Effect of mercury on NADH and the protective role of oxalacetate

Abstract

Because of the many biochemical roles of heavy metals experiments were done to examine the nature of the interaction between NADH and mercuric chloride. The comparative effects of HgCl/sub 2/ and methylmercury chloride on beef heart lactate dehydrogenase were studied spectrophotometrically following the oxidation of NADH by pyruvate. When 0.003 x 10/sup -6/ M MM was present in the 3 ml reaction mixture LDH activity was not inhibited. When the mixture contained 0.03 or 0.3 x 10/sup -6/ M MM, the enzyme activity was inhibited 11.7 and 41.7 percent compared to the control. When 0.0003 x 10/sup -6/ HGCl/sub 2/ was added to the reaction mixture in place of MM there was no inhibition of LDH activity. The enzyme was completely inhibited by 0.15 x 10/sup -6/ M HgCl/sub 2/. The addition of 0.2 x 10/sup -6/ M HgCl/sub 2/ to Na-phosphate buffer containing 0.2 x 10/sup -6/ M NAD had no effect on the normal NAD spectra. When the NADH-HgCl/sub 2/ reaction mixture was incubated 2 min prior to the addition of LDH enzyme, it was noted that the absorbance at 340 nm decreased rapidly due to interaction between NADH and HgCl/sub 2/. Various levels of other intermediate metabolitesmore » such as L-malate, succinate, tartarate and oxaloacetate or pyruvate were added to the NADH-HgCl/sub 2/ complex in the reaction mixture. Only OAA restored, in part, the absorbance at 340 nm. The data revealed that HgCl/sub 2/ inhibited LDH more than did MM, probably due to the monofunctional nature of MM, which forms one ligand (R-Hg-L), whereas HgCl/sub 2/ is bifunctional and can react with two ligands (L-Hg-L). The results indicate that OAA protects NADH by reaction with Hg/sup 2 +/. (MU)« less