J. Plant Biochemistry & Biotechnology VoL 7,121-124, July 1998 Short Communication Kinetics of Thermal Inactivation and Molecular Asymmetry of Urease from Oehusked Pigeongea (Cajanus cajan L.) Seeds Arvind M Kayastha* and Nilanjana Das School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India The purified urease from pigeonpea was moderately stable at -lo·e. The shelf-life of the enzyme on storage in 0.1 M Tris-acetate buffer, pH 6.5, at -lo·e showed a single exponential decay with a t.,.. of approx. 30 days. In the presence of additives like 5mM dithiothreitol the t.,.. increased to 223 days at the same temperature, in a single exponential decay. The Arrhenius plot of the kinetics of the pigeonpea urease catalysed urea hydrolysis over the temperature range 27 to 77·e, was IinE-.ar. The Ql0 value was found to be 1.46. The energy of activation calculated from the Arrhenius equation was found to be 5.1 kcaVmole active site. The thermal denaturation of pigeopea urease at 65 and 70·e was found to obey biphasic kinetics in which half of the activity is destroyed faster than the remaining half. The time course of thermal inactivation can be by the following equation, consisting of two first order terms: At = Afast.e-k'asft + Aslow.e-kslow·t where, At is the residual activity at time t, A fast and A slow , krast and kslow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. The data suggests the existence of site-site heterogeneity in oligomeric urease molecule from pigeonpea. Key words: urease, thermal inactivation, molecular asymmetry, pigeonpea, Cajanus caJan. Urease (Urea aminohydrolase, EC 3.5.1.5), a nickel- dependent metalloenzyme, catalyzes the hydrolysis of urea to form ammonia and carbon dioxide (1) with a rate approximately 10 14 times the rate of the uncatalysed reaction. In 1926, urease was isolated from the seeds of the jack bean plant (Canavalia ensiformis) as a pure, crystalline enzyme by Sumner (2). For unknown reasons, some seeds are particularly rich sources of urease. Urease has been extensively studied in seeds of various leguminosae and cucurbitaceae plants (3-5). The urease from pigeon pea has been purified to apparent homogeneity and partially characterised (6) and has been shown to be an important enzyme for analytical purposes (7,B). The urease from pigeopea has been shown to be a hexameric enzyme of identical subunits (subunit molecular mass BO kO). Like other ureases, pigeonpea urease has also been shown to be a nickel containing enzyme (6). Kinetics of thermal inactivation has been successfully used as a potential technique for studying molecular asymmetry of some important oligomeric enzymes from plants (9-17). The results of thermal inactivation from pigeonpea urease have been described in the present communication and its significance has been discussed in the light of molecular asymmetry in the enzyme. *Corresponding,author. e-mail: kayastha@banaras ernet in Urease was isolated from dehusked seeds of pigeonpea (Cajanus cajan L.) procured from the locar market. Seeds were soaked overnight in 0.025 M Tris- acetate buffer, pH 6.5, at 4°C. Purification involved crushing and extraction of the enzyme in the same buffer, two acetone fractionation steps, a pH-change step followed by Sephadex G-200 and OEAE-cellulose chromatography steps,. as described earlier (6). These steps brought about a 200-fold purification with a final specific activity of 4000-5000 units/mg of protein. The isolated enzyme was more than 95% pure as judged by native and SOS-PAGE (6). For measurements of urease activity, ammonia liberated in a fixed time interval at an enzyme-saturating concentration of urea was determined using Nessler's reagent as described earlier (6,B). An enzyme unit has been defined as the amount of enzyme required to liberate 1 of ammonia per minute under our test conditions (0.1 M urea, 0.05 M Tris-acetate buffer, pH 7.3, at 3rC). Protein was assayed by the method of Lowry et al (1 B) using boving serum albumin as standard. The storage stability was studied at -10°C. The enzyme in small aliquots in 0.1 M Tris acetate buffer, pH 6.5 and in the presence of 5 mM OTT was kept in vanous Eppendorf tubes at - 10°C and assayed on different days at 3rc for the residual activity, taking the day of preparation of the enzyme as zero day and activity to be 100%.