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NAPHTHOL AS-BI beta-d-GLUCOSIDURONIC ACID; ITS SYNTHESIS AND SUITABILITY AS A SUBSTRATE FOR beta-GLUCURONIDASE

WILLIAM H. FISHMAN 1, YASUO NAKAJIMA 1, C. ANSTISS 1, and SIDNEY GREEN 1

1 Department of Pathology (Oncology), Tufts University School of Medicine and the Cancer Research Department, New England Center Hospital, Boston, Mass.

1. Naphthol AS-BI beta-d-glucosiduronic acid has been prepared by first reacting potassiumnaphtholate AS-BI with acetobromomethyl glucuronate in absolute alcohol and then by removing the protecting acetyl and methyl groups with barium hydroxide and hydrochloric acid. A stable crystalline product gave the elemental and group analytical values expected for C24H22O9NBr·0.6 H2O and yielded the expected equimolar amounts of naphthol AS-BI and glucuronic acid on complete hydrolysis with purified rat liver beta-glucuronidase. Contaminants were found absent after examining the product by thin layer chromatography. The glucuronide is stable at room temperature (20°C) as a solid or in aqueous solution.

2. Purified rat liver beta-glucuronidase hydrolyzes the glucosiduronic acid at a rate somewhat more rapidly than it does phenolphthalein-, p-nitrophenyl- and 8-hydroxyquinoline beta-d-glucosiduronic acids. The Michaelis constant is 0.000021 M, the optimum pH is 4.5. There is a linear relationship to enzyme concentration. Saccharolactone and the presence of a second substrate effectively inhibit the reaction.

3. Incidentally to the main objectives of the study, a simple colorimetric method was devised for measuring enzymically-liberated naphthol AS-BI in the kinetic studies.

4. It is concluded that naphthol AS-BI beta-d-glucosiduronic acid is a suitable substrate for beta-glucuronidase and that it lacks none of the desirable attributes of the classical biochemical and histochemical substrates. Moreover, it is now possible to undertake definitive studies on the design of enzymorphologic techniques for beta-glucuronidase utilizing this new substrate.

Submitted on November 22, 1963


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