Spectrophotometric determination of phenolphthalein with sulfuric acid and its applications to the assay of phosphatases

SUZUKI Masao, KANBE Kunio, ASABE Yoshihiro, TAKITANI Shoji

doi:10.2116/bunsekikagaku.27.8_495

The following new method for the determination of phenolphthalein(PP) with sulfuric acid was established: To sample which contains (525)μg of PP in 0.5 ml of ethanol is added 75%(v/v) sulfuric acid-ethanol solution with cooling in an ice water bath and the total volume is adjusted to 5 ml. The absorbance is measured at 506 nm (reference: 75% sulfuric acidethanol solution). The color obtained by this reaction was stable at least for 4 h at room temperature and this method was twice as sensitive as the usual alkaline coloration method. Then, the proposed method was applied to the assay of alkaline phosphatase (ALP) and acid phosphatase (ACP) activities; the enzymatic reactions of ALP and ACP were according to the Babson's and Huggins-Talalay's methods, respectively. The reaction products were measured by two-wave-length spectrophotometric method with masking the color due to the substrate, phenolphthalein monophosphate. The standard procedure for the assay of both activities is as follows: Five ml of 80% sulfuric acid-ethanol solution is added to the mixture resulted from the enzymatic reaction of ALP (in ACP activity, the reagent solution is added to the mixture until the volume is 5 ml). After mixing, the absorbance is measured at λ₁ 413 nm, λ₂ 506 nm for ALP activity and at λ₁ 427 nm, λ₂ 501 nm for ACP activity, respectively. The correlation coefficient between this method and Babson's method (Huggins-Talalay's method) for the control serum was 0.992 (n=39) [0.984 (n=31)].

Spectrophotometric determination of phenolphthalein with sulfuric acid and its applications to the assay of phosphatases

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