The binding of NADH to horse liver alcohol dehydrogenase at subzero temperatures. A three-step reaction.

The binding reaction of NADH to liver alcohol dehydrogenase was investigated at temperatures to -45 degrees C using a cryosolvent composed of 50% aqueous dimethylsulfoxide. At temperatures below -30 degrees C, the reaction produced biphasic changes in the protein fluorescence. The slow phase caused a 15 to 20% quenching of protein fluorescence, reached a maximum at 50 to 100 micro M NADH, and had an energy of activation of 27.3 kcal/mol. The fast phase appeared to be linearly dependent upon NADH concentration, produced a 45 to 50% quenching of the protein fluorescence, and had an energy of activation of 19.6 kcal/mol. The results are interpreted in terms of a three-step binding reaction, corresponding to initial complexation followed by conformational changes.