Якісний аналіз пептидного та білкового складу цитозольних фракцій фетальних тканин людини

Abstract

Проведено препаративний розподіл тотального цитозоля фетальних тканин людини (мезенхімально-мезодермального походження) шляхом хроматографії у полівініловому гелі та горизонтального ультратонкого електрофорезу на поліакриламідному гелі. Під час хроматографії було отримано 6 фракцій, одна з яких білкова, 5 – пептидні. При електрофоретичному розподілі білків було одержано 13-15 білкових фракцій з молекулярною масою від 9 до 110 кДа ***************************************************************************************************

Actual problem of present-day stem and progenitor cell biology and regenerative medicine is a search of biologically active substances in tissues enriched by stem/progenitor cells. Area of our interest is the fetal tissues mainly of mesenchymal and mesodermal origin that demonstrated a wide spectrum of biological activity. In this study low molecular weight peptides and proteins of human fetal tissue cytosol (FTC) obtained by two-steps high-speed ultracentrifugation of tissue homogenate was analyzed by gel-chromatography and gel-electrophoresis. Gel-chromatography was performed using polyvinyl gels TSK-Gel Toyopearl HW-40, HW-60 Fine (Toyosoda, Japan) and tris-HCl buffer (25 mM, pH 7.5) contained 1M urea and 100 mM NaCl as an eluent. Obtained fractions were estimated spectrophotometrically, wave-length 254 and 280 nm. Horizontal ultrathin gel-electrophoresis was performed using polyacrylamide gel with pore gradient 4-22.5%. Sodium dodecyl sulfate was used as a main detergent. During gel-chromatography 6 pronounced peaks were obtained. One of them was a protein fraction, the rest 5 – peptide ones with molecular weights 2990, 2410, 1570, 1150, 900 Da approximately. Protein fraction was about 34000 Da. After gel-electrophoresis of total FTC 13 protein fractions were discovered. Two of them fit with standards – chemotrypsinogen A and albumin. For protein fraction obtained during chromatographic separation 15 lines were shown, 3 of them fit with standards, additional one was cytochrome c. Protein molecular weight range was between 9000 and 110000 Da.