A number of methods for determination of &glucuronidase (EC 321.31) activity based on the chromogenic or fluorometric properties of aglycones as well as their absorbance in ultraviolet have been published (1,2) ; among them the ~henolphthalein method is the most widely used. Methods that measure the simultaneously liberated glucuronic acid have been developed to a much lesser extent. The Fishman-Green naphthoresorcinol method (2) measures total glucuronic acid in the mixture and that remaining after hypoiodite oxidation at pH 10.1; the value for free glueuronic acid is then obtained by difference in uranic acid content before and after oxidation. Using the same oxidation procedure, Yuki and Fishman (3) have applied the carbazole method (1,4) to the differential analysis of free and conjugated glucuronic acid mixtures. The naphthoresorcinol reaction has been adapted (5) to direct determinat,ion of glucuronic acid in the presence of difficultly hydrolyzable phenolic conjugates; t,he procedure involves treatment with concentrated orthophosphoric acid at 70" for more than 1 hr. Separation of free and conjugated glucuronic acid by solvent extraction of the enzymic digest was also reported (6) ; nonext,ractable glucuroni~ acid was then determined by a cerimetric method.
In our studies dealing with the synthesis and structure of I-O-acyl-LY-and P-n-glucopyranuronic acids (7,8), a quantitative method that would enable to follow t'he rate of enzymic hydrolysis of the ester-type /3-D-glucosiduronic acids was required. Glucuronides in which the Dglucuronic. acid residue is conjugated with the carboxyl group of an organic acid are labile compounds, particularly in alkaline solutions (1,9) ; under drastic conditions used in analytical procedures mentioned above, glucuronic esters decompose spontaneously to glucuronic acid and the corresponding aglycone. Thus, the above methods, although very 164