Partial Purification and Biochemical Characterization of Horse Gram Arginase

Abstract

Plant arginase that catalyses the hydrolysis of arginine to ornithine and urea is known to play an important role in nitrogen metabolism. Recently, we reported a highly stable arginase from cilantro and its sensitivity to biotic and abiotic stress. During this investigation, we found horse gram also possessing a stable arginase among legumes. Hence, we partially purified arginase from horse gram seedlings by conventional chromatographic techniques with 869-fold purity, and a specific activity of 13752 nmoles of urea formed/mg of protein/min. The enzyme is relatively heat stable and requires Mn2+ for its activity and is sensitive to reducing agents and EDTA similar to cilantro arginase. The optimum pH and temperature for partially purified horse gram arginase was found to be 7.87 and 37° C - 60° C respectively. Arginine-derived polyamines and amino acids can regulate horse gram arginase in vitro. Partially purified arginase hydrolyses L-arginine and is incapable of hydrolysing other arginine analogues except L-homoarginine, a property that distinguishes horse gram arginase from cilantro arginase. The Km for partially purified arginase was found to be 5.47 ± 0.34 mM with respect to L-arginine. As plant arginases are not stable and their subunit organization differs from source to source, for further purification and biochemical characterization horse gram serves as an ideal and easily available source.