Effect of thiols on the activity of urease from dehusked seeds of watermelon (Citrullus vulgaris)

Abstract

Urease from seeds of watermelon (Citrullus vulgaris) was purified to apparent homogeneity, using two acetone fractionation steps, heat treatment at 48°C and gel filtration through Sephadex G-200. The effect of various thiols like 2-mercaptoethanol (ME), DL-dithiothreitol (DTT), and L-cysteine was investigated. The enzyme was significantly activated by thiol and the order of effectiveness as activator was ME > DTT > cysteine. All the thiols exhibited two peaks of activation, one at lower concentration and another at higher concentration. Time-dependent activation of urease exhibited very rapid activation; about 5.3-fold with 8.0 mM ME and 4.9-fold with 30 μM DTT within 10 min of treatment. Moreover, the inhibition caused by high urea concentration was completely abolished by ME. Cysteine and DTT were ineffective in abolishing high substrate inhibition. Enzyme pretreated with ME also exhibited elevated activity in the urea concentration range of 10-2000 mM. The significance of these observations is discussed. © 2002 Elsevier Science Ireland Ltd. All rights reserved.