THE JOURNAL BIOLOGICAL OF CHEMISTRY
Vol. 236, No. 4, April 1961 Printed in U.S. A.
Comparative Ability of P-Glucuronidase Preparations (Liver, Escherichia coli, Helix pomatia, and Patella vzdgata) to Hydrolyze Certain Steroid Glucosiduronic Acids*
MASAO From Tufts University WAKABAYASHI AND WILLIAM H. FISHMAN Center Hospital,?
School of Medicine and the New England Boston,Massachusetts for publication, December 1, 1960)
An important aim of the steroid analyst is to accomplish complete and safe hydrolysis of steroid conjugates before the application of the available micro and ultramicro methods of steroid separation and of analysis. At the present time reliance is placed on preparations of P-glucuronidase and sulfatase to liberate steroids from theirglucosiduronic acids and sulfate esters, respectively. This practice is fraught with empiricism, each laboratory advocating its own brand of enzyme and widely differing conditions of incubation. Adding to the complexity of this first step in the analysis of urinary steroids, in particular, is the frequent presence of ,&glucuronidase inhibitors and the resulting uncertainty of the completeness ofhydrolysis. Moreover, a crude enzyme preparation, e.g. the digestive juice of the edible snail, Helix pomatia, may contribute pigments which can The difficulty of deciding find their way into steroid fractions. whether certain steroid glucosiduronic acids are themselves resistant to /3-glucuronidase, or that the effectiveness of the enzyme is reduced by inhibitors has led some investigators (l-3) toattempt to separate and purify the conjugated steroids from urine and blood. It would seem desirable, therefore, to ascertain the susceptibility to hydrolysis by &glucuronidase of individual pure steroid glucosiduronic acids, at the optimal pH for each. Moreover, for purposes of economy of both enzyme and glucosiduronic acid, it does not seem necessary to hydrolyze more than 100 pg of steroidglucosiduronic acid, especially since the analytical methods to be applied to the products are so sensitive. Accordingly, four P-glucuronidase preparations (beef liver, Escherichia coli, Patella uulgata, and Helix pomatia) were adjusted to the same potency at their respective optimal pH values with respect to the hydrolysis of 100 pg of the standard substrate, phenolphthalein glucosiduronic acid. With suchpreparations the optimal pH of hydrolysis of similar quantities of each of eight steroid glucosiduronic acids was determined, and subsequently the time course of hydrolysis at the optimal pH was established. Moreover, a convenient process of preparing highly * Aided m part by grants (P-108, P-107) from the American Cancer Society and (C-3213) from the National Cancer Institute, NationalInstitutes of Health, United States Public Health Service. t Mailing address, Cancer Research Department, 30 Bennet Street, Boston 11, Massachusetts. 996
purified /‘%glucuronidase, free from pigment and sulfatase, from the digestive juice of Helix pomatia has been developed. An account of these experiments is given in this paper. Aside from the practical significance of these results, the datacontribute to the knowledge of the action of the enzyme on steroid glucosiduronic acids as a function of pH.
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“Ketodase ?” which Enzymes-The beef liver P-glucuronidase, was supplied as a generous gift by the Warner-Chilcott LaboraAt this enzyme concentratories, was diluted 1 to 5 with water. tion, 100 pg of phenolphthaleinglucosiduronic acid are hydrolyzed completely at the maximal pH of the acid (pH 5.0), after 1 hour of incubation at 37” (86 Fishman units (4) per 0.1 ml). The limpet (Patella vulgata) P-glucuronidase, kindly supplied This powder by Dr. A. E. Kellie of London, was a dry powder. (10 mg) was suspended in 3 ml of water and was ground with a The mixglass homogenizer previously chilled with ice water....
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