60] Purification of 5,10-methenyltetrahydrofolate cyclohydrolase from Clostridium formicoaceticum

Abstract

This chapter focuses on purification of 5, l0-methenyltetrahydrofolate cyclohydrolase from Clostridium formicoaceticum. Methenyltetrahydrofolate cyclohydrolase activity has been demonstrated in animal and plant tissues and in many bacteria. The 5, 10- methenyltetrahydrofolate cyclohydrolase activity is assayed by following the decrease in absorbance at 350 nm of 5,10-methenyltetrahydrofolate as it is hydrolyzed to 10- formyltetrahydrofolate. The enzyme is most active around pH 7. At this pH 5,10-methenyltetrahydrofolate is hydrolyzed nonenzymatically at a measurable rate, which is deducted from the rate of hydrolysis in the presence of the enzyme. The enzymatic and the nonenzymatic reactions are strongly affected by the nature of the buffer system that is used. The enzymatic rate is high with triethanolamine, morpholinopropane, and maleate buffers, in which the nonenzymatic rate is relatively slow. All purification steps are performed at 5° with potassium phosphate buffers of pH 7.2. The purified enzyme is stable for at least 1 week when stored at 5° in 50 mM phosphate buffer (pH 7.0). When in this buffer the enzyme can be frozen in an acetone-dry ice bath and lyophilized without loss of activity. At 55°, 70% of the activity is lost within 2 min.