COUPLING OF 1-NAPHTHOL WITH FAST-RED-TR .1. STUDIES ON THE OPTIMIZATION OF A CONTINUOUS DETERMINATION OF ACID-PHOSPHATASE

  • 1 January 1980
    • journal article
    • research article
    • Vol. 18  (10) , 603-610
Abstract
In the course of studies on the optimization of a method for the continuous determination of acid phosphatase, the coupling of 1-naphthol with fast-red-TR was investigated. In the presence of detergents or protein, the detection reaction gives a linear response for spectral absorption in the range 0-1 against concentration. The reaction between naphtol and fast-red-TR salt is 1st order with respect to 1-naphthol. The t1/2 for the coupling must be less than 0.5 min to keep the lag phase of the reaction below 2 min. Shortening of the coupling t1/2 (increase in the rate of the indicator reaction) was achieved by increasing the fast-red concentration and pH-value, and by alterations, with certain limits, of ionic strength and the concentration of protein and/or detergents. Development of the chromophore depends on time and the presence of proteins and/or detergents. Reliable measurements are only possible at the isosbestic point of the chromophore. In the presence of albumin, these are 460 nm and 390 nm. Reproducible formation of the chromophore requires the presence of protein (albumin); replacement by detergents is possible to a limited extent. To avoid a time-dependent bathochromic effect, buffer materials should be present in the lowest possible concentration: citrate buffer 0.2 mol/l; acetate buffer 0.4 mmol/l. By coupling with fast-red-TR salts, serum proteins result in increased positive values (apparent enzyme activity) up to 2.6 U/l, depending on the measurement wavelength for the chromophore. Molar absorption coefficients for wavelengths 390, 405 and 460 nm were determined. Recommendations are given for the optimization of the indicator reaction in the determination of acid phosphatase by hydrolysis of naphthyl phosphate.

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