vestichИзвестия Национальной академии наук Беларуси. Серия химических наукProceedings of the National Academy of Sciences of Belarus, Chemical Series1561-83312524-2342The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"vestich-257Research ArticleБИООРГАНИЧЕСКАЯ ХИМИЯBIOORGANIC CHEMISTRYПОЛУЧЕНИЕ ВЫСОКОАКТИВНОЙ ГЛУТАТИОН-S-ТРАНСФЕРАЗЫ Р1-1 ИЗ ЭРИТРОЦИТОВ ЧЕЛОВЕКА С ПОМОЩЬЮ АФФИННЫХ МЕМБРАН И СВОЙСТВА ОЧИЩЕННОГО ФЕРМЕНТАPREPARATION OF HIGHLY ACTIVE HUMAN ERYTHROCYTE GLUTATHIONE S-TRANSFERASE P1-1 USING AFFINITY MEMBRANES, AND PROPERTIES OF THE PURIFIED ENZYMEГилевичС Н.GilevichS. N.

канд. хим. наук, вед. науч. сотрудник

Ph. D. (Сhemistry), Leading Researcher

gilevich@iboch.bas-net.byБречкоЮ. В.BrechkaYu. V.

мл. науч. сотрудник

Junior Researcher

brechko@yandex.ruРипинскаяК. Ю.RipinskayaK. Yu.

мл. науч. сотрудник

Junior Researcher

kristina.ripinskaya@gmail.comИнститут биоорганической химии НАН БеларусиInstitute of Bioorganic Chemistry of the National Academy of Sciences of Belarus201707082017026679Copyright © Гилевич С.Н., Бречко Ю.В., Рипинская К.Ю., 20172017Гилевич С.Н., Бречко Ю.В., Рипинская К.Ю.Gilevich S.N., Brechka Y.V., Ripinskaya K.Y.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestichem.belnauka.by/jour/article/view/257

Глутатион-S-трансфераза Р1-1 (GSTP1-1) человека является диагностически важным ферментом, гиперэкспрессия которого в опухолях и лимфомах приводит к формированию множественной лекарственной устойчивости опухолевых клеток. Небольшие количества фермента можно выделить из эритроцитов, однако известные методы очистки не позволяют получить препараты с высокой удельной активностью и хорошим выходом. С целью разработки более эффективной процедуры очистки эритроцитарной GSTP1-1 в настоящей работе впервые получены и охарактеризованы глутатионсодержащие аффинные мембраны на основе химически модифицированной целлюлозной бумаги. Мембраны успешно использованы для очистки фермента вместо традиционных агарозно-гелевых адсорбентов. Разработан новый метод выделения и очистки GSTP1-1 из гемолизата эритроцитов, включающий предварительное удаление гемоглобина на анионообменнике и аффинную хроматографию фермента на картридже с глутатионсодержащими мембранами. По эффективности (выход фермента 76,5 %, степень очистки 23589 раз, удельная активность 104,5 Ед/мг белка) предложенный метод значительно превосходит ранее опубликованные. Гомогенность полученного препарата, по данным гель-электрофореза и масс-спектрометрии, составляет не менее 95%. Найдены стационарные кинетические параметры очищенной GSTP1-1 в реакции конъюгации глутатиона (GSH) и 1-хлор-2,4- динитробензола (CDNB) при рН 6,5 и 25 °С: для GSH Km = 0,19 мМ, kcat = 47,8 с–1; для CDNB Km = 0,68 мМ, kcat = 54,3 с–1. Результаты работы представляют интерес для скрининга новых ингибиторов фермента с противоопухолевой активностью. Полученные аффинные мембраны могут быть использованы при выделении нативных и рекомбинантных глутатион-S-трансфераз (GST) из различных источников, а также гибридных белков с GST-доменом. 

Human glutathione S-transferase P1-1 (GSTP1-1) is an important enzyme in clinical diagnostics since its overexpression in solid tumours and lymphomas largely contributes to multiple drug resistance of cancer cells. Small amounts of the enzyme can also be isolated from erythrocytes. However, known isolation methods are disadvantageous and do not allow to obtain the purified enzyme in good yield and with high specific activity (≥ 100 U/mg protein). In order to elaborate more effective purification procedure for the erythrocyte GSTP1-1, we have for the first time synthesized and investigated glutathione-containing affinity membranes based on chemically modified cellulose paper. The membranes have been shown to successfully replace conventional glutathione-agarose affinity gels in the enzyme purification. To isolate and purify GSTP1-1 from erythrocyte hemolysate, a novel two-step method has been developed involving preliminary hemoglobin removal on a small anion exchange column and further affinity chromatography on the cartridge with glutathione-containing membranes. In terms of activity yield (76.5%), purification factor (23589-fold), and specific activity of the purified enzyme (104.5 U/mg), the method is notably superior to previously published procedures. Gel electrophoretic and MALDI-TOF mass spectrometric analyses reveal apparent homogeneity (≥ 95%) of the obtained preparation. Steady-state kinetic parameters have been determined for the purified GSTP1-1 in the conjugation reaction between glutathione and 1-chloro-2,4-dinitrobenzene at pH 6.5 and 25 °С: for the former substrate, Km = 0.19 mМ, kcat = 47.8 s–1; for the latter, Km = 0.68 mМ, kcat = 54.3 s–1. The results of the present work may be useful for screening new enzyme inhibitors with possible antitumour activity. The affinity membranes may also find application in isolating native and recombinant glutathione S-transferase (GST) isoforms from various sources, as well as fusion proteins with GST tag. 

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The authors declare that there are no conflicts of interest present.