Re-evaluation of the kinetics of lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by superoxide radicals in the presence of ethylenediaminetetraacetate.

The chain oxidation of lactate dehydrogenase-bound NADH initiated by superoxide radicals and propagated by oxygen was studied with pulse radiolysis. The kinetic parameters were re-evaluated in a system with carefully purified reagents (water and other chemicals) and in the presence of EDTA. The rate constant for the oxidation of the enzyme-bound NADH by O2- is calculated from the observed pseudo-first order disappearance of NADH and the chain length (molecules of NADH oxidized per O2- anion generated in the pulse). It is (1.0 +/- 0.2) X 10(5) M-1 S-1, consistent within a 13-fold variation in lactate dehydrogenase. NADH complex concentration and with varying chain length up to 6.1. Based on experiments with varying pH values from 4.5 to 9.0, the rate constant for oxidation of enzyme-bound NADH by HO2 is estimated to be 2.0 X 10(6) M-1 S-1.

The chain oxidation of lactate dehydrogenase-bound NADH initiated by superoxide radicals and propagated by oxygen was studied with pulse radiolysis. The kinetic parameters were re-evaluated in a system with carefully purified reagents (water and other chemicals) and in the presence of EDTA. The rate constant for the oxidation of the enzyme-bound NADH by O,m is calculated from the observed pseudo-first order disappearance of NADH and the chain length (molecules of NADH oxidized per O,-anion generated in the pulse). It is (1.0 + 0.2) x lo5 M-' s-l, consistent within a 13.fold variation in lactate dehydrogenase NADH complex concentration and with varying chain length up to 6.1. Based on experiments with varying pH values from 4.5 to 9.0, the rate constant for oxidation of enzyme-bound NADH by HO, is estimated to be 2.0 x 10B~-ls-l.
In an aqueous solution superoxide and hydroperoxyl radicals are in equilibrium with a pK = 4.8 (1).
HO l=H+ + O,-K,.-, = 1.6 x 10-S~ U-1) Our earlier reports (2-4) have shown that the two radical forms HO, and O,-react with lactate dehydrogenasel-bound NADH at different rates, and that the nucleotide radical produced in either Reaction 3 or Reaction 4 reacts in turn with molecular oxygen (Reaction 5) to generate another superoxide radical. The chain oxidation mechanism was proposed as follows: LDH.NADH + HO, + LDH.NAD.
X + O,-+ product(s) (7) where X represents an unknown scavenger for HOJO,-. Most of the experiments in this study were carried out in the neutral pH range, in which the ionized form of the oxygen radical is the predominant species; therefore Reaction 3 is of primary interest in this study. In the discussion that follows, for simplicity, the sum of the radicals (O,-+ HO,) are referred to as superoxide radicals. The reason for our reinvestigation of the kinetics of this chain mechanism (Reactions 2 to 6) is that although we did observe good pseudo-first order kinetics for the disappearance of NADH over a 4-fold LDH.NADH concentration range (4), at that time we were unable to establish a consistent value of the chain propagation rate constant (k, or k,).  (19). The disappearance of NADH was followed at 380 nm because of the high absorbance of NADH at 340 nm in a 6.1.cm light path. The superoxide radicals were generated with a lO+s pulse of 1.9 MeV electrons delivered from a Van de Graaff generator.
The superoxide radical concentration at a given pH was determined from a calibration curve as a function of energy input. The experimental data are summarized in Table I.
The main objective of this study was the re-evaluation of k, at the physiological pH, hence the bulk of the data were determined at pH 7.35 and 7.5 (Table I)  Thus the contribution of Reaction 4 to the overall rate of disappearance of NADH was of the order of 6 and 4%, respectively.
As is apparent from the data in Table I, statistically, one cannot differentiate between the two series of r l .  Table I; n , average value for Runs 15 to 37 in Table I

experiments.
The average value of all the runs listed in Table I