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 abil-ities [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 spectrome-try 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 pre-parative 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 devel-opment of chemical methods for obtaining
2H-labeled
aromatic amino acids, the Russian industry of
individ-ual 2H-labeled membrane proteins has not
received wide acceptance.
This work was designed to obtain sernipreparative
quantities of 2H-labeled BR for reconstruction of
artifi-cial 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 bacteri-orhodopsin followed with further semipreparative
iso-lation were performed. The deuteration level was deter-mined 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
chromatogra-phy (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 micro-organisms (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
Sci-ences, 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
vita-min 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
Erlenmeyers 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
syn-thetic medium and grown for five to six days (similarly to
obtaining of the inoculum). All further manipula-tions 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 resus-pended 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 mix-ture 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 concentra-tion 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 super-natant 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 decreas-ing the concentration of SDS to 0.2% and
0.1%. Crys-talline protein (8-10 mg) was washed with cold
dis-tilled water and centrifuged at 1200 g for 15 min.
Purification of BR. This procedure
was performed by gel-permeation chromatography on a calibrated
col-umn (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
contain-ing 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) con-taining 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 superna-tant 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 (Ger-many) equipped with a Knauer
pump, 2563 UV detec-tor, and C-R 3A integrator (Shimadzy, Japan).
The col-umn 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 vol-ume was 10 mkl. The
following systems of solvents were used: (A) acetonitrile and
trifluoroacetic acid (at a vol-ume ratio of 100 : 0.1-0.5) and (B)
acetonitrile. Gradi-ent 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 prepa-rations 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 incorpo-ration, 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 opti-mum growth conditions
(exponential growth on a syn-thetic medium with 4.3 M NaCl at
35-37°C and illumi-nation), 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
con-taining 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
admix-tures, cellular RNA, carotenoids, and lipids; fraction-ation
of solubilized (in 0.05% SDS) protein by metha-nol; and
purification on Sephadex G-200. Low-molec-ular-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 cel-lular
homogenate was then treated with RNase I (two-three units of
activity) to induce the maximum destruc-tion of cellular RNA. The
PM fraction obtained con-tained the complex of the desired protein
with Hpids and polysaccharides, as well as admixtures of fixed
car-otenoids and foreign proteins. Therefore, it was neces-sary to
use special methods of protein fracdonation, which would not damage
the native structure of the pro-tein 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 neces-sary (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 caro-tenoids. Figure 3 shows (curves b,
c) these bands at vari-ous 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
DNS chloride, 2 M
NaHCO3, 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
remov-ing carotenoids was reached. The formation of the
reti-nal-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
num-ber was determined by the presence of trans-retinal
res-idue 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
absorp-tion 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 sol-vents 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 com-plex with molecules of lipids and detergent)
was in the supernatant. Another high-molecular-weight admix-tures
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 per-formed by
decreasing the detergent concentration 2.5 and 5 times,
respectively. The final stage of BR purifi-cation 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) con-taining 0.1%
SDS and 2.5 mM EDTA. The method designed for fractionation of the
protein made it possi-ble 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
empha-sized 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 deu-terium-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 treat-ment, 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 com-plete 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
notice-able weakness. Processes of the isotopic exchange (of a high
rate) of aromatic protons (deuterons) in mole-cules 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 con-ditions of acid hydrolysis. Thus, the data on
incorpora-tion of deuterium into the protein can not be derived
from the hydrolysis performed even in deuterium-con-taining
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 proce-dure 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 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.
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