The estimation of serum acid phosphatase using alpha-naphthyl phosphate as substrate: observations on the use of Fast Red TR salt in the assay.
The estimation of acid phosphatase (EC 3.1.3.2) in serum by reaction rate analysis, utilising alpha-naphthyl phosphate as substrate and a diazonium salt (Fast Red TR) for colour development is examined. In the absence of substrate the occurrence of a pseudo catalytic activity is shown to be due to the hydrolysis of the Fast Red TR with the subsequent production of 4-chloro-2-methylphenol. The 4-chloro-2-methylphenol reacts with the Fast Red TR to form an azo compound which absorbs at a wavelength of 340 nm. The production of 4-chloro-2-methylphenol during the reaction is first order with respect to the Fast Red TR concentration, is temperature dependent and is accelerated in the presence of human albumin or Triton X-100. Monitoring the reaction at a wavelength of 585 nm eliminates the need for substrate independent blank correction. The final reaction conditions are summarised, together with a comparison of the method with that of the Kind and King [9] procedure.