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Electron-transfer dissociation (ETD) and electron-transfer and higher-energy collision dissociation (EThcD) spectra of short tryptic peptides with leucine/isoleucine residues in neighboring positions demonstrate intensive w-ions. On the contrary, u-ions possess very low intensities (if present at all). Therefore radical site migration is negligible in the applied conditions while ETD (EThcD) spectra allow for the reliable discrimination of the isomeric residues in the sequencing process. The presence of a fragment ion 43.055 mass units lower than z 2 -ion of peptides with IK sequence at their C-termini was shown to be a result of alternative fragmentation starting from the loss of propylammonium ion from the doubly protonated peptide molecule and formation of an oxazole fragment ion.

The polypeptide complex of the epiphysis and the peptide AEDG, constructed on the basis of its amino acid analysis, exert similar biological effects. Both bioregulators normalize melatonin synthesis in the pineal gland, functioning of the brain, eye retina, cardiovascular, endocrine, and immune systems; they also act as antioxidants, stress-protectors, and geroprotectors. Within the epiphysis polypeptide complex, free amino acids (3.26%), dipeptides (23.19%), tripeptides (50.72%), tetrapeptides (22.10%), and pentapeptides (0.72%) were revealed by mass spectrometry and HPLC. Peptide AEDG was detected among the tetrapeptides of the epiphysis polypeptide complex by selective reaction monitoring method. The biological effects of the epiphysis polypeptide complex are determined by the effect of its component AEDG.

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.

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu)n-Lys(Arg)-(downward arrow)-B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G5DK-F(NO2)G (kcat/Km = 2380 mM(-1) x min(-1)) is more efficient than the fusion protein PrAD4K-P26 (kcat/Km = 1260 mM(-1) x min(-1))., (Copyright 2004 MAIK)

The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140-180 degrees C was analyzed. This reaction was used for preparing [(3)H]enkephalins such as [(3)H]DALG with specific activity of 138 Ci/mmol and [(3)H]LENK with specific activity of 120 Ci/mmol at 180 degrees C. The analogues of [(3)H]ACTG(4-10) with specific activity of 80 Ci/mmol, [(3)H]zervamicin IIB with specific activity of 70 Ci/mmol and [(3)H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [(3)H]Peptide analysis using (3)H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the beta-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [(3)H] beta-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.