Continuous Spectrophotometric Assay for β - Glucuronidase

A continuous spectrophotometric assay has been developed for detecting β-glucuronidase activity. In the assay, Para-nitrophenyl β-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is highly sensitive and able to detect less than 1.4 × 10-4 U/mL of the enzyme activity in solution. Such a new assay offers significant advantages over the existing discontinuous methods and should be useful for both routine enzyme assay and accurate kinetic studies. INTRODUCTION β-glucuronidase (GUS, EC 3.2.1. 31) catalyzes the hydrolysis of β-linked D-glucopyranosiduronic acids (β-glucuronides) to glucopyranosiduronic acid (glucuronic acid) and aglycone: βD-glucuronide + H2O → D-Glucopyranosiduronic Acid + R-OH. This enzyme is present in animals and some bacteria but is absent from higher plants (1,2,10). In animals, GUS degrades natural glucuronide substrates, such as heparan sulphate, dermatan sulphate, and chondroitin sulphate, to glucuronic acid and aglycone residues (5). GUS enzyme deficiency in humans leads to a disease known as mucopolysaccharidosis type VII that results from lysosomal storage of undegraded glycosaminoglycans (6,21,22). In enteric bacteria, which normally inhabit the vertebrate gut, the GUS activity is involved in the hydrolysis of glucuronide compounds derived from conjugation of endogenous and xenobiotic organic compounds with glucuronic acid in the liver. The released glucuronic acid residue is used as a carbon source for bacterial cells (25). GUS assays are extensively performed in various fields. Polymorphonuclear leukocytes are quantitated by the release of lysosomal enzyme GUS (7). The biochemical analysis of the GUS gene activity in gingival crevicular fluid offers a noninvasive means of assaying periodontal disease (13). GUS assays are routinely used as diagnostic purposes for the specific detection of E. coli and Shigella species in clinical and environmental samples (11,15,18,25). The GUS gene is the most widely used reporter gene in plant molecular biology because of the absence of background activity in higher plants. Cell-, tissue-, and organ-specific gene expression is now routinely studied using GUS fusions in transgenic plants (9,10,26). Numerous applications demonstrate the importance of developing a simple, sensitive, and high-throughput assay for GUS activity. During the last three decades, a number of convenient substrates have been synthesized for the assay, taking advantage of the lack of structural specificity for the glucuronides (10,12,16,17,20,23,27). All of these synthetic substrates contain the sugar D-glycopyranosiduronic acid attached by glycosidic linkage to a hydroxyl group of a chromophoric, fluorogenic, or other detectable molecule. A variety of discontinuous assay protocols now exist for histochemical, spectrophotometric, or fluorometric analysis. However, these procedures are tedious and generate kinetic data that may not be accurate (24). Here, we describe an improved GUS assay procedure by continuous spectrophotometric analysis and demonstrate the utility of this assay with the enzyme from E. coli. Research Report 846 BioTechniques Vol. 30, No. 4 (2001) Continuous Spectrophotometric Assay for β-Glucuronidase BioTechniques 30:846-850 (April 2001)