ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 2, pp. 166–173. © Pleiades Publishing, Inc., 2006. Original Russian Text © Yu.A. Zolotarev, A.K. Dadayan, O.V. Dolotov, V.S. Kozik, N.V. Kost, O.Yu. Sokolov, E.M. Dorokhova, V.K. Meshavkin, L.S. Inozemtseva, M.V. Gabaeva, L.A. Andreeva, L.Yu. Alfeeva, T.S. Pavlov, K.E. Badmaeva, S.E. Badmaeva, Z.V. Bakaeva, G.N. Kopylova, G.E. Samonina, B.V. Vaskovsky, I.A. Grivennikov, A.A. Zozulya, N.F. Myasoedov, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 2, pp. 183–191.
Evenly Tritium Labeled Peptides in Study of Peptide In Vivo and In Vitro Biodegradation Yu. A. Zolotareva,1, A. K. Dadayana, O. V. Dolotova, V. S. Kozika, N. V. Kostb, O. Yu. Sokolovb, E. M. Dorokhovaa, V. K. Meshavkinb, L. S. Inozemtsevaa, M. V. Gabaevab, L. A. Andreevaa, L. Yu. Alfeevaa, T. S. Pavlovc, K. E. Badmaevac, S. E. Badmaevac, Z. V. Bakaevac, G. N. Kopylovac, G. E. Samoninac, B. V. Vaskovskyd, I. A. Grivennikova, A. A. Zozulyab, and N. F. Myasoedova a
Institute of Molecular Genetics, Russian Academy of Sciences, pl. akademika Kurchatova 2, Moscow, 123182 Russia b National Research Center for Mental Health, Russian Academy of Medical Sciences, Kashirskoe sh. 34, Moscow, 115522 Russia c Biological Faculty, Moscow State University, Vorob’evy gory, Moscow, 117234 Russia d Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia Received July 4, 2005; in final form, July 5, 2005
Abstract—Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a µg scale of biological samples both in vitro and in vivo. Key words: enkephalinases of blood plasma, peptidase inhibitors, peptide biodegradation, peptides labeled with tritium, Selank, Semax DOI: 10.1134/S1068162006020099
INTRODUCTION An essential stage in the development of new drugs is elucidation of their action mechanisms.2 The preparations labeled with radioactive isotopes are usually used in the study of drug distribution in tissues, pathways of their biodegradation, and determination of their specific binding to target proteins. The preparations labeled with tritium are most versatile, convenient 1
Corresponding author; phone: +7 (495) 196-0213; fax: +7 (495) 196-0221; e-mail:
[email protected] 2 Abbreviations: HSCIE, high temperature solid phase catalytic isotope exchange; SH, spillover hydrogen.
in handling, and safe. As a rule, they are obtained by catalytic hydrogenation and dehalogenation of precursors with gaseous tritium. The use of these reactions imposes limits on the values of molar radioactivity of labeled preparations, requires big spending to the development of technology for each compound, and restricts the list of labeled ligands in catalogues. It is well known, that, in solid state, isotope atoms can be substituted for hydrogen atoms in amino acids under the action of thermally atomized tritium [1, 2]. This method was successfully used for studying the spatial interaction of proteins in protein–protein com-
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plexes and in viral particles [3, 4]. However, it does not allow a high incorporation of tritium label into the preparations. Highly tritium-labeled compounds with retained physiological activity can be obtained by the isotope exchange with gaseous tritium using the HSCIE reaction occurring in solid organic compounds under the action of SH [5–7]. A virtually complete absence of racemization makes this reaction a valuable preparative method. The HSCIE reaction is carried out at 120–200°ë in a solid mixture consisting of an organic compound applied onto an inorganic carrier and a highly dispersed catalyst of platinum group. By this reaction, deuterium and tritium were practically entirely substituted for protium in some amino acids. The data on reactivity of amino acids were obtained using NMR spectroscopy [8]. The mechanism of HSCIE reaction can be represented as a synchronous one-center substitution at the saturated carbon atom characterized by the formation of pentacoordinated carbon and a three-center bond with involvement of the incoming and outgoing hydrogen atoms in the transition state [9]. The HSCIE reaction in amino acids preliminarily applied onto an inert inorganic support occurs on the acidic catalytic centers formed in the solid mixture under the action of SH [10]. Quantum-chemical calculations showed that