用4-硝基-1-萘基磷酸酯为底物测定碱性磷酸酶活性

Assay of alkaline phosphatase with 4-nitro-1-naphthyl phosphate

摘要:

目的：合成4-硝基-1-萘基磷酸酯 (4-nitro-1-naphthyl phosphate，NNPP)为显色底物测定牛小肠粘膜碱性磷酸酶 (alkaline 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12?mol/L而p-硝基苯基磷酸酯的Km约35?mol/L，产物磷酸相对NNPP的竞争性抑制常数接近20?mol/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。

Abstract:

Objective: 4-nitro-1-naphthyl phosphate (NNPP) as a chromogenic substrate was synthesized for the assay of alkaline phosphatase (ALP). 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: The hydrolysis of NNPP by ALP gave the product with a maximum absorbance peak around 460 nm and an isoabsorbance wavelength around 405 nm. In comparison to the absorptivity of 4-nitrophenol, the absorptivity of 4-NNP is at least three-times at pH from 6.0 to 7.0, and is about twice at pH over 7.0. Km for NNPP is about 12 ?mol/L while that for 4-nitrophenylphosphate is about 35 ?mol/L; the competitive inhibition constant of phosphate against both substrates was about 20 ?mol/L. Activity of ALP on NNPP is about 40% of that on 4-nitrophenylphosphate. Conclusion: NNPP is an effective chromogenic substrate for the assay of ALP and is promising for simultaneous assay of two enzymes in one reaction solution by using a derivative of 4-nitrophenol as the chromogenic substrate of another enzyme.