Horse‐liver alcohol dehydrogenase requres Zn2+ for enzymatic activity. Reactivation experiments after dissociation and denaturation of the enxyme in 6 M guanidinium hydrochloride and subsequent separation of zinc prove thaty the effect of the metal on the rate and yield of reconstitution is complex. In the absence of Zn2+ no reactivation is detecatble, while excess of Zn2+ leads to inactive aggregates. Optimum reactivation yields are obtained at 10 μM Zn2+ after short incubation in the denaturant; increasing zinc concentration causes a decreasae of the rate of reactivation.The refolding of the zinc‐free enxyme is characterized by consecutive first‐order processes which may be separated from second‐order dimer formation. Addition of 10 μM Zn2+ during refolding may be used to block side reaction scompeting with the reconstitution. The transition from sigmoidal kinetics to secodn‐order profiles by adding Zn2+ after completion of the aforementioned first‐order process corroborates the proposed uni‐bimolecular reactivation mechanism which implies the involvement of inactive monomers. These gain their enzymatic function as a consequence of dimerization.The effect of Zn2+ may be explained by a side reaction in the overall reaction scheme of reactivation and renaturation which allows the kinetic measurements to be quantitatively described.

RUDOLPH, R., GERSCHITZ, J., & JAENICKE, R. (1978). Effect of Zinc(II) on the Refolding and Reactivation of Liver Alcohol Dehydrogenase. European Journal of Biochemistry, 87(3), 601–606. Portico.