the participation of electron donors (O and N atoms) in the stabilization of the transition state of HSCIE is a prerequisite for the proceeding of this reaction in amino acids and peptides. These atoms in peptides may belong to both the amino acid involved into the exchange reaction and the amino acid residue spatially close to this amino acid. The reactivity of amino acid fragments depends on the structure of peptides [11, 12]. A number of tritium-labeled peptides with completely retained biological activity were obtained using HSCIE. The molar radioactivities of these peptides were many times higher than those of labeled preparations obtained by traditional methods, which is particularly important for studying the specific binding. The evenly tritiumlabeled peptides obtained using the HSCIE reaction were used for studying the receptor binding to opioid and nicotinic acetylcholine receptors [13–15]. Unlike the label in peptides obtained by traditional methods, the isotope label introduced in peptides using HSCIE is distributed throughout the entire peptide molecule, which allows the monitoring of all the products of peptide biodegradation. The evenly tritium-labeled peptide [G-3H]MEHFPGP was used for studying the Semax biodegradation by peptidases from the rat forebrain cellular membranes [16] and fetal serum [17]. The inhibitory action of Selank on the [Leu]enkephalin biodegradation in human blood plasma was analyzed using the evenly tritium–labeled peptide [G-3H][Leu]enkephalin [18, 19]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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Table 1. The concentration (nM) of Semax and the products of its in vitro degradation by peptidases of the rat forebrain neuronal cells* Peptide EHFPGP FPGP HFPGP MEHFPG MEHFP MEHFPGP (Semax)
Incubation time, h 0.5
1
2
3
24
0 0 9 4 7 418
0 0 15 6 13 397
1 1 29 8 17 371
2 4 44 10 20 336
3 10 79 16 45 116
* The starting concentration of [3H]MEHFPGP in the incubation mixture was 440 nM.
RESULTS AND DISCUSSION An analogue of a fragment of adrenocorticotropic hormone ACTH-(4–10) (Semax), the heptapeptide MEHFPGP completely devoid of the hormonal activity but exhibiting properties of a nootropic, has been synthesized in the Institute of Molecular Genetics of the Russian Academy of Sciences [20]. Semax has been shown to be capable of increasing the lifetime of nerve cells in primary cultures by reducing their death [21], which is probably associated with another important property of Semax: it is an efficient agent in the therapy of brain ischemia. Peptide preparations undergo a rapid biodegradation in tissues of organism. The resulting peptide fragments may essentially contribute to the pharmacological activity of preparation. Therefore, the study of kinetics and pathways of transformation of a biologically active peptide in tissues of organism can be regarded as an important stage in elucidation of molecular mechanism of its action. One of the possible mechanisms of neuroprotective and nootropic action of Semax may be the regulation of expression of some neurotrophins and their receptors in brain [22, 23]. We used the peptide MEHFPGP evenly labeled with tritium by HSCIE [12] for the study of Semax biodegradation in the presence of rat brain neuronal cells. The cell culture was incubated with the peptide [G-3H]MEHFPGP, and the composition of a mixture of labeled peptides formed upon its biodegradation was studied by HPLC (Table 1). The results show that the degradation of Semax in the presence of cultures of neuronal cells from rat forebrain basal nuclei proceeds from both the C- and N-termini at comparable rates. The predominant process is the formation of five-aa fragments. This may indicate that dipeptidyl peptidases that cleave the N- and C-terminal fragments ME and GP are mainly involved in the Semax biodegradation. Note that the degradation of Semax in the presence of cellular membranes of rat forebrain predominantly pro-
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Table 2. Pharmacokinetics of PGP in human blood plasma* Time, min 5 10 15 30 60 120 240
Peptide concentration, nM PG
GP
PGP
Degradation degree, %
10 50 70 60 50 40 40
520 700 900 1090 910 530 460
2960 2160 1400 860 490 370 290
20 42 62 77 87 90 92
* The starting concentration of PGP was 3710 nM.
