Fractionation of Echinacea purpurea L. (Moench) proteins and assessment of Glycosylated proteins distribution in fractions

Abstract

Plant lectins, non-immune origin glycoproteins, got their attention for possible beneficial effects to health just 3 decades ago and are quiet new group of compounds with pharmacological interest. Echinacea purpurea L. (Moench) is a well-known medical plant. However, pharmacologically interesting proteins in this plant aren’t well characterized. In this study we extracted and fractionated proteins of Echinacea purpurea L. (Moench) roots and checked estimation of glycosylated proteins in all fractions. Materials and Methods 30 g of fresh roots were homogenized in mortar with liquid nitrogen and extracted in phosphate buffer saline pH 7.4 in a ratio 1:5 for 2 hours at 4 °C. Proteins were precipitated from crude extract by adding 4 volumes of cold (-20 °C) 10 % Trichloracetic acid/acetone solution, containing 0.2 % β - mercaptoethanol. Protein pellets were washed with acetone solution, containing 0.2 % β – mercaptoethanol for 3 times and resuspended in phosphate buffer 7.4. Resuspended protein sample was loaded on HiTrap Q FF 1 mL column with gradient elution from 0 M to 1 M NaCl. 5 mL fractions were collected, dialyzed in phosphate buffer 7.4 and immunoblotted with anti-xylose antibody. Results After anion exchange fractionation we’ve collected 18 protein fractions, 11 of them contained glycosylated proteins. Glycoproteins started eluting from the column when sodium chloride concentration in eluent reached 0.4 M. Elution of glycosylated proteins decreased when sodium chloride concentration reached 0.7 M. Conclusions Protein fractions extracted from Echinacea purpurea L. (Moench) roots contained glycosylated proteins. Glycosylated protein pattern