Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Yu A. Zolotarev, A. K. Dadayan, V. S. Kozik, E. V. Gasanov, I. V. Nazimov, R. Kh Ziganshin, B. V. Vaskovsky, A. N. Murashov, A. L. Ksenofontov, O. N. Kharybin, E. N. Nikolaev, N. F. Myasoedov

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

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 (B1-13) 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.

Original languageEnglish
Pages (from-to)26-35
Number of pages10
JournalRussian Journal of Bioorganic Chemistry
Volume40
Issue number1
DOIs
Publication statusPublished - Jan 2014
Externally publishedYes

Keywords

  • insulin
  • mass spectrometry
  • solid-phase reactions of isotope exchange
  • tritium and deuterium labeling

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