Abstract
Incubation of NADH at neutral and slightly alkaline pH leads to the gradual absorption of 1 mol of H+. This uptake of acid requires oxygen and mainly yields anomerized NAD+ (NAD+), with only minimal formation od acid-modified NADH. The overall stoichiometry of the reaction is: NADH + H+ + 1/2O2 leads to H2O + NAD+, with NADH peroxide (HO2-NADH+) serving as the intermediate that anomerizes and breaks down to give NAD+ and H2O2. The final reaction reaction mixture contains less than 0.1% of the generated H2O2, which is nonenzymically reduced by NADH. The latter reaction is inhibited by catalase, leading to a decrease in the overall rate of acid absorption, and stimulated by peroxidase, leading to an increase in the overall rate of acid absorption. Although oxygen can attack NADH at either N-1 or C-5 of the dihydropyridine ring, the attack appears to occur primarily at N-1. This assignment is based on the inability of the C-5 peroxide to anomerize, whereas the N-1 peroxide, being a quaternary pyridinium compound, can anomerize via reversible dissociation of H2O2. The peroxidase-catalyzed oxidation of NADH by H2O2 does not lead to anomerization, indicating that anomerization occurs prior to the release of H2O2. Chromatography of reaction mixtures on Dowex 1 formate shows the presence of two major and several minor neutral and cationic degradation products. One of the major products is nicotinamide, which possibly arises from breakdown of nicotinamide-1-peroxide. The other products have not been identified, but may be derived from other isomeric nicotinamide peroxides.
Highlights
Incubation of NADH at neutral andslightly alkaline uatedampules from Sigma,andstored a t room temperature in a pH leads to the gradual absorptiofn 1mol of H+
The overall stoichiometry of the reaction is: NADH + + H+ ‘/zOZ + Hz0 NAD+, with NADH peroxide (H02-NADH+)serving as theinml of 0.15 M NaCl. pH Measurement a n d Titration-pH was measured with a Metrohm, ModelE-512 pHmeterand EA-147 electrode(Brinkmann Instruments, Cantiague, NY), and the output (100 mV/14 pH units) recorded on a Linear, Model 555 recorder (Linear Instruments Corp., Irvine, CA), adjusted to spansof 2 to 7 pH units
The latter reaction Titrant (0.1 M HCI in 0.15 M NaCI) was delivered with a Model E-415 is inhibited by catalase, leading to a decrease in the Multi-Dosimat (Brinkmann), equipped with a 1-ml burette and strip overall rate of acid absorption, and stimulatedby peroxidase, leading to an increase in the overall rate of acid absorption
Summary
The peroxidase-catalyzed oxidationof NADH by HzOzdoes not lead to anomerization, indicating that anomerization occurs prior to the reloefaHsezOz.Chromatography of reaction mixtures on Dowex 1formate shows the presence of two major and several minor neutral and cationic degradation products. NADH-All experiments were conducted with thesamelot of NADH, which was obtained preweighed (100 mg; 128 pmol) in evacknown concentration of P-NADH in the starting mixture or from analysis of A:MandA 2 e 1 , describe dabove The latter analysis is valid whenNADHand NAD’ arethe onlyuv-absorbingspecies; the accumulationof 260 nm-absorbing degradation products. To determine /?-NADHin reaction mixtures initially containing 50 to 100 mM /?-NADH, 1.5- to 30-p1aliquots were added to 1 ml of 100 mM Na Bicine, pH 7.8, containing 5 mM Na pyruvate, and the A340 recorded versus the appropriate blank. To determine /?-NAD+in reaction mixtures initially containing 50 to 100 mM /3-NADH, 1.5- to 2 .
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