-aminoet-hyl)-indole, as well as 3 M 2-mercaptoethanesulfonic acid [18], are the potent agents for retaining tryptophan (to 93% [17]). However, these methods are not suitable for working the problem, because they have a noticeable weakness. Processes of the isotopic exchange (of a high rate) of aromatic protons (deuterons) in molecules of tryptophan, tyrosine, and histidine [19], as well as the exchange of protons at C3 atom of aspartic acid and C4 atom of glutamic acid [20], proceed under conditions of acid hydrolysis. Thus, the data on incorporation of deuterium into the protein can not be derived from the hydrolysis performed even in deuterium-containing reagents (2HC1,2H2SO4, and 2H2O).

Reactions of the isotopic hydrogen exchange are nearly undetected (except for the proton (deuteron) at C2 atom of histidine), and tryptophan is not degraded under conditions of alkaline hydrolysis (4 N Ba(OH)2 or NaOH, 110C, 24 h). Thus, this method of hydroly: sis was used in our study. Simplification of the procedure for isolating the mixture of free amino acids (due








Fig. 4. El mass spectrum of the mixture of methyl esters of /V-DNS derivatives of amino acids of the BR hydrolysate. Cultivation was performed on synthetic medium containing [2,3,4,5,6- Hslphenylalanine, [3,5- H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan. Images of molecular ions of arnino acids correspond to their derivatives (here and on Fig. 5). Ordinate: relative intensity of the peak /)-

to neutralization with H2SO4) was the cause of selection of 4 N Ba(OH)2 as a hydrolyzing agent. Possible racemization of amino acids during alkaline hydrolysis did not affect the results of further mass-spectrometry assay showing the deuteration level of molecules of amino acids.

Study of incorporation of [2,3,4,5,6-2H5]phenylala-nine, [3,5-2H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan into the molecule ofBR. El mass spectrometry following the modification of the mixture of free amino acids of the protein hydrolysate into methyl esters of N-DNS derivatives of amino acids was used for studies of incorporation of 2H-labeled aromatic amino acids. Total El mass spectrum of the mixture of methyl esters of N-DNS derivatives of 2H-labeled amino acids was recorded to obtain reproducible data on the incorporation of 2H-labeled aromatic amino acids. The deuteration level of molecules was determined by calculating the difference between the values of heavy peaks of molecular ions [M]+ enriched with deuterium of derivatives of aromatic amino acids and their light unlabeled analogues. Methyl esters of N-DNS derivatives of aromatic amino acids were separated by reverse-phase HPLC, and El mass spectra of individual-amino acids were obtained. The El mass spectrum of the mixture of methyl esters of N-DNS derivatives of amino acids (scanning at m/z 50-640, the base peak of m/z 527, 100%) was of the continuous type (Fig. 4). The peaks (in the range from 50 to 400 on the scale of mass numbers) were represented by fragments of metastable ions, low-molecular-weight admixtures, and products of chemical modification of amino acids. 2H-labeled aromatic amino acids with mass numbers in the range

from 414 to 456 on the scale of mass numbers were the mixtures of molecules containing various numbers of deuterium atoms. Therefore, their molecular ions [M]+ were polymorphously split (depending on the number of hydrogen atoms in the molecule) into individual clusters displaying static sets of m/z values. Taking into account the effect of isotopic polymorphism, the deuteration level was determined from the most commonly encountered peak of the molecular ion [M]+ (which value was mathematically averaged by mass spectrometer) in each cluster (Fig. 4). Phenylalanyne had a peak of a molecular ion that corresponded to [M]+ and was 13% at m/z 417 (instead of [M]+ at m/z 412 for unlabeled phenylalanine; peaks of unlabeled amino acids are not represented here). Tyrosine had the peak of molecular ion that corresponded to [M]+ and was 15% at m/z 429 (instead of [M]+ at m/z 428). Tryptophan had a peak of a molecular ion that corresponded to [M]+ and was 11 % at m/z 456 (instead of [M]+ at m/z 451). Levels of deuteration corresponding to the increase in molecular weights were one (for tyrosine) and five (for phenylalanine and tryptophan) atoms of deuterium. These results showing deuteration levels of phenylalanine, tyrosine, and tryptophan are in agreement with data on the deuteration levels of initial amino acids. This indicates a sufficiently high potency of incorporation of 2H-labeled aromatic amino acids into the protein molecule. Thus, incorporation of 2H-labeled amino acids into the BR molecule was of a specific type. Deuterium was detected in all residues of aromatic amino acids. However, it should be stressed that there were [M]+ peaks of protonated and semideuterated analogues of phenylalanine with [M]+ at m/z 414 (20%), 415 (18%), and 416


170. 234.A 353 B81


Fig, 5. El mass spectrum of the mixture of methyl esters of N-DNS phenylalanine under various experimental conditions: (a) unla-beled methyl ester of N-DNS phenylalanine and (b) methyl ester of /V-DNS [2,3,4,5,6-2H5] phenylalanine isolated by reverse-phase HPLC.

(11%); tyrosine with [M]+ at m/z428 (12%); and tryp-tophan with [M]+ at m/z 455 and 457 (9%) displaying various contributions to the deuteration level of molecules. This suggests that small part of minor pathways of their biosynthesis de novo leading to the dilution of a deuterium label was retained. The presence of these peaks probably depended on conditions of biosynthetic

incorporation of 2H-labeled aromatic amino acids into the protein molecule.

The analysis of scan El mass spectrum showed that peaks of