Evenly Tritium-Labeled Peptides in Study of Peptide In Vivo and In Vitro Biodegradation

Yu. A. Zolotarev ^a , ^# A. K. Dadayan ^a , O. V. Dolotov ^a , V. S. Kozik ^a , N. V. Kost ^b , O. Yu. Sokolov ^b , E. M. Dorokhova ^a , V. K. Meshavkin ^b , L. S. Inozemtseva ^a , M. V. Gabaeva ^b , L. A. Andreeva ^a , L. Yu. Alfeeva ^a , T. S. Pavlov ^c , S. E. Badmaeva ^c , Z. V. Bakaeva ^c , G. N. Kopylova ^c , G. E. Samonina ^c , B. V. Vaskovsky ^d , I. A. Grivennikov ^a , A. A. Zozulya ^b , and N. F. Myasoedov ^a

^# Phone: +7 (495) 196-0213; fax: +7 (495) 196-0221; e-mail: zolya@img.ras.ru

^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 @[mu]g 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

Russian Journal of Bioorganic Chemistry 2006, 32 (2):166-173