Some kinetic and physical properties of biliverdin reductase.

unable to bind NADPH. The results obtained so far suggest that the modified histidine residue is in, or near, the coenzyme site, and the protection by inhibitors competitive with NADPH suggest that it is in the region of the binding site for adenosine ribose and distal to the site of hydride transfer. Thiol groups. The role of thiol groups in the activity of mammalian aldehyde reductases is not at all clear. From the amino acid composition, depending on the source of the enzyme, three to six cysteine residues are present per molecule of enzyme (Davidson & Flynn, 19796). Flynn et al. (1975) found that four of the five cysteine residues in pig kidney aldehyde reductase reacted with pchloromercuribenzoate (PCMB) at pH 7.0 with no resulting loss of catalytic activity. The addition of high concentrations (a 50-fold excess over thiol groups) of PCMB were required to cause complete inhibition. In studies with the human liver enzyme, Wermuth et al. (1977) found that reaction of PCMB with the most reactive thiol group did not cause inactivation. The addition of concentrations of PCMB in excess of 2 or more equivalents per mol of SH groups resulted in turbidity and an inability to monitor the reaction spectrophotometrically. It is noteworthy that in a re-examination of the role of thiol groups in the activity of pig kidney aldehyde reductase we have encountered the same phenomenon. We have been exploring the possibility that the denaturation that occurs is due to intramolecular disulphide-bond formation, which has been suggested to occur with other oxidoreductases when they are titrated with disulphide-interchange reagents (Wassarman & Mayor, 1969; Rippa et al., 1978). In a recent paper, Morpeth & Dickinson (1980) reported that reaction of pig kidney aldehyde reductase with high and low concentrations of 5,5’-dithiobis(2-nitrobenzoate) (Nbs,) results in modification of one thiol group per molecule of enzyme. The inhibition of the enzyme at high concentrations of Nbs, is attributed to the reagent acting as a reversible inhibitor separate from its thiol-modifying role.