Fluorescence Detection of the Chemical Relaxation of the Reaction of Lactate Dehydrogenase with Reduced Nicotinamide Adenine Dinucleotide

Abstract

SUMMARY The combination of lactate dehydrogenase from rabbit skeletal muscle with reduced nicotinamide adenine dinucleotide was in- vestigated fluorimetrically with the temperature jump method. Best agreement with the experimental results and the data of Zewe and Fromm is obtained by assuming a fast bimolecular step which is followed by a slow monomolecular interconversion. The dissociation constant of the former is 6 pM equilibrium con- stant of the latter is 0.3, resulting in an over-all dissociation con- stant of 1.4 PM. The bimolecular velocity constant is estimated near log M-’ set-I; the consecutive monomolecular constant is evaluated at 1000 se@. The enthalpy of the bimolecular reac- tion step is near 7 kcal per mole; that of the monomolecular one is nearly one-fifth of the former. The signal height of the chemi- cal change is generally only 10% of the total change, the re- mainder being caused by the change of fluorescence yield with temperature. Acknowledgments-We would like to express our high apprecia- 7 The reasoning for assuming this condition is quite similar to the reasoning outlined in footnote 5.