Kadhum Mohammed Alaa, Hadwan Mahmoud Hussein
Department of Chemistry, College of Science, University of Babylon, Hillah City 51002, Iraq.
Biol Methods Protoc. 2024 Sep 18;9(1):bpae069. doi: 10.1093/biomethods/bpae069. eCollection 2024.
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.
乙二醛酶II（Glo II）是乙二醛酶系统中的一种关键酶，在解毒有害代谢产物和维持细胞氧化还原平衡方面发挥着至关重要的作用。Glo II的失调与包括癌症和糖尿病在内的各种健康状况有关。本研究介绍了一种使用2,4-二硝基苯肼（2,4-DNPH）测量Glo II活性的新方法。该方法的原理是2,4-DNPH与Glo II催化反应产生的丙酮酸形成一种有色腙络合物。Glo II催化S-D-乳酰谷胱甘肽（SLG）水解，生成D-乳酸和还原型谷胱甘肽（GSH）。然后，D-乳酸被乳酸脱氢酶转化为丙酮酸，再与2,4-DNPH反应形成棕色的腙产物。在430nm处测量该络合物的吸光度，可对Glo II活性进行定量。该研究严格验证了2,4-DNPH方法，证明了其稳定性、灵敏度、线性以及对各种生化物质干扰的抗性。与现有的紫外法相比，这种2,4-DNPH-Glo II测定法显示出很强的相关性。使用2,4-DNPH测量Glo II活性的新方案简单、经济高效且准确，使其成为研究人员和医学专业人员的宝贵工具。它在从学术研究到临床诊断的各种实验室环境中广泛应用的潜力，为未来的研究和医学应用提供了重要机遇。
