A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

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Abstract
A convenient method of preparation of jack-bean urease (EC 3.5.1.5) involving covalent chromatography by thiol-disulfide interchange is described. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 .+-. 1.47 katal/kg, Km = 3.32 .+-. 0.05 mM; kcat. [catalytic rate constant] = 2.15 .times. 104 .+-. 0.05 .times. 104 s-1 at pH 7.0 and 38.degree. C). Titration of the urease thiol groups with 2,2''-dipyridyl disulfide (2-Py.sbd.S.sbd.S.sbd.2-Py) and application of the method of Tsou Chen-Lu suggests that the urease molecule (assumed to have MW 483,000 and .epsilon.280 [emission at wavelength 280] = 2.84 .times. 105 l .cntdot. mol-1 .cntdot. cm-1) contains 24 unessential thiol groups of relatively high reactivity (class-I), 6 essential thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6 M-guanidinium chloride. The reaction of the class-I thiol groups with 2-Py.sbd.S.sbd.S.sbd.2-Py was studied in the pH range 6-11 at 25.degree. C (I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. The class-I thiol groups consist of at least 2 sub-classes whose reactions with 2-Py.sbd.S.sbd.S.sbd.2-Py are characterized by pKa = 9.1, ~k = 1.56 .times. 104 M-1 .cntdot. s-1 and pKa = 8.1, ~k = 8.05 .times. 102 M-1 .cntdot. s-1, respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and ~k = 1.60 .times. 102 M-1 .cntdot. s-1. At pH values 7-8 the class-I thiol groups consist of approximately 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases as the pH is raised according to a pKa value .gtoreq. approximately 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. The reactivity of the class-II thiol groups towards 2-Py.sbd.S.sbd.S.sbd.2-Py is insensitive to the nature of the group used to block the class-I thiols. All the essential thiol groups in urease appear to be reactive only as uncomplicated thiolate ions. The implications of this for the active-center chemistry of urease relative to that of the thiol proteinases are discussed.