Simplified and economic measurement of glyoxalase I activity using 2,4-dinitrophenylhydrazine: A valuable tool for researchers

• Published in: Biology methods and protocols Vol. 10; no. 1; p. bpaf013
• Main Authors: Kadhum, Mohammed Alaa, Hadwan, Mahmoud Hussein
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 2025
• Subjects: Advanced glycosylation end products; Enzymatic activity; Enzymes; Glutathione; Lactoylglutathione lyase; Metabolic disorders; Methods; Pyruvaldehyde; spectrophotometry; methylglyoxal; S-D-lactoylglutathione; 2,4-dinitrophenylhydrazine; glyoxalase I
• ISSN: 2396-8923; 2396-8923
• DOI: 10.1093/biomethods/bpaf013

Glyoxalase I (Glo I) is an enzyme essential for detoxifying methylglyoxal, a toxic compound associated with advanced glycation end products. Given Glo I’s multifaceted roles in various physiological and pathological processes, accurately measuring its activity is crucial for understanding its implications in metabolic disorders. The current assay utilizes 2,4-dinitrophenylhydrazine (2,4-DNPH) to measure Glo I activity. This reagent has previously been employed to evaluate a group of enzyme protocols. The procedure involves incubating Glo I enzyme samples in a controlled phosphate buffer at pH 6.6, optimizing conditions for enzymatic activity. Glutathione and methylglyoxal serve as substrates, with Glo I catalyzing the conversion of the hemithioacetal adduct into S-D-lactoylglutathione. Unreacted methylglyoxal is quantified by forming a colored hydrazone complex with 2,4-DNPH. The 2,4-DNPH method is rigorously validated for linearity, stability, resistance to interference, and sensitivity from several chemicals. It strongly correlates with the existing ultraviolet method, offering enhanced simplicity and cost-effectiveness. The protocol allows precise quantification of Glo I activity, with potential in research and diagnostics. Intra- and inter-day analyses confirm accuracy as percentage relative error, ensuring reliable measurement activity. The DNPH-Glo I method exhibited excellent sensitivity, with low limits of detection and quantification at 0.006 U/L and 0.018 U/L, respectively. This research provides valuable insights into the quantification of Glo I, highlighting significant implications for future studies in metabolic disorders and related health fields. This study contributes to a deeper understanding of its role in health and disease management by advancing the methods available for measuring Glo I activity.