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1.A sytematic study has been made using electron paramagnetic resonance spectroscopy of the effects of enzyme concentration and relative proportions of active and inactivated enzyme on the anaerobic reduction of xanthine oxidase a t pH 8.2 by the substrates xanthine, purine and NADH. Changes in intensity of two molybdenum (V) signals (earlier designated Rapid and Slow) and iron-sulphur and flavin radical signals were monitored over the time-range 0.1 sec to 2 days. A few measurements with the inhibitory substrate allopurinol were also made.2. Reduction can occur in as many as four phases, all of which are clearly seen with xanthine as substrate. The Rapid type of signals from active enzyme first appear in a time comparable to the turnover time (phase I), then decrease in intensity over a somewhat longer time (phase 11). This is followed by a further increase in intensity over periods of up to a few hours (phase 111), to levels higher than those attained in phase I. Finally with a half-life of several hours (phase IV) the Slow signal, which is known to be derived from inactivated xanthine oxidase, appears also. Fewer phases can be clearly distinguished with the other substrates.3. I n accordance with other work, results are interpreted in terms of redox equilibria among different enzyme species with molybdenum signal intensity controlled by interconversions between the paramagnetic Mo(V) and the diamagnetic Mo(IV) and Mo(V1) oxidation states. The observed kinetics indicate that Mo(V) produced in the catalytic phase I is "over reduced" in phase I1 to Mo(1V) in a bimolecular reaction in which inactivated enzyme is not involved and €or which the reducing agent may possibly be a substrate radical. This Mo(1V) of active xanthine oxidase is then slowly reoxidised to Mo(V) by inactivated enzyme, which remains inert in the catalytic phase but undergoes reduction in the slower phases. The reducing equivalents appear in the inactivated form, first in the flavin and iron-sulphur and then in the molybdenum. However, with NADH, where catalytic reduction is known to occur via flavin instead of via molybdenum, both active and inactivated enzymes are reduced simultaneously, thus confirming that the difference between these enzyme forms is minimal.I n studies of enzyme catalysis in two-substrate reactions it is often possible to observe, by some spectroscopic method, changes in the enzyme when treated with the first substrate in the absence of the second. Only if the observed changes are shown to occur within the turnover time for catalysis, can it reasonably be assumed that they are relevant to the enzymatic mechanism. Slower reactions between enzyme and substrate may still provide useful information about the chemical nature of groups in the active site but the interpretation of such results must be approached with caution. I n particular, consideration must be given to the possibility of the substrate reaction with groups other than the active site, or of reaction involving catalytically inactive variants of the enzyme.Abbrevi...

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1. Inhibition of collagenase from rabbit bone cultures by the chelating agents 1,10-phenanthroline and EDTA is almost completely reversed by Zn2+; other metal cations are less effective in reversing the inhibition. Optimal restoration of activity is achieved at Zn2+ concentrations below that of the chelator, but excess of Zn2+ is inhibitory. 2. Prolonged incubation of collagenase with either chelator causes irreversible inactivation. This inactivation is prevented by Zn2+ at the same concentrations needed to reverse the primary inhibition. 3. Collagenase incorporates 65Zn by exchange when incubated with 1,10-phenanthroline and Zn2+ containing this radioactive isotope. The 65Zn2+ can be removed from its binding site in collagenase by 1,10-phenanthroline or EDTA. Irreversible inactivation of collagenase by chelators destroys its ability to incorporate 65Zn2+. 4. Latent collagenase, the inhibited form in which collagenase first appears in culture, behaves similarly to the active enzyme in 65Zn2+-exchange experiments, but is resistant to irreversible inactivation by chelators. 5. It is concluded that collagenase is a zinc metalloenzyme that forms an inactive and unstable apoenzyme on treatment with chelators. The bound inhibitor component of latent collagenase evidently stabilizes the apoenzyme.