[Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin ß-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the ß-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

[The study of peptide stability during hydrolysis by rat gastroenteric tract fragments].

The hydrolytic stability of therapeutic peptides such as dalargin, stemokin and some others, including cyclic tripeptides modified by ibuprofen and aspirin, was studied. Two experimental systems were used, one containing purified enzymes pepsin, trypsin and chymotrypsin and other based on fragments of rat stomach and ileum. It was found that linear peptides without D-aminoacids are hydrolyzed by fragments of stomach and ileum but resistant to hydrolysis with purified enzymes. The peptides with D-aminoacids and cyclic peptides are stable in all experimental conditions used, however, peptides modified with aspirin lost acetyl moiety of aspirin residue in acidic medium, the process is accelerated in presence of pepsin.

Sulfation of N-acyl dopamines in rat tissues.

Sulfation of N-acyl dopamines has been shown for the first time in cytosolic fractions of rat liver and nervous system. Sulfation of dopamine amides of docosahexaenoic and oleic acids occurred in all tissues studied, N-arachidonoyl dopamine was sulfated in the liver and spinal cord, and N-stearoyl dopamine was sulfated only in the liver. Depending on the substrate and tissue, the sulfation activity varied from 0.5 to 3.5 nmol/min per mg total protein. Kinetic parameters of N-docosahexaenoyl dopamine sulfation in the brain were determined. The findings characterize the sulfation system as the most productive metabolic pathway of N-acyl dopamines, but the role of this system in the body is unclear because of high K(m) value.

[Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes].

A reaction of high-temperature solid-phase catalytic isotope exchange (HSCIE) was studied for the preparation of tritium- and deuterium-labeled ligands of glutamate and dopamine receptors. Tritium-labeled (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclopenten-5,10-imine ([G-(3)H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-(3)H]-7-OH-DPAT) were obtained with a specific activity of 210 and 120 Ci/mol, respectively. The isotopomeric distribution of deuterium-labeled ligands was studied using time-of-flight mass-spectrometer MX 5310 (ESI-o-TOF) with electrospray and orthogonal ion injection. Mean deuterium incorporation per ligand molecule was 11.09 and 3.21 atoms for [G-(2)H]MK-801 and [G-(2)H]-7-OH-DPAT, respectively. The isotope label was shown to be distributed all over the ligand molecule. The radioreceptor binding of tritium-labeled ligands [G-(3)H]MK-801 and [G-(3)H]-7-OH-DPAT was analyzed using the brain structure of Vistar rats. It was demonstrated that [G-(3)H]MK-801 specifically binds to hippocampus membranes with K(d) 8.3 +/- 1.4 nM, B(max) being 3345 +/- 300 fmol/mg protein. The [G-(3)H]-7-OH-DPAT ligand specifically binds to rat striatum membranes with K(d) 10.01 +/- 0.91 nM and B(max) 125 +/- 4.5 fmol/mg protein. It was concluded that the HSCIE reaction can be used for the preparation of highly tritium-labeled (+)-MK-801 and 7-OH-DPAT with retention of their physiological activities.

[Fragments of functional proteins in the primary culture of human erythrocytes].

According to previously reported data, the supernatant of a primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic beta-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, as well as the amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age or blood group of donors. The dynamics of accumulation in the supernatant (20-80 min of incubation) of the 14 hemoglobin fragments with the most reliably reproducible contents was obtained. The content of six peptides increased more than twofold between 20 and 40 min of incubation: the maximum increase in concentration was observed between 40 and 80 min (140%). The level of peptides that had the maximum concentration at the end of incubation was about 1000 pmol/ml of sedimented erythrocytes. The biological effects of the peptides identified in the supernatant of erythrocytes involve the stimulation of proliferation and hemopoiesis, suppression of proliferation, a bactericide effect, etc. These effects indicate the physiological importance of peptide release by erythrocytes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[Arachidonoyl amino acids and arachidonoyl peptides: synthesis and properties].

