Zolotarev Iu A, Dadaian A K, Dolotov O V, Kozik V S, Kost N V, Sokolov O Iu, Dorokhova E M, Meshavkin V K, Inozemtseva L S, Gabaeva M V, Andreeva L A, Alfeeva L Iu, Pavlov T S, Badmaeva K E, Badmaeva S E, Bakaeva Z V, Kopylova G N, Samonina G E, Vas'kovskiĭ B V, Grivennikov I A, Zozulia A A, Miasoedov N F
Bioorg Khim. 2006 Mar-Apr;32(2):183-91.
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 tg scale of biological samples both in vitro and in vivo.
用均匀标记氚的生物活性肽研究肽的体外和体内生物降解。通过高温固相催化同位素交换（HSCIE）与溢出氚获得比放射性为50 - 150 Ci/mmol的氚标记肽。这些肽的所有氨基酸残基间的同位素标记分布使得能够同时测定其酶促水解的几乎所有可能产物。所开发的分析方法包括从生物组织中提取氚标记肽以及从降解产物混合物中色谱分离单个标记肽。根据液体闪烁计数结果计算所研究肽及其生物降解产物的浓度。该方法用于研究七肽TKPRPGP（司来吉兰）和三肽PGP在血浆中的生物降解途径。还通过鼻内体内给药该肽在脑组织中研究了抗焦虑肽司来吉兰的药代动力学。测定了标记肽的浓度，结果表明五肽TKPRP、三肽TKP以及二肽RP和GP是司来吉兰生物降解的主要产物。在神经细胞存在下对七肽MEHFPGP（赛美克斯）生物降解的研究表明，其生物降解的主要产物是五肽HFPGP和三肽PGP。使用均匀氚标记的[亮氨酸]脑啡肽研究了血浆的脑啡肽酶活性。结果表明，赛美克斯对血浆脑啡肽酶的高抑制作用源于其对氨肽酶的作用。基于使用均匀氚标记肽的方法能够在体外和体内以微克规模的生物样品测定肽浓度及其降解相关酶的活性。
