摘要
目的：合成4-硝基-1-萘磷酸酯（4-nitro-1-naphthyl phosphate,NNPP）为显色底物测定牛小肠黏膜碱性磷酸酶（alka-line phosphatase,ALP）。方法：4-硝基-1-萘酚（4-nitronaphthol,4-NNP）与三氯氧磷反应,经硅胶柱纯化制得NNPP;检测产物吸收跟踪水解过程测定初速度,双倒数法测定米氏常数（Michaelis-Menten constant,Km）。结果：ALP水解NNPP产物最大差吸收峰接近460 nm,等吸收波长接近405 nm。与p-硝基苯酚相比,在pH 6.0~7.0间4-NNP消光系数为其3倍以上,在pH 7.0以上为其2倍以上。ALP对NNPP的Km约12μmol/L而p-硝基苯基磷酸酯的Km约35μmol/L,产物磷酸相对NNPP的竞争性抑制常数接近20μmol/L。ALP催化NNPP水解效率接近水解p-硝基苯基磷酸酯的40%。结论：NNPP可用于测定ALP活性,且有望与作用于p-硝基苯酚类显色底物的其它酶联用实现单通道两种酶同步测定。
Objective：To detect alkaline phosphatase（ALP） in mucosa of cattle small intestine using synthesized 4-nitro-1-naphthyl phosphate（NNPP） as a chromogenic substrate.Methods：4-nitronaphthol（4-NNP） was reacted with phosphorus oxychloride to yield NNPP.By recording the absorbance of 4-NNP during the hydrolysis of NNPP,initial rates of ALP were estimated.Michaelis-Menten constant（Km） was estimated by Lineweaver-Burk plot.Results：Maximum absorbance peak and absorbance wave length of hydrolysis product of ALP against NNPP were around 460 nm and 405 nm.Absorptivity of 4-NNP was at least three times of p-nitrophenol at pH from 6.0 to 7.0 and twice at pH over 7.0.Kmof ALP against NNPP was about 12 μmol/L while that of p-nitrophenyl phosphate was about 35 μmol/L.Competitive inhibition constant of phosphate against both substrates was about 20 μmol/L.Activity of ALP against NNPP was about 40% of that against p-nitrophenyl phosphate.Conclusions：NNPP is an effective chromogenic substrate for assay of ALP and is promising for simultaneous assay of two enzymes in one reaction solution by using a derivative of p-nitrophenol as the chromogenic substrate of another enzyme.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2013年第4期413-416,共4页
Journal of Chongqing Medical University
基金
国家重大科技专项“863”资助项目(编号:2011AA02A108)
重庆市教委资助项目(编号:KJ100313)