Table 3. The concentration (nM) of Selank and the products of its in vitro degradation by peptidases of the rat blood plasma Peptide* 1 2 3 4 5 6 7 8 9
TKP RP PRPGP TKPR TKPRPG TKPRP TKPRPGP RPGP KPRPGP
Incubation time, min 0.33
1
1.5
2
2.5
4
23
0 0 12 0 0 109 895 0 0
39 38 37 0 0 151 731 0 0
82 69 46 0 0 171 555 0 0
125 123 53 0 0 164 410 0 0
137 185 35 0 0 145 321 0 0
166 207 37 0 0 118 158 0 0
19 31 5 0 0 6 8 0 0
* The order of peptides in the table corresponds to the order of their chromatographic elution (Fig. 1). The starting concentration of [3H]TKPRPGP was 1210 nM.
ceeds from its N-terminus and leads to the formation of the only peptide, HFPGP [16]. The in vivo experiments carried out on the models of ulcer formation in white rats have shown [24] that the peptide PGP defends the gastric mucosa by exerting a protective effect and ulcer-curing properties. This peptide has been shown to be effective in the stress model of ulcer formation where the mechanisms are realized with involvement of the central nervous system. The efficiency of PGP has also been demonstrated in the models of gastric mucosa lesions associated with peripheral mechanisms of ulcerogenesis, which are realized upon direct action of ethanol or glacial acetic acid on gastric mucosa [25, 26]. The evenly tritiumlabeled peptide PGP (80 mCi/mmol) obtained by HSCIE was used to study its biodegradation in human blood plasma (Table 2). This peptide is highly stable and present in significant amounts even after several hours of incubation. The predominant pathway of its
biodegradation is a cleavage of N-terminal Pro under the catalysis by aminopeptidase. The peptide Selank (TKPRPGP) was designed in the Institute of Molecular Genetics of the Russian Academy of Sciences in collaboration with the Research Institute of Pharmacology of the Russian Academy of Medical Sciences. It exhibits a stable anxiolytic effect and is devoid of all the side effects characteristic of common anxiolytic tranquilizers. Selank is advantageous, because its anxioselective action is combined with the activating effect on the mnestic and cognitive functions of brain [27]. The pharmacokinetics of Selank was studied using its evenly tritium-labeled analogue (110 Ci/mmol), obtained by the HSCIE reaction at 180°ë and containing isotope label at all its amino acid residues (Table 3). The composition of products of Selank hydrolysis by the multienzyme system of blood plasma peptidases was chromatographically analyzed at a certain time (Fig. 1). The concentration of [G-3ç]Selank and its fragments in blood plasma were determined by radioactivity of the corresponding HLPC fractions. We found that 90% of peptidase activity of blood plasma in vitro falls on the dipeptidyl carboxypeptidases responsible for the formation of pentapeptide TKPRP and the most long-living tripeptide TKP and dipeptides RP and GP. The dipeptidyl aminopeptidases account for 10% of the activity, whereas aminopeptidases and carboxypeptidases that cleave amino acid residues from the N- and C-termini do not substantially contribute to the in vitro biodegradation of Selank. The peptides evenly labeled with tritium serve as effective tools for studying the in vivo distribution of peptides and their fragments in organs and tissues. One can see from the in vivo distribution of Selank and the products of its peptidase degradation in rat organs 3 min after intranasal introduction (Table 4) that their content in brain parts is several times higher than that in blood. The highest concentration of Selank in olfactory bulbs is 5–10 times higher than in blood upon its intranasal introduction. The composition of the products of in vivo Selank degradation is similar in brain structures and in blood; it is mainly determined by the action of dipeptidyl carboxypeptidases (~70%). The