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

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.