PURIFICATION AND PROPERTIES OF A NEW 8-DcGLUCURONlDASE Thesis for tho Degree of Ph. D. MICHIGAN STATE UNNERSETY Julie Vista; Faber: 2963 / /LL? 2% 7726 This is to certify that the thesis entitled PURIFICATION AND PROPERTIES OF A NEW fl-D-GLUCURONIDASB presented by Julio Victor Pabon has been accepted towards fulfillment of the requirements for Ph.D. Chemistry degree in Major professdr MCML}\ Date August 19, 1963 LIBRARY Michigan State University 'J . . O ' 7. E . “WWW“ "’ . PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5’08 K'lProlecc8Pres/CIRC/Dateoue‘indd ABSTRACT PURIFICATION AND PROPERTIES OF A NEW B-D—GLUCURONIDASE By Julio V. Pabon A new B-D-glucuronidase of high activity has been prepared from the digestive tract of the aquatic snail, Ampullaria cupina. The prep— aration of the enzyme involved (1) extraction with 20% saturated anmxnr- ium sulfate, (2) heat denaturation, (3) precipitation with ammoniUhl sulfate, (h) fractional precipitation with ammonium sulfate, (S).frac- tional elution from DEAE-cellulose, and (6) ammonium sulfate precipi- tation. This procedure gave a BOO-fold purification of the material.ir1 the first extract. The final fraction was colorless and amorphous. It was free of arylsulfatase activity and probably was free of cellulase activity also. Repeated precipitations of the preparation with control— led ammonium sulfate concentrations yielded an electrophoretically homogeneous fraction of very high activity. Examination of a purified preparation in the analytical ultracentrifuge allowed the approximation of the molecular weight of the enzyme. The sedimentation data obtained at top speed of the rotor indicated that the preparation obtained by the purification procedure (steps 1—6) was about 63% pure. The catalytic properties of the enzyme have been investigated. The enzyme hydrolysed readily phenolphthalein, pregnanediol, l—menthyl, and p—nitrophenyl glucuronides. The enzyme exhibited a sigmoid pH- activity curve with inflection point about pH h.7. The Michaelis cons- tant, determined for phenolphthalein glucuronide, was found to vary Julio V. Pabon with the pH of the assay mixtures. The constant, however, was inde- pendent of pH when determined for p-nitrophenyl glucuronide. The enzyme was inhibited strongly by mercuric ion, and moderately by silver and cupric ions. Sulfhydryl group reagents caused no inhibi- tion. The effect of other parameters, such as temperature, nature of buffering ions, stability of the enzyme and nature of the aglycons, on the enzyme activity has been determined also. PURIFICATION AND PROPERTIES OF.A NEW B-D-GLUCURONIDASE By Julio Victor Pabon A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1963 {>\ 9c) ACKNOWLEDGMENT The author wishes to express his sincere thanks to Dr. John C. Speck, Jr., whose inspiration, great interest, guidance and co-oper— ation made possible the progress of the present investigation. Grateful acknowledgment is also due to Mr. Harold Swangood for running the ultracentrifugation experiments and making the necessary calculations. He is also indebted to Mr. Richard Dardas for his kind help in checking the homogeneity of one of the purified enzyme preparations. Acknowledgment is also due to the Department of Chemistry, the Department of Biochemistry and the National Institutes of Health for funds provided in support of this work. ii TABLE OF CONTENTS I. INTRODUCTION Historical Background . . . . . . . . . Occurrence Assay of Enzyme Activity variation of the Activity with pH . pH-Stability and the Effect of Temperature Interfering substances Specificity and Inhibition . . . . . . II. EXPERIMENTAL . . . . . . l. O\\J'LJ:"\» <1 10. ll. 12. Apparatus . Materials . Preparation of p-nitrophenyl E-D-glucuronide Determination of Protein Assay of Enzyme Activity . . . . . . . . Purification of the Enzyme Zone Electrophoresis . . . . . . Analytical Ultracentrifugation Stability of Enzyme versus pH . . . . . . . Effect of Temperature . . . . . . . . Inhibition by Cations . Inibition by Sulfhydryl-Group Reagents iii Page \TO\O\ 10 13 13 1h 16 17 18 21 23 25 25 26 26 27 TABLE OF CONTENTS (Cont.) III. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 1. Purification of the Enzyme . . . . . . . . . 2. Test of Homogeneity (Zone Electrophoresis) . . . 3 Analytical Ultracentrifugation . . . ... . . . h Stability as Function of pH . . . 5. Effect of Enzyme Concentration . . . . . . . . 6 . Time Course for the Hydrolysis of Various Substrates . . . . . . . . . . . . . . . 7. Effect of Temperature on the Enzyme Activity 8. Effect of Ph (and Buffers) on the Enzyme Activity 9. Effect of Substrate Concentration 10. Inhibition IV. SUMMARY . . . . . . . . . . . . . . . . . . . . V. APPENDIX . . . . . . . . . . . . . . . . . . . . . . l. Calculations for the Analytical Ultracentrifuge Experiment . . . . . . . . . . . . . . . . .’. 2. variation ofthe Enzyme Activity with Substrate Concentration at Various pH Values . . . . . VI. REFERENCES . . . . . . . . iv Page 29 29 36 37 39 hl h1 h2 uh A6 52 5b 56 S6 62 LIST OF TABLES TABLE Page I. Effect of heating the enzyme extract for h minutes at 70-7110 0 C C O O O O O C O O C O C O C C O O O O O O 3() II. Extent of enzyme purification given by steps l-h . . . . 31 III. Fractional elution of the enzyme from DEAE- resins . . . 31 IV. Fractionation of DEAE-eluates by ammonium sulfate precip; : itation o o o o o o a o o o o o o o o o o a o o o o 0 3h V. Purification of B-D-glucuronidase from the aquatic snail, Ampullaria cupina . . . . . . . . . . . . . . 3S VI. Paper electrophoresis in L.K.B. apparatus . . . . . . . 37 VII. Effect of pH on the stability of the enzyme. . . . . . . 39 VIII. Effect of enzyme concentration on the hydrolysis of phenolphthalein glucuronide . . . . . . . . . . . hl IX. Effect of temperature on the B—D-glucuronidase activity. h2 X. Effect of heavy cations on the enzyme activity . . . . . 53 IIST OF FIGURES FIGURE 1. Elution of the enzyme from DEAE-cellulose . . . . . . . . 2. Sedimentation of the enzyme in the analytical ultracentrifuge at 59,780 rpm . . . . . . . . . . . . 3. Sedimentation of the enzyme in the analytical ultracentrifuge at 10,589 rpm . . . . . . . . . . . . b. