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

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

Applied Biochemistry and Microbiology, Vol.
35, ffo.
/. 1999, pp.
29-17. Translated from Prikladnayti Biokhimiya i Mikrobialogiya, Vol. 35, No.
1,@  1999, pp. 34-42. Original Russian Text
Copyright
© /999 hy Mosin,
Skluclnev, Shvatz.

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. V. Mosin*, D. A. Skladnev**, and V. I.
Shvets*

* Lotnonosov Moscow
State Academy of Fine Chemical Technology, Moscow, 117571 Russia

** State Center of
Genetics and Selection of Industrial Microorganisms (GNU GENETICA), Moscow,
113515 Russia

Received
September 25, 1997

Abstract—Incorporation of
[2,3A5,6-2H5]phenylalanine, [3,5-2H2]tyrosine,
and [2,4,5,6,7-2H5]tryptophan into the
bacteriorhodopsin molecule followed by semipreparative isolation of
bacteriorhodopsin resulted in a yield of 8-10 mg per g bacterial biomass. This method is
based on the growth of the strain of halophilic bacteria Halobacterium halobium
on a
synthetic medium containing 2H-labeled aromatic ammo acids and
fractionation of solubilized (in 0.5% sodium dodecyl sulfate) protein by methanol,
including purification of carotenoids. lip-ids, and high-molecular-weight and
low-molecular-weight compounds, as well as gel-permeation chromatog-raphy on Sephadex
G-200. Incorporation of 2H-labeled amino acids was analyzed by
electron impact mass spectrometry after hydrolysis of the protein in 4 N
Ba(OH)2 and separation in the form of methyl esters of /V-DNS derivatives of
amino aids by re versed-phase high-performance liquid chromatography.


The retinal-containing protein (a chromophore, pro-tonated
aldimine of retinal containing Lys-216 e-amino group) bacteriorhodopsin (BR),
functioning as an ATP-dependent translocase in cell membranes of halophilic bacteria Halobacterium
halobium
was initially described by Oesterhelt [1]. In spite of the fact that
the structure and functions
of this protein were studied in detail, it is still a focus of interest. This
protein is used in practice as a biological
photochromic material because of its
high photosensitivity and resolution abil­ities [2]. Moreover, BR is
attractive as a model object for studies of the functional activity and
structural properties of membrane proteins
hi the composition of artificially designed energy-transforming
membranes.

The introduction of isotopic labels into molecules of membrane proteins is appropriate for
studies of these proteins. Isotopic labels allow using the method of
high-sensitivity electron impact (El) mass spectrome­try for further analysis of isotopic incorporation [3, 4]. Thus,
studies of BR labeled with the hydrogen isotope (deuterium) at residues of functionally important amino acids
(phenylalanine, tyrosine, and tryptophan) involved
in hydrophobic interaction of the protein polypeptide chain with the
lipid bilayer of the cell membrane are
important for practice [5, 6]. Raw 2H-labeled amino acids can
be readily synthesized in pre­parative
quantities by a reverse isotopic 1H-2H exchange in molecules of protonated amino acids, [2,3,4,5,6-2H5]phenylalanine
(in 85% 2H2SC>4 at50°C), [3,5-2H2]tyrosine (in 6 N 2H2SO4
at slight boiling), and [2,4,5,6,7-2H5]tryptophan
(in 75% [2H]trifluoroacetic acid
at 25°C) [7]. However, in spite of the rapid devel­opment of chemical
methods for obtaining 2H-labeled


aromatic amino acids, the Russian industry of individ­ual 2H-labeled
membrane proteins has not received wide acceptance.

This work was designed to obtain sernipreparative quantities of 2H-labeled
BR for reconstruction of artifi­cial membranes. Processes of incorporation of [2,3,4,5,6-2H5]phenylaIanine,
[3,5-2H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan into the molecule of
bacteri­orhodopsin followed with
further semipreparative iso­lation
were performed. The deuteration level was deter­mined by means of El mass spectrometry performed after separation
of the protein hydrolysate in the form of
methyl esters of /V-DNS derivatives of amino aids by reverse-phase
high-performance liquid chromatogra­phy
(HPLC).

MATERIAL AND METHODS

Objects of studies. The
carotenoid-contain ing strain of extreme halophilic bacteria Halobacterium halo-bium ET 1001 from the
collection of cultures of micro­organisms (Moscow State University) was used. The strain was
maintained on solid peptone medium (2% agar) containing 4.3 M NaCl.

Preparation of growth media. DL-amino acids (Reanal, Hungary), adenosine monophosphate
(AMP) and uridine monophosphate (UMP)
(Sigma, USA), were used.
5-[Dimethylamino]naphthalene-l-sulfonyl chloride (DNS chloride; Sigma, USA) and diaz-omethane obtained
from JV-nitroso-Af-methylurea (Merck, Germany) were applied for the synthesis
of amino acid derivatives. [2,3,4,5,6-2H5]Phenylalanine (90 at. % 2H), [3,5-2H2]tyrosine
(96 at. % 2H), and



[2,4,5,6,7-2H5]tryptophan (98 at. % 2H)
(methods for obtaining are described in [8, 9]) were supplied by A.B. Pshenichnikova
(Candidate of Chemical Sci­ences,
Lomonosov Moscow State Academy of Fine Chemical
Technology).

2H-Labeled BR. 2H-Labeled BR was obtained on a synthetic medium, in
which protonated ammo acids (phenylalanine, tyrosine, and tryptophan) were replaced by their deuterium-containing
analogues ([2,3,4,5,6-2H5]phenylalanine,
[3,5-2H2]tyrosine, and [2,4,5,6,7-2HJtryptophan). The medium contained 0.43
g/1 DL-alanine, 0.4 g/1 L-arginine,0.45 g/1 DL-aspartic acid, 0.05 g/1
L-cysteine, 1.3 g/1 L-glutamic acid, 0.06
g/1 L-glycine, 0.3 g/1 DL-histidine, 0.44 g/1 DL-isoleucine, 0.8 g/1 L-leucine, 0.85 g/1 L-lysine, 0.37 g/1
DL-methionine, 0.26 2/1 DL-phenylalanine, 0.05
g/1 L-proline, 0.61 g/1 DL-serine, 0.5 g/1 DL-thre-onine, 0.2 g/1 L-tyrosine, 0.5 g/1 DL-tryptophan,
1.0 g/1 DL-valine, nucleotides (0.1 g/1 AMP and 0.1 g/1 UMP), salts (250 g/I Nad, 20 g/1 MgSOa x 7H2O,
2 g/1 KC1, 0.5 g/1 NH4C1,
0.1 g/1 KNO3, 0.05 g/1 KH2PO4, 0.05 g/1 KoHPO4, 0.5 g/1 sodium citrate, 3 x 10
-4 g/1 MnSO4 x 2H2O, 0.065 g/1 CaCl2
— 6H2O, 4 x 10 -5 g/l ZnSO4 x 7H2O, 5 x 10 -5FeSO4
— 7H2O, and 5 x 10 -5 g/1 CuSO4 x 5H2O),
1 g/1 glycerin, and growth factors (1 x 10 -4 g/1 biotin, 1.5 x l0 -4 g/1 folic acid,
and 2 x 10 -5 g/1 vita­min
B!2).

Cultivation of bacteria. The growth medium
was autoclaved for 30 min at 0.5 atm (pH was brought to 6.5-6.7 using 0.5 N
KOH). The inoculum was grown in 750-ml Erlenmeyer’s flasks (the medium volume was 100 ml) on a 380-S orbital shaker (Biorad,
Hungary) at 35-37°C under conditions of intensive aeration and illumination (three LDS-40 lamps of 1.5 Ix each).
After 24 h, the inoculum (5-10%) was
transferred to the syn­thetic medium
and grown for five to six days (similarly to obtaining of the inoculum). All further manipula­tions for BR isolation were performed with the use
of a dimming lamp equipped with an ORZh-1
orange light filter.

Isolation of the fraction of purple membranes
(PM).
The biomass (1 g) was washed with distilled water and precipitated on a
T-24 centrifuge (Carl Zeiss, Germany) at 1500 g for 20 min. The precipitate
was suspended in 100 ml of distilled water and kept at 4°C. After 24 h, the reaction mixture was
centrifuged at 1500 g for 15 min. The precipitate was resuspended in 20 ml of distilled water,
disintegrated by sonication (2 kHz, three times per 5 min) on a water bath
containing ice (0°C), and centrifuged at
1500 g for 20 min. After washing with distilled water, the cellular homogenate was resus­pended in 10 ml of buffer containing 125 mM NaCl, 20 mM MgCl2, and 4 mM Tris-HCl (pH
8.0). RNase (5 u,g, two-three units
of activity) was added. The mix­ture was incubated at 37°C. The same
buffer (10 ml) was added 2 h later. The
mixture obtained was kept at 4°C for 14-16 h. The water fraction was removed by
centrifugation at 1500 g for 20 min. The precipitate of


PMs was treated (five times) with 7 ml of 50% ethanol at -5°C. The solvent
was removed by centrifugation at 1200 g and cooling for 15 min. The
protein concentra­tion was measured on a DU-6 spectrophotometer (Beckman, USA)
calculating the D280/D56S ratio [10]. Regeneration of PMs
was conducted as described in [11].