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, gamma-amino butyric acid (GABA), dihydroxyphenylalanine, tyrosine, tryptophan, and alanine) and peptides (Semax, MEHFPGP, and PGP) were synthesized in order to study the biological properties of acylamino acids. The mass spectra of all the compounds at atmospheric pressure electrospray ionization display the most intense peaks of protonated molecular ions; the detection limits for these compounds are 10 fmol per sample. AA-Gly showed the highest inhibitory activity toward fatty acid amide hydrolase from rat brain (IC50 6.5 microM) among all the acylamino acids studied. AA-Phe, AA-Tyr, and AA-GABA exhibited a weak but detectable inhibitory effect (IC50 55, 60, and 50 microM, respectively). The acylated amino acids themselves, except for AA-Gly, were stable to the hydrolysis by this enzyme. All the arachidonoylamino acids inhibited cabbage phospholipase D to various degrees; AA-GABA and AA-Phe proved to be the most active (IC50 20 and 27 microM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue showed no formation in vitro of either this acylamino acid or AA-dopamine and AA-Phe, the products of its metabolism. The highest contents of these metabolites were detected in liver homogenate and in the brain homogenate, respectively. Acylamino acids exert no cytotoxic effect toward the glioma C6 cells. It was shown that N-acylation of Semax with arachidonic acid results in enhancement of its hydrolytic stability and increases its affinity for the sites of specific binding in rat cerebellum membranes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

[A comparative study of antistroke activity of the new drug "cerebral" and its fractions in rats].

The effect of the new drug "cerebral" and its fractions 1-3 on the model of bilateral hemorrhagic stroke in white rats was studied with reference to the action of cebrolysin and cerebrolysate-M. With respect to the general functional state, behavioral activity restoration, and morphological data, the most pronounced antistroke action was observed for the cerebral-1 fraction. This fraction was further separated into three subfractions. The most promising test results were obtained for the 1.2 subfraction, which was selected for the further investigation.

Determination of biologically active low-molecular-mass thiols in human blood. III. Highly sensitive narrow-bore isocratic reversed-phase high-performance liquid chromatography with fluorescence detection.

The fast isocratic narrow-bore reversed-phase high-performance liquid chromatographic method employing fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine, cysteine and glutathione in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. Optimized sample preparation conditions as well as chromatographic conditions allowed to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm and pathology. The detection limit was approximately 70 amol for all labeled aminothiols. The proposed method for these compounds analysis includes simple sample preparation, high selectivity, good linearity (r2>0.998), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<1.8% for three times consequently injected sample); high reliability and the small sample volume (1 microl) required for analysis make it suitable for clinical studies.

Monitoring of recombinant human insulin production by narrow-bore reversed-phase high-performance liquid chromatography, high-performance capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry.

An analytical scheme for monitoring recombinant human insulin (rhI) production is suggested. The scheme includes high-performance separation micro-techniques (narrow-bore RP-HPLC, HPCE) based on different separation mechanisms and matrix-assisted laser desorption ionisation time-of-flight MS, and allows one to obtain unambiguous information about purity and primary structure of all intermediates of the rhI production. The use of this scheme at all production steps provided optimisation of certain technological parameters [conditions for a fusion protein (FP) refolding, temperature and duration of the FP cleavage with trypsin, conditions for carboxypeptidase B digestion of di-ArgB31-B32-insulin] and achievement of a high purity of the end-product. The proposed scheme may be used for solving various problems in monitoring production of other recombinant proteins.

Determination of biologically active low-molecular-mass thiols in human blood. I. Fast qualitative and quantitative, gradient and isocratic reversed-phase high-performance liquid chromatography with photometric and fluorescence detection.

The fast isocratic and gradient reversed-phase high-performance liquid chromatographic methods employing photometric and/or fluorescence detection are described for the precise reproducible simultaneous measurement of total homocysteine, cysteine, and glutathione in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions), as well as by chromatographic conditions to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm and pathology. Its sensitivity allows the detection of aminothiol quantities >2 pmol.

Determination of biologically active low-molecular-mass thiols in human blood. II. High-performance capillary electrophoresis with photometric detection.

A new high-performance capillary electrophoresis assay for aminothiols in human blood, including homocysteine, a marker of several human metabolism disorders, has been developed. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fused-silica capillary (65 cm x 50 microm I.D.) at 30 degrees C. With the electric field of 250 V cm(-1), separation of homocysteine, glutathione and cysteine occurred at less than 10 min. Detection at 250 or 234 nm was used to confirm the monobimane-thiols peaks. The detection limit was approximately 5 nmol/ml for all labeled aminothiols. The proposed method for these compounds' analysis included simple sample preparation, high selectivity, good linearity (r2>0.999), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<5% for three times consequently injected sample); high reliability and the small volumes required for analysis made it suitable for clinical studies.

Direct determination of amino acids and carbohydrates by high-performance capillary electrophoresis with refractometric detection.

