Abstract
A simple method, based on the colorimetric reaction of glucuronic acid with benzidine in the presence of acetic acid, has been developed for the direct measurement of glucuronic acid in mixtures containing glucuronic acid and labile glucosiduronic acids. The color is developed at 100° within 15 min and the absorbance is read at 410 nm. Data on the specificity, sensitivity, and precision of the method are presented. Standard procedure I allows analysis of glucuronic acid samples buffered with acetate at pH 5.0, and standard procedure II is designed for samples buffered with phosphate at pH 6.8; procedure I is superior to II. The method has been tested by measurement of glucuronic acid in mixtures containing known amounts of 1- O-benzoyl- and 1- O-veratroyl-β- d-glucopyranuronic acids, respectively; it has been shown that under mild conditions of the assay quantitative determination of glucuronic acid in the presence of glucuronic esters is possible. Application of the assay for determination of liberated glucuronic acid in enzymic digests was tested on phenolphthalein-β-glucuronide: results obtained by the phenolphthalein method agreed closely with those obtained by the present assay. Initial experiments with glucuronic esters indicate the applicability of the method for enzymic kinetic studies with ester-type glucosiduronic acids as substrates.
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