products of dipeptidyl aminopeptidases and aminopeptidases account for approximately 20% and 10%, respectively. Thus, the role of aminopeptidases in the in vivo proteolysis of Selank is somewhat higher than in its in vitro biodegradation in blood plasma. The endogenous opioid system is one of the most important regulatory systems of organism. It comprises opioid peptides and the corresponding opioid receptors, as well as enkephalinases and inhibitors of enkephalinases. Most functions of the endogenous opioid system are regulatory and directed to the maintenance of homeostasis in organism. This system regulates the pain sensitivity, emotions, learning processes, memory, thermoregulation, hormone secretion, and the functions
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169
1
7
3
9
2 4
6
5
8
ch2
1
2
3
4
5
6
7
8 min
9
10
11
12
13
14
15
Fig. 1. The biodegradation of Selank in rat organs in vivo and in vitro. The chromatography of a mixture of Selank, its peptide fragments (5 µg each), a peptide fraction extracted from the sample of plasma containing [3H]Selank, and the products of its degradation. A Kromasil column (4 × 150 mm), a gradient elution with acetonitrile in 0.1% heptafluorobutyric acid; 1, TKP; 2, RP; 3, PRPGP; 4, TKPR; 5, TKPRPG; 6, TKPRP; 7, TKPRPGP; 8, RPGP; and 9, KPRPGP.
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Peptide
Midbrain
Cerebral cortex
Cerebellum
The enzymatic hydrolysis was carried out up to 20−80% conversion of the initial [Leu]enkephalin. The radioactive products are formed from [Leu]enkephalin in four pathways. The main way is the action of aminopeptidases; they account for ~90% of the activity (Table 5). This biodegradation type is in accord with the
Table 4. The concentration (pM) of Selank and the products of its in vivo peptidase degradation in rat tissues upon intranasal introduction of the tritium-labeled Selank (500 µCi) Olfactory bulbs
Here, we present the data on the inhibitory effect of Semax on the activity of rat blood plasma enkephalinases. The enkephalinase activity was determined by the rate of accumulation of the products of enzymatic degradation of [Leu]enkephalin (YGGFL) [18]. The concentrations of [Leu]enkephalin and the products of its peptidase degradation were determined using the evenly tritium-labeled [Leu]enkephalin; the data on the distribution of label in the tritium-labeled [Leu]enkephalin were obtained by the NMR spectroscopy [12]. The composition of the mixture of labeled peptides formed upon its biodegradation was analyzed by HPLC (Fig. 2).
results of [Leu]enkephalin biodegradation in human blood plasma, which had previously been obtained by the same method [16]. The rate of [Leu]enkephalin hydrolysis is substantially reduced in the presence of Semax. The rate of its hydrolysis in diluted rat blood plasma is reduced approximately twofold at 40-µM
Blood
of respiratory, cardiovascular, digestive, and immune systems. Moreover, the enzymes hydrolyzing endogenous opioid peptides, in particular, enkephalinases, are known to one or other extent participate in the degradation of other regulatory peptides, such as substance P, bradykinin, and angiotensin. Therefore, the inhibitors of enkephalinases may affect the physiological functions, which are not directly connected with the endogenous opioid system, e.g., blood pressure or heartbeat rate.
1 2 3 4 5 6 7 8 9
TKP (C) RP (C) PRPGP (N) TKPR (C) TKPRPG (C) TKPRP (C) TKPRPGP RPGP (N) KPRPGP (N)
1.96 1.63 0.23 0 1.28 2.40 5.92 0.12 0.94
9.8 6.3 0 0 7.8 22 56 0 4.1
1.8 1 0 0 2.2 5.4 11.6 0 0.9
0.5 0.5 0 0 0.7 2.5 3.1 0 0.6
0.5 0.6 0 0 1.8 4.3 6.1 0 1.7
* The order of peptides corresponds to the order of their chromatographic elution (Fig. 1); the direction of peptidase action on the starting peptide is indicated in parentheses (N- or C-end).
