Complex formation of 1,10-phenanthroline with zinc ions and the zinc of alcohol dehydrogenase of horse liver.

1, IO-Phenanthroline interacts with certain zinc metalloenzymes to form enzymatically inactive protein-zinc-phenanthroline complexes exhibiting ultraviolet absorption maxima analogous to those observed for zinc ions and phenanthroline in aqueous systems (1). Such spectral changes offer a means of studying directly the chemical mechanism of the enzymatic inhibition resulting from such interactions. Inhibition might result from the removal of zinc from the protein (Pr) by the chelating agent: [Pr .Zn] + OP1 * Pr + [Zn.OP]. Alternatively, the formation of a mized complex would have the same functional consequences: [Pr.Zn] + OP ti [Pr .Zn.OP]. If OP inhibits solely by interacting with the zinc of the enzyme, the system should be susceptible to physicalchemical approaches and interpretations akin to those which pertain to simple chelate systems. The stoichiometry of metal chelates can be established through Job’s method of continuous variations (2). In the present study, spectrophotometry has been employed to determine and compare the stoichiometry of the interaction between OP and zinc ions and between OP and the enzyme, horse liver alcohol dehydrogenase. This enzyme has been shown to contain 2 g atoms of zinc per mole of protein, and hence can be represented by the empirical formula [(LADH)Zm] (3). The zinc contained in this dehydrogenase will henceforth be referred to as Zn’. The molar extinction coefficients, el, e2, and e3, for [ZnOPi]*, [ZnOPz]++, and [ZnOP#+ were determined as was e’ the molar extinction coefficient, for [Zn’.OP]; this, in turn, allowed the calculation of the apparent dissociation constant, K, for the reaction Zn’ + OP ti [Zn’.OP].