Abstract
Glyoxalase II (Glo II) is a crucial enzyme in the glyoxalase system, and plays a vital role in detoxifying harmful metabolites and maintaining cellular redox balance. Dysregulation of Glo II has been linked to various health conditions, including cancer and diabetes. This study introduces a novel method using 2,4-dinitrophenylhydrazine (2,4-DNPH) to measure Glo II activity. The principle behind this approach is the formation of a colored hydrazone complex between 2,4-DNPH and pyruvate produced by the Glo II-catalyzed reaction. Glo II catalyzes the hydrolysis of S-D-lactoylglutathione (SLG), generating D-lactate and reduced glutathione (GSH). The D-lactate is then converted to pyruvate by lactate dehydrogenase, then reacting with 2,4-DNPH to form a brown-colored hydrazone product. The absorbance of this complex, measured at 430 nm, allows for the quantification of Glo II activity. The study rigorously validates the 2,4-DNPH method, demonstrating its stability, sensitivity, linearity, and resistance to interference from various biochemical substances. Compared to the existing UV method, this 2,4-DNPH-Glo II assay shows a strong correlation. The new protocol for measuring Glo II activity using 2,4-DNPH is simple, cost-effective, and accurate, making it a valuable tool for researchers and medical professionals. Its potential for widespread use in various laboratory settings, from academic research to clinical diagnostics, offers significant opportunities for future research and medical applications.