Lactate Dehydrogenase-B Oxidation and Inhibition by Singlet Oxygen and Hypochlorous Acid

Abstract

Alterations in cellular energy metabolism are a hallmark of cancer and lactate dehydrogenase (LDH) enzymes are overexpressed in many cancers regardless of sufficient oxygen and functional mitochondria. Further, L-lactate plays signaling roles in multiple cell types. We evaluated the effect of singlet oxygen and hypochlorous acid (HOCl) on pig heart LDH-B, which shares 97% homology with human LDH-B. Singlet oxygen was generated photochemically using methylene blue or the chlorophyll metabolites, pheophorbide A and chlorin e6. Singlet oxygen induced protein crosslinks observed by SDS-PAGE under reducing conditions and inhibited LDH-B activity. Ascorbate, hydrocaffeic acid, glutathione and sodium azide were employed as singlet oxygen scavengers and shown to protect LDH-B. Using fluorescein-modified maleimide, no changes in cysteine availability as a result of singlet oxygen damage were observed. This was in contrast to HOCl, which induced the formation of disulfides between LDH-B subunits, thereby decreasing LDH-B labeling with fluorescein. HOCl oxidation inhibited LDH-B activity; however, disulfide reduction did not restore it. LDH-B cysteines were resistant to millimolar H2O2, chloramines and Angeli’s salt. In the absence of pyruvate, LDH-B enhanced NADH oxidation in a chain reaction initiated by singlet oxygen that resulted in H2O2 formation. Once damaged by either singlet oxygen or HOCl, NADH oxidation by LDH-B was impaired.

Keywords

• lactate dehydrogenase
• singlet oxygen
• hypochlorous acid
• cysteine oxidation
• reactive oxygen species
• disulfide