Scott D A, Goward C R, Scawen M D, Atkinson T, Price C P
Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK.
Ann Clin Biochem. 1990 Jan;27 ( Pt 1):33-7. doi: 10.1177/000456329002700107.
A method for assaying glucose in serum or plasma samples using a thermostable glucokinase was developed. Glucokinase from Bacillus Stearothermophilus was coupled with glucose-6-phosphate dehydrogenase to produce NADPH, which reduced the tetrazolium dye MTT to its formazan. Detection of the product at 660 nm allowed samples containing up to 30 mmol/L glucose to be assayed with an endpoint method. Use of the optimal wavelength for formazan detection, 570 nm, increased sensitivity for NADPH detection by over threefold compared to UV detection. The stability of glucokinase assay mixtures was extensively studied, with variation in buffers, salt and enzyme stabilizers. Maximal half life for reagent stability at room temperature was approximately 30 days, with storage of assay mixtures in two solutions. Various drugs and metabolites were tested for interference in the method and no significant interferences were found.
开发了一种使用热稳定葡萄糖激酶测定血清或血浆样品中葡萄糖的方法。嗜热脂肪芽孢杆菌的葡萄糖激酶与葡萄糖-6-磷酸脱氢酶偶联产生NADPH，NADPH将四唑盐染料MTT还原为其甲臜。在660nm处检测产物可使用终点法测定葡萄糖含量高达30mmol/L的样品。使用甲臜检测的最佳波长570nm，与紫外检测相比，NADPH检测的灵敏度提高了三倍多。对葡萄糖激酶测定混合物的稳定性进行了广泛研究，包括缓冲液、盐和酶稳定剂的变化。在室温下试剂稳定性的最大半衰期约为30天，测定混合物储存在两种溶液中。测试了各种药物和代谢物对该方法的干扰，未发现明显干扰。
