N-乙酰-β-D-氨基葡萄糖苷酶的基质合成及其动力学法测定

Synthesis of Substrate And KineticI Rate Assay of N—Acetyl—β—D—Glucosaminidase

本文应用本室合成的底物2-氯-4-硝基苯基N-乙酰-β-D 氨基葡萄糖苷(CNP-NAG),用动力学法测定N-乙酰-β-D 氨基葡萄糖苷酶(NAG),酶促反应最适pH 值为4.8～5.2,Km 为0.66～0.70mmol/L。用N.N-二甲基甲酰胺和乙二醇等量混合液使基质浓度达到4mmol/L,同时提高了反应的灵敏度和基质液的稳定性.用该法测定不受尿色的干扰.批内CV<3.57%,批间CV>4.04%,与PNP 终点法比较相关系数为0.933,100例尿参考值调查为0.75～23.9u/g.Cr。

In this paper,we have assayed thd activity of N-acetyl-β-D-glucosaminidase(NAG)by kinetic method with a new synthetic substrate 2-chloro-4-nitrophenyl-N-acetyl-β-D-glucosaminide(CNP-NAG).Optimal pH for NAG enzymatic hydrolyzation waspH 4.8～5.2 and the Km value of NAG was 0.66～0.70 m mol/L.To increase the solubility andstability of this substrate and reaction sensitvity,we used N,N-dimethylcarboxylamide andethylene glycol.The colour of urine do not interfere.Within-run coeffcients of variationwas 3.57%,and between-run was 4.04%.It was compared with the PNP-endpoint method withgood correlation,r=0.933.Thereference reang of urinary NAG activities was 0.75～23.9u/gcreatinine in 100 normal subjects.