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5
1
8
2
3
ch1 4
6 7
ch2
5
10
20
15
25
30
min
Fig. 2. The biodegradation in vitro of [Leu]enkephalin in blood plasma in the presence of the heptapeptide Semax (40 µM). A chromatography of a mixture of [Leu]enkephalin, its peptide fragments (5 µg each), and the peptide fraction extracted from a plasma sample and containing (G-3ç)[Leu]enkephalin and products of its biodegradation. A gradient elution with acetonitrile in 0.1% mixture of heptafluorobutyric acid and TFA; ch1, 254 hm; ch2, 280 nm; 1, YGG; 2, Y; 3, YG; 4,GGF; 5, YGGF; 6, GGFL; 7, GFL; and 8, YGGFL.
Semax concentration. The inhibition of aminopeptidase makes the main contribution to the change in the enkephalinase activity of rat blood plasma, since the concentrations of [3H]YGGF and tyrosine formed by the action of this enzyme were also approximately twofold reduced in the presence of Semax. Judging from a negligible reduction of the concentrations of other
labeled peptides, Semax exerts a slight effect on the activity of other enzymes of blood plasma involved in the hydrolysis of [Leu]enkephalin. Thus, we can conclude that the evenly tritiumlabeled peptides allow the effective study of the kinetics and the pathways of in vivo and in vitro transformations of peptides. A high molar radioactivity of labeled pep-
Table 5. The effect of Semax on the rate of biodegradation of [Leu]enkephalin (YGGFL) in rat blood plasma according to the chromatographic analysis of [G-3H]enkephalin and its fragments* Incubation time, min Peak number 1 2 3 4 5 6 7 8
Peptide**
YGG Y YG GGF YGGF GGFL GFL YGGFL [Leu]enkephalin Conversion YGGFL, %
8
16
32
A
B
A
B
A
B
0.07 1.26 0.02 0.05 0.03 1.15 0.18 5.64 20
0.07 0.76 0.02 0.01 0.03 0.74 0.06 6.23 11
0.24 3.02 0.10 0.44 0.15 2.28 0.15 3.65 48
0.32 1.18 0.12 0.02 0.09 1.08 0.07 5.25 25
0.15 5.44 0.07 0.17 0.12 4.48 0.38 1.45 79
0.25 2.73 0.12 0.04 0.15 2.27 0.28 4.27 39
Notes: * A, the concentration (µM of [Leu]enkephalin and the products of its peptidase biodegradation in the plasma fivefold diluted with buffer; B, the same in the presence of 40 µM Semax. ** The order of peptides corresponds to the order of their chromatographic elution (Fig. 2). RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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tides obtained using the HSCIE reaction permits the effective use of these peptides in studying the peptidergic systems. EXPERIMENTAL Solid phase isotope exchange of hydrogen by tritium in peptides. A peptide (1.0 mg) was dissolved in water (1 ml) and mixed with aluminum oxide (10 mg, Serva). Water was removed in a vacuum at 20°ë. The aluminum oxide with applied peptide was mixed with 5% Rh/Al2O3 catalyst (10 mg, Fluka). The resulting solid mixture was placed in an ampoule of 10 ml volume. The ampoule was evacuated, filled with gaseous tritium up to the pressure of 1.84 Pa, and kept for 10– 60 min at 140–190°ë. The ampoule was then cooled, evacuated, and flushed with hydrogen. The peptide was desorbed with 20% ethanol and twice dissolved in 20% ethanol and evaporated to dryness in order to remove the labile tritium. The labeled peptide was purified on Kromasil C18 (10 × 250 mm) and Nucleosil C18 (4 × 250 mm) columns with a concentration gradient of acetonitrile in 0.1% aqueous TFA. The molar radioactivity of peptide was calculated using a liquid scintillation counter. The evenly tritium-labeled peptides TKPRPGP and YGGFL with the molar radioactivity of 110 and 50 Ci/mol, respectively, were obtained at 180°ë. The evenly tritium-labeled peptides MEHFPGP and PGP with the molar radioactivities