Incorporation of 23456-2H5Phenylalanine 35-2H2Tyrosine and 24567-2H5Tryptophan into the Bacteriorhodopsin (стр. 2 из 3)

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

Incorporation of 23456-2H5Phenylalanine 35-2H2Tyrosine and 24567-2H5Tryptophan into the Bacteriorhodopsin

Disintegration by ultrasound

Water-soluble productsof cellular content,inorganic salts,and other low-molecular-weightcompounds

Incorporation of 23456-2H5Phenylalanine 35-2H2Tyrosine and 24567-2H5Tryptophan into the BacteriorhodopsinDistilled H2O

RNase I,

125 mM NaCl, 20 mM MgCl,

4 mM Tris-HCl

Distilled H2O

Isolation of the biomass

Raw biomass
________ t

Osmotic shock

Culture liquid

4.3 M NaCl, and other

inorganic salts

and metabolites

50% ethanol

1.0.5%SDS-Na 2. Methanol



PM fraction



Delipidation + BR precipitation

— Extract of carotenoids

_._ Residuals of cellular walls, lipids, and other high-molecular-weight compounds

Crystalline BR

Gel-permeation chromatography on Sephadex G-200

4NBa(OH)7 UO°C,24h

1. DNS chloride, 2 M
NaHCO3, and ethyl acetate

2. jV-Nitroso-N- methyl-_

urea, 40% KOH

diethyl ester, and diazomethane

Purified BR ±

Incorporation of 23456-2H5Phenylalanine 35-2H2Tyrosine and 24567-2H5Tryptophan into the Bacteriorhodopsin

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 reached. The formation of the reti­nal-protein complex induced a bathochromatic shift in the absorption band of PMs (Fig. 3). The major band recorded at the maximum absorption of 568 nm and induced by the light isomerization of chromophore at

bonds positioned at C13=C14 or multiples of this num­ber was determined by the presence of trans-retinal res­idue of retinal (BR568). The additional low-intensity band recorded at 412 nm characterized the presence of a minor admixture of the M412 spectral form (produced in light) containing the deprotonated aldirnine bond

Incorporation of 23456-2H5Phenylalanine 35-2H2Tyrosine and 24567-2H5Tryptophan into the Bacteriorhodopsinbetween the residue of trans-retinal and the protein. The band recorded at 280 nm depended on the absorp­tion of aromatic amino acids of the polypeptide chain of this protein (the D2%0/D56% ratio was 1.5 : 1 for pure BR).

Fractionation and careful chromatographic purifica­tion of the protein were the next necessary stages. BR is a transmembrane protein with a molecular weight of 26.7 kDa that penetrates the lipid bilayer in the form of seven a-helixes. Therefore, the use of ammonium 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 (-5°C) 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, 110°C, 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, 110°C, 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

Incorporation of 23456-2H5Phenylalanine 35-2H2Tyrosine and 24567-2H5Tryptophan into the Bacteriorhodopsin








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

Incorporation of 23456-2H5Phenylalanine 35-2H2Tyrosine and 24567-2H5Tryptophan into the Bacteriorhodopsin


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 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 [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 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