This is an initial report to propose a novel approach in high-performance capillary electrophoresis (HPCE) for the direct detection of compounds without natural absorbance in the UV and visible spectral range, such as amino acids and carbohydrates. A refractometry detector with the 2 nl cell (Applied Systems, Minsk, Belarus) was employed to identify amino acids and carbohydrates without derivatization. The first results are provided on separation of seven free amino acids in the phosphate running buffer and three free carbohydrates in the borate-sodium dodecyl sulfate running buffer and detection by refractometer. Fused capillaries of 50 or 75 microm internal diameter and separation voltage (10-23 kV) were applied. Detection limits ranged typically from 10 to 100 fmol and the response was linear over two orders of magnitude for most of the amino acids and carbohydrates. The HPCE system demonstrated good long-term stability and reproducibility with a relative standard deviation, less than 5% for the migration time (n=10).

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity.

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome-c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin-derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions.

[Analytical biotechnology of recombinant peptides and proteins. II. Primary structure of the fusion protein containing human proinsulin and optimization of its proteolysis by trypsin]. / Analiticheskaia biotekhnologiia rekombinantnykh peptidov i belkov. II. Kontrol' pervichnoi struktury gibridnogo belka, soderzhashchego chelovecheskii proinsulin, i optimizatsiia ego proteoliza tripsinom.

The kinetics of trypsin proteolysis of the fusion protein (FP) containing human proinsulin was studied by a set of analytical micromethods. These were the microcolumn reversed-phase HPLC and the qualitative identification by MALDI-TOF mass spectrometry and amino acid sequencing. The first stage of the proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off of C-peptide from proinsulin results in the formation of ArgB31-ArgB32-insulin. The effect of temperature on the formation of de-ThrB30-insulin, a by-product, was also studied. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain no N-terminal Met residue.

GABA-induced changes of the tissue-specific peptide pool of white rat brain.

Internasal administration of gamma-aminobutyric acid (GABA) induced prolonged behaviour changes and the appearance of three new compounds absent in the brain extracts of control rats. Two peptides associated with GABA administration were isolated and sequenced: Thr-Tyr-Thr-Phe, which corresponds to a gamma-immunoglobulin segment, and Val-Leu, which is present in a great number of proteins, hence its precursor could not be established. The third compound was not amenable to the Edman degradation technique. The data obtained show that the introduction of a neurotransmitter could cause specific changes in the levels of tissue-specific peptide components.

[Analytical biotechnology of recombinant peptides and proteins. I. Analysis of the purity, composition and structure of human, porcine and bovine insulins]. / Analiticheskaia biotekhnologiia rekombinantnykh peptidov i belkov. I.Analiz chastoty, sostava i struktury insulinov cheloveka, svin'i i krupnogo rogatogo skota.

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 from Staphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.

Identification of unusual (modified and non-encoded) amino acid residues in peptides by combinations of high-performance liquid chromatography and high-performance capillary electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The techniques for micro-level analysis of some widespread unusual amino acids (phosphorylated and hydroxylated ones) as well as of some genetically non-encoded amino acids were developed for their subsequent identification in the peptide and protein amino acid sequence by narrow-bore column high-performance liquid chromatography (approximately 10 pmol of the sample), high-performance capillary electrophoresis (approximately 1-10 pmol), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (approximately 1-10 pmol), and automatic protein gas phase sequencing (approximately 1-50 pmol).

Qualitative and quantitative determination of biologically active low-molecular-mass thiols in human blood by reversed-phase high-performance liquid chromatography with photometry and fluorescence detection.

The reversed-phase high-performance liquid chromatographic method employing photometry and fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine (tHcy), cysteine (Cys), and glutathione (GSH) in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with trichloroacetic acid, conjugation of the thiols with monobromobimane (mBrB). The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions) to obtain reliable quantitative results within the concentration range corresponding to normal and pathological levels of these thiols in human blood.

A protein residing at the subunit interface of the bacterial ribosome.

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.

[Proteolytic degradation of hemoglobin in erythrocytes results in formation of biologically active peptides]. / Proteoliticheskaia degradatsiia gemoglobina v éritrotsitakh privodit k obrazovaniiu biologicheski aktivnikh peptidov.

The formation of biologically active hemoglobin fragments in human erythrocytes was studied. The structures of 33 peptide products of intraerythrocytic hemoglobin cleavage were determined. Based on an analysis of these sequences, a model of the stepwise degradation of the hemoglobin alpha- and beta-chains was suggested. The processes of peptide formation in a cell-free erythrocyte lysate system were studied. The involvement of an enzymatic complex of the cell membrane fraction was demonstrated. It was found that the cells of a surviving human erythrocyte culture secrete short (of 5-20 amino acid residues) peptides, and the structures of 36 peptides were determined. The dynamics of peptide secretion was investigated, and preliminary data on the energy-dependence of this process were obtained. Based on the experimental results, a model describing erythrocytes as an endocrine gland was suggested.