of 45 and 80 Ci/mol, respectively, were obtained at 160°ë. Preparation of samples for chromatographic analysis of biodegradation products of labeled peptides. The in vivo study of the pharmacokinetics of tritium-labeled peptides was carried out on male outbred rats (200–250 g of body mass). A solution of peptide analyzed was evaporated to dryness and dissolved in isotonic solution. The resulting solution with label concentration of 25 mCi/ml (20 µl) was intranasally introduced to rats. The rats were killed by decapitation, the tissues under investigation were taken out during 1−1.5 min and frozen with liquid nitrogen. The fractions containing labeled peptides were isolated by extraction with organic solvents upon disintegration of the tissues in a cell homogenizer. To this end, the frozen tissue (1–2 g) was placed in a homogenizer, mixed with 20% acetonitrile (20 ml) containing 1% TFA, and ground for 10 min. The mixture was centrifuged at 12 000 g for 15 min at 4°ë. The supernatant was taken and evaporated in a rotary evaporator. The dry residue was dissolved in methanol (1 ml) and centrifuged under the conditions described above. The supernatant was dried on a rotary evaporator. The dry residue was dissolved in 0.1% TFA (100 µl), frozen, and stored at –20°ë. For studying the in vitro biodegradation of tritiumlabeled peptides, the sample under study was evaporated to dryness and dissolved in isotonic solution. A solution with concentration of 10 mCi/ml was used for RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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incubation. After incubation, a tenfold volume of 90% acetonitrile containing 1% TFA was added to the sample (10–200 µl). The mixture was centrifuged at 12000 g for 15 min at 4°ë. The subsequent treatment was carried out as described for the in vivo analysis of pharmacokinetics of the labeled peptides. To analyze the biodegradation of PGP, [3H]PGP (50 µCi) was incubated in human blood plasma (150 µl). Then the proteolysis was stopped, the peptide fraction was extracted from plasma, and PGP, GP, and PG were isolated by HPLC. In vitro study of the Semax biodegradation in the presence of neuronal cells. A primary dissociated culture of rat forebrain basal nuclei neurons was obtained from 16–18-day embryos according to procedure [28]. Pregnant rats of the Sprague-Dowly line (body mass 200–300 g) were killed by carbon dioxide asphyxia (15 min) and placed in aqueous 80% ethanol for 1 min. A brain isolated from the embryo under aseptic conditions was mechanically dissociated to individual cells by pipetting. The primary neurons were cultured in 24-well plates (500 × 103 cells per well) in a medium containing D-glucose (6 g/l), progesterone (2 mM), insulin (25 mg/l), putrescine (100 mg/ml), and sodium selenite (30 nM). The cells were incubated in a ëé2 incubator at 37°ë in an atmosphere containing 5% ëé2 and 95% air. [3H]Semax (440 nM) with the specific radioactivity of 45 Ci/mol was added into the incubation mixture. The aliquots of incubation mixture were taken, cooled to 4°C, and centrifuged at 15 000 g for 15 min at 4°ë. The supernatants were stored at –20°ë. HPLC analysis of the biodegradation of peptides. The procedure for determination of the content of labeled peptide and its metabolites is based on the measurement of radioactivity of chromatographic fractions of the corresponding unlabeled fragments preliminary added to the sample under study before separation and isolated using UV detection (the mass of the labeled peptide and its [3H]metabolites subjected to chromatographic separation according to