Purification and characterisation of urease from ruminal fluid of camel (Camelus dromedarius)

Urease (EC 3.5.1.5) from ruminal fluid of the camel (Camelus dromedarius) was purified to homogeneity and characterised. The enzyme was purified 96 fold with specific activity of 215.05 units/mg protein. The molecular weight of the enzyme, as determined by gel filtration on Sephadex G-200, was estimated to be 316,000 +/- 1200 and it was composed of five identical sub units with molecular weight of 63,000 as found by using SDS-PAGE electrophoresis. The enzyme exhibited maximal activity at pH 7.0 in potassium phosphate buffer recorded and Km was 0.3 mM. The enzyme was found to be stable upto 40 degrees C, while a loss of 55% and 100% was recorded at 70 and 80 degrees C, respectively. The enzyme was capable of hydrolysing hydroxyurea, allantoic acid and allantoin with the relative rates with 10 mM of each of the substrate being 28.85, 45.20 and 48.09% of that with urea. No enzymatic activity could be detected with benzamide, glyoxylurea, semicarbazide and thiourea. Although Ca2+ at low concentrations (5 and 10 mM) caused an activation of the enzyme, at high concentrations (20 and 30 mM) it caused an apparent inhibition. On the other hand, Ba2+ at the concentrations ranging from 10 to 30 mM and Mn2+ at low concentrations ranging from 0.05 to 0.2 mM caused an activation of the enzyme. The effectiveness of the heavy metals as inhibitors of camel rumen urease at the concentration of 0.005 mM was in order Hg2+>Cu2+>Zn2+>Ni2+>Co2+ with 97, 94, 90, 61, 7% inhibition, respectively. Thiols (dithiothreitol, glutathione, L-cysteine and beta-mercaptoethanol) at low concentrations (0.0001-0.01 mM) enhanced urease activity, while the higher concentration (10 mM) caused a complete inhibition. The pKa values determined for catalytic ionisable groups were 6.75, 7.65 and 8.65 which were consistent with the ionisation of histidine, a-ammonium group and cysteine-SH, respectively. Inactivation studies supported the kinetic experiments. The enzyme was found to be competitively inhibited by acetohydroxamic acid and N-ethylmaleimide with Ki values of 0.24 x 10(-4) M and 0.8 x 10(-4) M, respectively.