A scheme for analyzing the mass spectra of fragmented peptides, including chemical modification of the N-terminal amino group of the peptide by 5-(N,N-dimethylamino)naphthalene-1-sulfonyl chloride followed by ionization of the dansylated peptide by electrospray and its fragmentation at the low vacuum zone of the sample solution input unit into the mass spectrometer (ESI-o-TOF scheme), has been proposed. It is shown that the resulting ions are fragmented with the predominant formation of b-ions bearing a Dns-group. In this way, it is possible to obtain high-intensity b-ions (peak/noise intensity ratio of 10/1 to 100/1), including b1 and b2 ions that are especially valuable for determining the structure. In combination with the previously proposed method of obtaining informative y-ions, this approach allows one to reliably determine the complete amino acid sequence of peptides containing up to 10 amino acid residues, without using computer programs for analyzing the fragment composition of peptides.

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B 1–13 ) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius , and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.

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 β-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 β-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.

Solid-state catalytic isotope exchange of hydrogen under the action of spillover hydrogen in organic compounds applied on a nonorganic carrier has been studied. The deuterium-labeled peptide [D]dalargin containing 14 deuterium atoms, [D]melatonin, and [D]histamine with the 72-92% substitution degree of all C H bonds were prepared by this reaction. The activation energy of hydrogen isotope exchange with T-2 and D-2 in glycine and alpha-aminoisobutyric acid was obtained experimentally. It was shown that for the studied reaction the kinetic isotopic effect is 1.2-1.4 by the Hartree-Fock method, which is several times smaller than one in the liquid-phase reactions. Quantum chemical calculations of the isotope shift values in the electronic spectra of deuterium-labeled metal ion complexes were performed using the RB3LYP/LanL2DZ and CIS/LanL2DZ methods for calculation of the ground and the excited states. It was shown for deuterium-labeled histamine complexes [D-12]Pd(him)(2)Cl-2 and [D-12]Cu(him)(2)Cl that the isotopic effect of UV spectra was 1600 and 1800 cal/mol, respectively. The measurement of isotopic shifts for electronic transitions potentially can be utilized as a new informative method for the investigation of complex formation. The deuterium-labeled compounds were shown to be useful as internal standards for quantitative mass spectroscopy (MS) analysis.

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.

The hydrolytic stability of a range of cyclic tripeptides, including the therapeutically important dalargin and stemokin, as well as peptides modified by ibuprofen and aspirin, has been studied. The first two experimental systems used utilized purified enzymes (pepsin, trypsin, and chymotrypsin), while the second one utilized fragments of the stomach and small intestine of rats. The linear peptides containing only L -amino acid residues were shown to be hydrolyzed by stomach and intestine fragments, although some of these peptides were resistant to hydrolysis by individual enzymes. The peptides containing D -amino acid residues and cyclic peptides were stable under all of the conditions used, but the peptides modified by aspirin lost the acetyl group of the aspirin moiety in acidic media, this process being accelerated in the presence of pepsin.

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.

The separation of mixtures of free genetically coded amino acids by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) using UV and refractometric detection was studied. Mixtures of 16 and 14 amino acids were separated by CZE and MEKC, respectively.

The method of structure determination of the recombinant glycosylated peptides and proteins has been elaborated by the example of the gene-spliced human insulin.

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-5,1-imine ([G-3H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-3H]-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-3H]MK-801 and [G-3H]-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-3H]MK-801 and [G-3H]-7-OH-DPAT was analyzed using the brain structure of Vistar rats. It was demonstrated that [G-3H]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-3H]-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.

The recombinant modified peptides and proteins method of structure and properties determination has been considered by the example of the glycosylated gene-spliced human insulin with disaccharide.

According to the previously reported data, the superntant of the 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 β-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, and amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age and blood group of donors. The dynamics of accumulation in the supernatant (20–80 min of incubation) of 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 the peptide release by erythrocytes.

Для изучения биологических свойств ациламинокислот синтезированы N-арахидоноильные (АА) производные аминокислот: Gly, Phe, Pro, Val, у-аминомасляной кислоты (GABA), диоксифенилала-нина, Туг, Тгр и Ala, а также пептидов MEHFPGP и PGP. В масс-спектрах ионизации при атмосферном давлении распылением в электрическом поле все соединения дают протежированный молекулярный ион максимальной интенсивности, предел детектирования составлял 10 фмоль вещества на образец. Из всех изученных ациламинокислот наибольшую ингибиторную активность по отношению к гидролазе амидов жирных кислот из мозга крысы показал AA-Gly (IC5o 6.5 мкМ). Слабое, но регистрируемое ингибирование проявляли AA-Phe, АА-Туг и AA-GABA (1С5о 55, 60 и 50 мкМ соответственно). Сами ацилированные аминокислоты, за исключением AA-Gly, были устойчивы к гидролизу этим ферментом. Все арахидоноиламинокислоты в той или иной степени ингибировали фос-фолипазу D из капусты, наиболее активными оказались AA-GABA и AA-Phe (IC5o 20 и 27 мкМ соответственно). Опыты по выяснению возможности биосинтеза АА-Туг в гомогенатах печени и нервных тканях крысы не показали образования этой ациламинокислоты in vitro, однако были обнаружены продукты ее метаболизма: АА-дофамин и АА-(З-О-метилдофамин); наибольшее количество первого метаболита наблюдали в гомогенате печени, а второго - в гомогенате головного мозга. Ациламинокислоты не оказывали цитотоксического действия на клетки глиомы С6. Показано, что N-ацилирование гептапептида MEHFPGP (семакс) арахидоновой кислотой приводит к повышению его гидролитической стабильности и увеличению сродства к местам специфического связывания на мембранах мозжечка крысы.

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, γ-aminobutyric 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 μM) 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 μM, respectively). The acylated amino acids themselves, except for AA-Glu, 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 μM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue in vitro were unsuccessful; however, AA-dopamine and AA-Phe, the products of its metabolism, were found. 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.

A current approach to determining low-molecular-weight amino thiols (cysteine, homocysteine, and glutathione) in model samples and blood plasma is considered. Procedures for determining 2–100 μM homocysteine in blood plasma with the use of microcolumn chromatography and capillary electrophoresis were developed. Photometric and fluorimetric detection techniques were used to identify amino thiols. Monobromobimane and 5-iodoacetamidofluorescein were used as labels. Mass spectrometry was used to confirm the structures of test amino thiol derivatives.