this procedure is too small for their direct observation using UV detection). A mixture of peptide fragments was separated by HPLC on a Kromasil C18 (5 µm) column (4 × 150 mm) at 20°ë. The detection was carried out at the wavelengths of 254 and 280 nm on a Beckman 165 spectrophotometer (Altex). The samples of peptide extracts obtained from tissues (40 µl) were mixed with a solution of unlabeled peptide under study (30 µl) and its metabolites (5 µg each) and were subjected to a gradient elution, and the radioactivities of products in each peak were determined. The contents of [G-3ç]peptide and its tissue metabolites were determined from the data on the content of radioactivity in peptide fraction, and the molar radioactivity of the corresponding fragment was calculated from the distribution of the label in peptide. Chromatographic separation of [G-3ç]Semax and peptide products of its biodegradation was carried
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out by HPLC on the same column at 20°ë as described in [16]. A gradient elution with 15–35% acetonitrile containing 0.1% heptafluorobutyric acid for 20 min was used for the chromatographic analysis of [G-3ç]Selank and its peptide fragments (Fig. 1). A chromatographic analysis of tritium-labeled [Leu]enkephalin was carried out in a mixture of eluent A (5% acetonitrile containing 0.05% TFA and 0.05% heptafluorobutyric acid) and eluent B (30% acetonitrile in eluent A). The following gradient of B in A was used: from 0 to 10% for 10 min and from 10 to 50% for 40 min at a flow rate of 1 ml/min (Fig. 2). A gradient elution with 2–15% acetonitrile containing 0.1% TFA for 20 min at a flow rate of 1 ml/min was used for the chromatographic analysis of [G-3ç]PGP. The effect of Semax on the enkephalinase activity of rat blood plasma peptidases. The enkephalinase activity was determined by the rate of accumulation of the products formed upon the enzymatic degradation of [G-3ç]enkephalin according to the modified procedure [16]. An incubation mixture (final volume of 125 µl) contained a fivefold diluted heparinized rat blood plasma (25 µl), 10 mM Tris-HCl buffer (pH 7.5, 100 µl) containing 150 mM NaCl, [G-3ç]enkephalin (44 µCi, the specific radioactivity 50 Ci/mmol), and the peptide under study (40 µM). The incubation was carried out at 37°ë, aliquots (10 µl each) were taken, and the biodegradation was stopped by addition of 90% acetonitrile (100 µl) containing 0.1% TFA. ACKNOWLEDGMENTS This work was supported by the Program Molecular and Cellular Biology of the Russian Acedemy of Sciences and the Russian Foundation for Basic Research (project no. 05-03-32411. REFERENCES 1. Shishkov, A.V., Filatov, E.S., Simonov, E.F., Unukovich, M.S., Gol’danskii, V.I., and Nesmeyanov, An.N., Dokl. Akad. Nauk SSSR, 1976, vol. 228, pp. 1237–1239. 2. Shishkov, A.V. and Baratova, L.A., Usp. Khim., 1994, vol. 63, pp. 825–841. 3. Agafonov, D.E., Kolb, V.A., and Spirin, A.S., Proc. Natl. Acad. Sci. USA, 1997, vol. 94, pp. 12 892–12 897. 4. Shishkov, A.V., Goldanskii, V.I., Baratova, L.A., Fedorova, N.V., Ksenofontov, A.L., Zhirnov, O.P., and Galkin, A.V., Proc. Natl. Acad. Sci. USA, 1999, vol. 96, pp. 7827–7830. 5. Zolotarev, Yu.A., Kozik, V.S., Zaitsev, D.A., Dorokhova, E.M., and Myasoedov, N.F., Dokl. Akad. Nauk SSSR, 1989, vol. 308, pp. 1146–1151. 6. Zolotarev, Yu.A., Kozic, V.S., Zaitsev, D.A., Dorokhova, E.M., and Myasoedov, N.F., J. Radioanal. Nucl. Chem. Art., 1992, vol. 162, pp. 3–14. 7. Zolotarev, Yu.A., Dadayan, A.K., and Borisov, Yu.A., Rus. J. Bioorg. Chem., 2005, vol. 31, pp. 1–17.
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