High-pressure stopped-flow fluorometry at subzero temperatures: application to kinetics of the binding of NADH to liver alcohol dehydrogenase.

A stopped-flow apparatus operating in fluorescence mode over temperature and pressure ranges of +30 to -30 degrees C and 10(-3) to 2 kbar, respectively, is described. The system was interfaced on a special spectrofluorometer. Its general design is an improvement of the previous instrument (C. Balny, J. L. Saldana, and N. Dahan, (1984) Anal. Biochem. 139, 178-189) in that the observation chamber and the driving mechanism have been modified. The application of the method to kinetics of the binding of NADH to horse liver alcohol dehydrogenase at subzero temperatures and as a function of hydrostatic pressure is described.