Incorporation of [2,3,4,5,6-2H5]Phenylalanine, [3,5-2H2]Tyrosine, and [2,4,5,6,7-2H5]Tryptophan into the Bacteriorhodopsin Molecule of Halobacterium halobium - рефераты, скачать рефераты, бесплатно рефераты

Рефераты. Incorporation of [2,3,4,5,6-2H5]Phenylalanine, [3,5-2H2]Tyrosine, and [2,4,5,6,7-2H5]Tryptophan into the Bacteriorhodopsin Molecule of Halobacterium halobium

o neutralization with H2SO4) was the cause of selec-tion 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 follow-ing 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 incorpora-tion of 2H-labeled aromatic amino acids. The deutera-tion level of molecules was determined by calculating the difference between the values of heavy peaks of molecular ions [M]+ enriched with deuterium of deriv-atives of aromatic amino acids and their light unlabeled analogues. Methyl esters of N-DNS derivatives of aro-matic 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 num-bers) 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 deutera-tion 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 deu-teration corresponding to the increase in molecular weights were one (for tyrosine) and five (for phenylala-nine and tryptophan) atoms of deuterium. These results showing deuteration levels of phenylalanine, tyrosine, and tryptophan are in agreement with data on the deu-teration 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. How-ever, it should be stressed that there were [M]+ peaks of protonated and semideuterated analogues of phenylala-nine with [M]+ at m/z 414 (20%), 415 (18%), and 416

(a)

170. 234. A 353 B81

100

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 mole-cules. 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 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 com-ponents were additive. Therefore, these mixtures can be analyzed only in the case of the presence of spectra of various components recorded under the same condi-tions. These calculations involve solution of the system of n equations in n unknowns for the mixture contain-ing n components. For the components, whose concen-trations 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 separa-tion of methyl esters of //-DNS derivatives of 2H-labeled amino acids of another microbial objects, e.g., methylotrophic bacteria and microalgae [21]), 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 per-formed. 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 success-fully 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 con-firms 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 phe-nylalanine) and the additional peak of the benzyl frag-ment 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 phenylala-nine. 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 incor-poration of 2H-labeled aromatic amino acids into the BR molecule. Completely deuterated protein prepara-tions 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 pro-vide the means for solving the problem of functioning of 2H-Iabeled BR in the composition of artificially con-structed membranes under conditions of deuterium-sat-urated 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.

REFERENCES

Oesterhelt, D. and Stoeckenius, W., Nature (London),
1971, vol. 233, no 89, pp. 149-160.

Spudich, J.L., Ann. Rev. Biophys. Chem, 1988, vol. 17,
no. 12, pp. 193-215.

Karnaukhova, E.N., Niessen, W.M.A., andTjaden, U.R.,
Anal Biochem., 1989, vol. 181, no. 3, pp. 271-275.

Mosin, O.V., Skladnev, D.A., Egorova, T.A., and
Shvets, V.I., Bioorg. Khim., 1996, vol. 22, nos. 10-11,
pp. 856-869.

Hardy, J.R, Knight, A.E.W., Ghiggino, K.R, Smith, T.A.,
and Rogers, P.J., Photochem. Photobiol, 1984, vol. 39,
no. 1, pp. 81-88.

Rosenbach, V., Goldberg, R., Gilon, C., and Ottolenghi, M.,
Photochem. Photobiol, 1982, vol. 36, no. 6, pp. 197-
201.

Mosin, O.V., Skladnev, D.A., Egorova, T.A., and
Shvets, V.I., Biotechnologiya, 1996, no. 10, pp. 24-40.

Griffiths, D.V., Feeney, J., Roberts, G.C., and Burgen, A.S.,

Biochim. Biophys. Acta, 1976, vol. 446, no. 4, pp. 479-585.

9. Matthews, H.R., Matthews, K.S., and Opella, S.J., Bio-
chim. Biophys. Acta, 1977, vol. 497, no. 23, pp. 1-13.

Oesterhelt, D. and Hess, B., Eur. J. Biochem., 1973,
vol. 37, no. 1, pp. 316-326.

Tokunada, F. and Ebrey, T, Biochemistry, 1978, vol. 17,
no. 10, pp. 1915-1922.

Pervushin, K.V. and Arsen'ev, A.S., Bioorg. Khim.,
1995, vol. 21, no. 10, pp. 83-111.

Zvonkova, E.N., Zotchik, N.V., Filippovich, E.I., Mitro-
fanova, T.K., Myagkova, G.I., and Serebrennikova, G.A.,
Khimiya biologicheski aktivnykh prirodnykh soedinenii

(Chemistry of Biologically Active Natural Compounds), Moscow: Khirniya, 1970, pp. 65-68.

Muromoto, K., Sunahara, S., and Kamiya, H., Agric.
Biol. Chem., 1987, vol. 51, no. 6, pp. 1607-1616.

Matsubara, H. and Sasaki, R.M., Biochim. Biophys. Res.
Com., 1969, vol. 35, no. 10, pp. 175-177.

Ng, L.T., Pascaud, A., and Pascaud, M., Anal. Biochem.,
1987, vol. 167, no. 2, pp. 47-52.

Liu, T.Y. and Chang, Y.H..J.Biol. Chem., 1971, vol. 246,
no. 2, pp. 2842-2848.

Simpson, R.J., Neuberger, MR., and Liu, T.Y., J. Biol.
Chem., 1976, vol. 251, no. 3, pp. 1936-1938.

Pshenichnikova, A.B., Karnaukhova, E.N., Zvonko-
va, E.N., and Shvets, V.I., Bioorgan. khimiya, 1995,
vol. 21, no. 3, pp. 163-178.

Cohen, J.S. and Putter, I., Biochim. Biophys. Acta, 1970,
vol. 222, no. 1, pp. 515-520.

2E. Egorova, T.A., Mosin, O.V., Eremin, S.V., Kar-naukhova, E.N., Zvonkova, E.N., and Shvets, V.I., Bio-technologiya, 1993, no. 8, pp. 21-25.

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