Modification of jack bean urease thiols by thiosulphinates contained in garlic extract: DTNB titration studies

Abstract

The reactivity of the thiol groups in urease with thiosulphinate contained in garlic extract was spectroscopically characterised. The enzyme was incubated with the garlic extract and the reaction progress curves were recorded in the presence of thiol-selective reagent 5,5′-dithiobis(2-nitrobenzoic acid). Simultaneously the enzyme residual activity was also determined. The process was studied in 50 mM phosphate buffer, at pH 7.8 and ambient temperature. It was found that thiosulphinates act as time- and concentration-dependent inactivators of urease. The observed decrease of the enzyme activity corresponds to the number of the urease thiols modified by thiosulphinates. The modification of half of all the urease thiols (18 of 36) causes a slight (only 8–10%) decrease of the catalytic activity. The modification of the remaining 18 thiols results in significant disturbance of urease action until complete loss of the catalytic function occurs. This provides the evidence that Cys 592, the critical residue for urease activity, belongs to the enzyme thiols, which are less reactive and more resistant to chemical modification than the other thiols.