ACETONE PRECIPITATION OF PROTEINS FOR DETERMINATION OF THE OF ENZYMES ACTIVITY IN MEDICINAL PLANTS

Abstract

Plant phenolic compounds are widely used in medicine and agriculture. The study of the mechanisms of their biosynthesis and accumulation is a complex task. It is also true for the determination of the activity of the key plant enzymes such as shikimate dehydrogenase. At the same time, the isolation of proteins and enzymes from plant is complicated by the presence of phenolic compounds and pigments. The aim of the research was to evaluate the feasibility of the acetone precipitation of proteins of leaf extracts of plants to determine the activity of shikimate dehydrogenase. The object of the study was the leaves of sea buckthorn (H. rhamnoides) and silky dogwood (C. sericea). The proteins were precipitated from the crude extract with cold (-20 °C) acetone in varying ratio followed by washing three times with acetone and final dissolution in buffer. The activity of shikimate dehydrogenase was determined at pH=10.0 and expressed in ncat per mg of protein. Protein fractions with different activity of shikimate dehydrogenase were isolated from the leaves of sea buckthorn (H. rhamnoides) and silky dogwood (C. sericea). It was shown that the protein fraction with a ratio of 1 : 1.5 of extract : acetone (60% saturation) is most convenient to work with, has a high enzyme activity and does not contain colored impurities. This method of protein isolation and pre-purification is fast, requires a small amount of sample, and suitable for screening studies of the activity of various enzymes in medicinal plants.