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Even though highly purified horse liver alcohol dehydrogenase (LADH) may contain from 3.1 to 4.3 g-atoms of Zn per mole, only 2 g-atoms of Zn are catalytically active. Further, 2 moles of the competitive inhibitors 1,10-phenanthroline (OP) and 2,2 '-bipyridine(BP)have been reported to bind to 1 mole of ertzyme. This stoichiometry has been confirmed by titrations employing the ehromophoric properties of these chelating agents, and the characteristic absorption, optical rotatory dispersion, and circular dichroic spectra of the LADH-Zn-OP(BP) complexes all yield this same value. In addition, spectral data confirm that OP and BP interact with the enzyme through coordination with Zn, not by nonspecific binding with amino acid side chains of the protein. The method of continuous variations was employed to resolve the statistical problem generated by the interaction of less than the total number of Zn atoms present in the enzyme. It also demonstrates that only 2 of the 3.5 g-atoms of Zn in the enzyme here employed interact with OP and BP. Moreover, both Zn atoms form 1:1 complexes with each agent. Since Zn complex ions containing both chemically T A he reversible inhibition of horse liver alcohol dehydrogenase, LADH,* 1 by the chelating agent, 1,10-phenanthroline, OP (Vallee and Hoch, 1957), first suggested that zinc plays a role in the catalytic function of this enzyme. Subsequent kinetic (Vallee et al., 1959; Plane and Theorell, 1961) and spectral studies (Vallee et al., 1958;Vallee and Coombs, 1959; Yonetanij 1963a,b) indicated that two molecules of OP interact with zinc atoms in LADH and compete with NADH, the coenzyme. These data were consistent with the presence of two active enzymatic sites (Theorell and Bonnichsen, 1951) each containing a single essential zinc atom.The more recent detection of zinc in excess of 2 g-atoms/ mole of more highly purified LADH (Ákeson, 1964;Oppenheimer et al., 1967;Drum et al., 1969a) occasioned a reexamination of the interaction of 1,10-phenanthroline and of 2,2'-bipyridine with this enzyme. Spectrophotometric titration

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The location of amino acid side chains-whether at the surface or in the interior of protein molecules-and their immediate chemical environment are thought to be critical to their chemical reactivity toward modifying agents. Certain catalytically essential amino acid residues of enzymes have proven particularly reactive chemically. Similarly, metal atoms of metalloproteins can exhibit differential reactivity related to their location and roles. This is exemplified by recent studies of horse liver alcohol dehydrogenase (LADH).'Materials and Mfethods.-Crystalline horse LADH isolated by a modification of the Dalziel procedure2 and homogeneous by ultracentrifugation was obtained from Boehringer M\Iannheim Corporation. By chromatography on carboxymethyl cellulose, about 95 per cent of the protein and enzymatic activity eluted together in a single symmetrical peak in a position corresponding to the major component described by Dalziel3 and contained 3.4-3.6 gm at. of Zn/mol wt 80,000. However, electrophoresis on cellulose acetate and starch gel at pH 8.5 revealed the presence of five bands, as demonstrated by staining techniques both for protein and enzymatic activity. About 75 per cent of the protein and the enzymatic activity were associated with the third and 25 per cent with the fourth cathodial bands, in accord with the findings of Pietruszko et al.4 5 Urea was recrystallized from ethanol and aqueous solutions prepared immediately before use. The control of metal contamination and the determinations of enzymatic and isotopic activities, protein concentration, and Zn content have been described.6 A Cary model 60 spectropolarimeter was employed for the optical rotatory dispersion studies. Velocity sedimentation and sedimentation equilibrium were performed in a Spinco model E ultracentrifuge.The apparent partial specific volume7 and density were determined pycnometrically.Results and Discussion.-Zinc content: LADH contains two active enzymatic centers, each binding one molecule of coenzyme.8 The Zn content of early preparations ranged from 1.7 to 2.8, with a mean of 2.1 gm at. of Zn based on a mol wt of 73,000.9' 10 Both by spectral' and by kinetic12 criteria, two moles of 1,10-phenanthroline (OP) competed with coenzyme and were thought to bind at or near the Zn1 atoms of the two active centers. However, the enzyme then employed contained substantial admixtures of impurities, largely eliminated by subsequent modifications of the isolation procedure as evidenced by specific enzymatic activity, sulfhydryl content, and chromatographic criteria.2 3 The enzyme prepared in this manner had a mol wt of 84,000.13 The amount of Zn found earlier9' 10 would be equivalent to 2-3.2 gm at. of Zn per mol wt 84,000. Carboxymethylation with C14-iodoacetate inactivates LADH while labeling the cysteinyl residues of two identical peptides,'4' 15 suggesting two active sites and two identical subunits per mole.The Zn content of more highly purified preparations has been found to be 4 gm at.1434