Jack bean urease inhibition by crude juices of Allium and Brassica plants. Determination of thiosulfinates.

The aim of this study was the elucidation of the inhibitory influence of Allium (garlic, onion, leek) and Brassica (cabbage, Brussels sprouts) plants juices, on jack bean urease activity. Concentrations of thiosulfinates, the compounds responsible for the inhibition, were determined in studied materials. The kinetics and mechanism of the inhibitions were investigated. Biphasic, time-dependent courses of the inhibition reactions were observed for all tested Allium and Brussels sprouts from Brassica. The cabbage material caused the monophasic course of the inhibition. In the presence of dithiothreitol, a total reactivation of the inhibited urease proceeded for the tested plants except for the onion. The onion juice modified urease, regained only half of the initial activity. The irreversible contribution was related to the presence of 1-propanethial-S-oxide, cepaenes and zwiebelanes formed in the onion juice. It was found that the thermal processing of the plant juices, results in the decrease of thiosulfinates concentration, as well as the efficiency of urease inhibition.

Influence of 2,5-dichloro-1,4-benzoquinone on jack bean urease activity. Inhibitory effect, total reducing capacity and DPPH radical scavenging activity.

Inhibition of jack bean activity by 2,5-dichloro-1,4-benzoquinone (DCBQ) was studied in phosphate buffer, pH 7.0. It was found that DCBQ acted as a strong, time and concentration dependent inactivator of urease. Under the experimental conditions obeyed the terms of pseudo-first-order reaction, urease was totally inactivated. Application of Wilson-Kitz method proved that the urease-DCBQ interaction followed a simple bimolecular process and the presence of intermediate complex was undetectable. The determined second order rate constant of the inactivation was 0.053 (µM min)(-1). Thiols such as l-cysteine, glutathione and dithiothreitol (DTT) protected urease from inhibition by DCBQ but DCBQ-modified urease did not regain its activity after DTT application. The thiol protective studies indicated an essential role of urease thiol(s) in the inhibition. The irreversibility of the inactivation showed that the process was a result of a direct modification of urease thiol(s) by DCBQ (DCBQ chlorine(s) substitution). The decomposition of DCBQ in aqueous solution at natural light exposure was monitored by visible spectrophotometry, determination of the total reducing capacity (Folin-Ciocalteu method) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability. The DCBQ conversion resulted in a decrease of the inhibition power and was well correlated with the increase of the total reducing capacity and DPPH scavenging ability. These findings were attributed to DCBQ transformation by photolysis and the hydrolysis effect was found to be negligible.

Heavy metal ions inhibition of jack bean urease: potential for rapid contaminant probing.

The kinetics of heavy metal ions inhibition of jack bean urease was studied by progress curve analysis in a reaction system without enzyme-inhibitor preincubation. The inhibition was found to be biphasic with an initial, small inhibitory phase changing over the time course of 5-10 min into a final linear steady state with a lower velocity. This time-dependent pattern was best described by mechanism B of slow-binding inhibition, involving the rapid formation of an EI complex that subsequently undergoes slow conversion to a more stable EI* complex. The kinetic parameters of the process, the inhibition constants Ki and Ki* and the forward k5 and reverse k6 rate constants for the conversion, were evaluated from the reaction progress curves by nonlinear regression treatment. Based on the values of the overall inhibition constant Ki*, the heavy metal ions were found to inhibit urease in the following decreasing order: Hg2+ > Cu2+ > Zn2+ > Cd2+ > Ni2+ > Pb2+ > Co2+ > Fe3+ > As3+. With the Ki* values as low as 1.9 nM for Hg2+ and 7.1 nM for Cu2+, 100-1000 times lower than those of the other ions, urease may be utilized as a bioindicator of the trace levels of these ions in environmental monitoring, bioprocess control or pharmaceutical analysis.