Studies in detoxication. 67. The biosynthesis of the glucuronides of umbelliferone and 4-methylumbelliferone and their use in fluorimetric determination of β-glucuronidase

Abstract

Umbelliferone (7-hydroxycoumarin) and its conjugates appear to be metabolites of coumarin in the rabbit (Mead, Smith & Williams, 1955). It was observed that, although umbelliferone fluoresced strongly in ultraviolet light at pH 9-10, its conjugates showed little or no fluorescence. The glucuronide and ethereal sulphate of umbelliferone were therefore made and the former was found to be practically non-fluorescent. The possibility that these conjugates could be used as substrates for the determination of ,-glucuronidase and arylsulphatase was therefore investigated. Since umbelliferone is highly fluorescent it appeared likely that these conjugates could be used for the deternination of the enzymes in very small amounts of material. The present communication deals with the use of the glucuronides of umbelliferone and the cheaper 4-methylumbelliferone (7-hydroxy-4methylcoumarin) for the determination of f,glucuronidase. (This work has been briefly reported by Mead, Smith & Williams, 1954.) The ethereal sulphates of -umbelliferone and 4-methylumbelliferone show a fluorescence which is considerably less than the corresponding hydroxycoumarins. We have attempted to use these compounds for the fluorimetric assay of arylsulphatase, but we do not consider them satisfactory because their fluorescence is sufficient to give relatively high blank values. The ,B-glucosides of umbelliferone and 4methylumbelliferone, however, have been found to be satisfactory for the determination of ,-glucosidase, and an account of these compounds will be given later. EXPERIMENTAL Materials. Umbelliferone, m.p. 224-226°, purchased from British Drug Houses Ltd., had a pale-reddish colour, and paper chromatography showed that it was not pure.In n-butanol-acetic acid-water (4:1:5 by vol.), besides a main fluorescent spot due to umbelliferone, three other much weaker fluorescent spots were present. As a standard for fluorescent purposes umbelliferone was purified by way of the sulphuric ester. The umbelliferone (4.5 g.) was * Part 66: Jondorf, Parke & Williams (1955).dissolved in pyridine (10 ml.) and a solution of chlorosulphonic acid (3 g.) in pyridine (10 ml.) added carefully with cooling. The mixture was then kept at room temp. for 24 hr. Water (100 ml.) was then added, followed by a slight excess of KHCO3. The precipitate which formed was collected by filtration, dissolved in the minimum of hot water (charcoal) and the solution filtered. On cooling the potassium umbeUiferone sulphate (potassium 2-oxo-1:2benzopyran-7-yl sulphate) separated and was recrystallized from 80 % (v/v) aqueous ethanol as colourless needles

Purification and specificity of pancreatic elastase

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1961little to choose between the p-nitrophenyl and methylumbelliferyl N-acetyl -, -glucosaminides, since, with the testicular enzyme, the ratio of the respective maximum velocities was 1: 0-95.The Laurence (1957) fluorimeter proved to be particularly suitable for the present kind of study.In agreement with the findings of Mead et al (1955) and Robinson (1956), the fluorimetric estimation of methylumbelliferone was found to be simple and convenient.Robinson (1956) pointed out that the fluorimetric method permitted the use of very dilute enzyme preparations and that the high dilution would tend to eliminate the effect of interfering substances. Before assaying very dilute preparations, it may be advisable to establish the form of the enzyme activity-concentration curve; it has been shown here that, with a methylumbelliferyl glycoside, this can be done conveniently with a combination of spectrophotometric and fluorimetric techniques.

The fluorimetric method permits the use of low substrate concentrations with the consequent advantage of conserving substrate; however, the advisability of using low substrate concentrations should be investigated, since results presented here show that characteristic properties of an enzyme (e.g.

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“…In agreement with the findings of Mead et al (1955) and Robinson (1956), the fluorimetric estimation of methylumbelliferone was found to be simple and convenient.…”

“…In agreement with the findings of Mead et al (1955) and Robinson (1956), the fluorimetric estimation of methylumbelliferone was found to be simple and convenient. Robinson (1956) pointed out that the fluorimetric method permitted the use of very dilute enzyme preparations and that the high dilution would tend to eliminate the effect of interfering substances.…”

Effects of Tumour Cell Culture Supernatants on Some Biochemical Activities of Macrophages

“…Acid phosphatase, i3-D-gIucuronidase and N-acetyl-D-glucosaminidase activities were assayed by the fluorimetric measurement of the hydrolysis of 4-methylumbelIiferyl (4-MU) derivatives to the parent 4-MU molecule (Mead, Smith and Williams, 1955;Leaback and Walker, 1961). The test samples were incubated with the appropriate derivative at 37° for a timed period.…”

Biology and the Exploration of Mars

“…The action of phosphatases on these phosphate esters can be readily followed by monitoring the emission at 450 mp ' [Moss, 1960]. Similarly, 7-hydroxycoumarin is highly fluorescent, while its glucoronide is non-fluorescent, permitting ready assay of the enzyme p-glucoronidase [Mead et al, 1955]. Rotman has shown that the use of fluorogenic substrates is sufficiently sensitive to detect single enzyme molecules such as p-D-galactosidase [Rotman, 1961].…”