Crystalline Papain Derivative Containing an Intramolecular Mercury Bridge

Abstract

Abstract A partially reduced derivative of papain, in which only the disulfide bond connecting the cysteine residues in positions 43 and 152 had been opened, was reacted with mercury chloride. The resulting crystallizable material contained 2 g atoms of mercury per mole of papain, and is denoted Pap·2Hg. One of the mercury atoms in this derivative is joined in complex with the free sulfhydryl group of papain which is involved in the catalytic site, whereas the second one is present in the form of an —S-Hg-S— bridge, which replaces the original —S—S— bond that had been reduced. Pap·2Hg is enzymatically active on a range of substrates; in respect to the hydrolysis of benzoyl-l-arginine ethyl ester its Vmax is similar to that of native papain, but its Km value is 0.042 compared to a value of 0.023 obtained for native papain. The internal mercury atom, in contrast to the one bound to the active sulfhydryl, is retained in the molecule under the conditions required for activating papain (0.005 m cysteine and 0.002 m EDTA). It is, however, totally removed upon extensive dialysis (48 hours) against 0.02 m EDTA, or when Pap·2Hg is exposed to the activating agents in the presence of 8 m urea. Fluorometric titrations of Pap·2Hg, in comparison to those of native papain and of commercial mercuripapain, show very high quenching effect of the 2 mercury atoms, but again indicate that during activation conditions 1 mercury atom is still present in the molecule. On the other hand, under the conditions leading to complete elimination of the mercury from Pap·2Hg, its titration curve is identical with that of native papain. The immunological reactivity of Pap·2Hg with antipapain serum is essentially identical with that of native papain.