Isolation of BR. The fraction of PMs
(1 mg/ml) was solubilized in 1 ml of 0.05% sodium dodecyl sulfate (SDS), kept at 37°C
for 7-9 h, and centrifuged at 1200 g for 15 min. The precipitate was removed.
Methanol (100
(ll) was added drop wise (three times) to the super­natant at 0°C. The mixture
was kept at -5°C for 14-15 h and then centrifuged at 1200 g and cooling for 15
min. Fractionation was performed three times with decreas­ing the concentration
of SDS to 0.2% and 0.1%. Crys­talline protein (8-10 mg) was washed with cold dis­tilled water and centrifuged at 1200 g for
15 min.

Purification of BR. This
procedure was performed by gel-permeation
chromatography on a calibrated col­umn
(150 x 10 mm). Sephadex G-200 (Pharmacia, USA) served as the stationary phase (bed volume: 30-40 ml per g). The
samples were taken manually. The column
was balanced with the buffer solution contain­ing 0.1% SDS and 2.5 mM EDTA. The protein sample was dissolved in 100 p.1 of the buffer solution and
eluted with 0.09 M Tris-borate buffer
(pH 8.5, / = 0.075) and 0.5 M NaCl at
a flow rate of 10 ml/cm2 per h. Combined protein fractions were subjected to lyo-philization.

Electrophoresis of the protein. The procedure was
performed in 12.5% polyacrylamide gel (PAAG) con­taining 0.1% SDS. The samples were
prepared for elec-trophoresis
by standard procedures (LKB protocol, Sweden). Electrophoretic gel stained with
Coomassie blue R-250 was scanned on a CDS-200
laser densitom-eter (Beckman, USA)
for quantitative analysis of the protein
level.

Hydrolysis of BR. The protein (4 mg)
was placed into glass
ampoules (10 x 50 mm in size), and 4 N Ba(OH)2
(5 ml) was added. The mixture was kept at 110°C for 24 h. The reaction mixture was suspended in 5 ml of hot
distilled water and neutralized with 2 N H2SO4
to pH 7.0. The sediment of BaSO4 was removed by centrifugation at 200 g for 10 min, and
the superna­tant was evaporated in a
rotor evaporator at 40°C.

Synthesis of N-DNS derivatives of amino acids. DNS chloride (25.6 mg) in 2 ml of acetone
was added gradually to 4 mg of dry
hydrolysate of BR in 1 ml of 2 M NaHCO3 (pH 9-10) under
conditions of constant mixing. The reaction mixture was kept at 40°C and mixing for 1 h, acidified with 2 N HCI to pH 3,
and extracted (three times) with 5 ml of ethyl acetate. The combined extract was washed with distilled water
to pH 7.0 and dried with anhydrous
Na2SO4. The solvent was removed at 10 mmHg.

Methyl esters of N-DNS derivatives of
amino acids
. Wet N-nitroso-.N’-methylurea (3 g) was added to 20 ml of 40% KOH in 40 ml of diethyl ether and
then mixed


on a water
bath with ice for 15-20 min for obtaining diazomethane.
After the completion of gas release, the ether layer was separated,
washed with distilled water to pH 7.0, dried
with anhydrous Na2SO4, and used for the treatment of
/V-DNS derivatives of amino acids.

Separation of the mixture of methyl esters
ofN-DNS derivatives of amino acids.
This was performed by the method of
reverse-phase high-performance liquid chro-matography on a Knauer liquid chromatograph
(Ger­many) equipped with a Knauer pump, 2563 UV detec­tor, and C-R 3A
integrator (Shimadzy, Japan). The col­umn of 250 x 10 mm in size was used. Separon
C18 (Kova, Czech) served as the stationary reverse phase. The diameter of
granules was 12 urn. The injection vol­ume was 10 mkl. The following systems of
solvents were used: (A) acetonitrile and trifluoroacetic acid (at a vol­ume ratio of 100 :
0.1-0.5) and (B) acetonitrile. Gradi­ent elution processes were performed at a
rate of 1.5
ml/min for 5 min (from 0% to 20% B), 30 min (from 20% to 100% B), 5 min (100% B), 2 min
(from 100% to 0% B), and 10 min (0% B).

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.

RESULTS AND DISCUSSION

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


Distilled 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


-5°C

-5°C


PM fraction

Decarotenoidation

±

Delipidation + BR
precipitation


—    Extract of
carotenoids

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


Crystalline
BR
       t

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


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
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


between
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
purifica­
tion
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


 

300

400         500         600         700

nm

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



527


200


100


300


400


500


600


 


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



(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 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


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.

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.

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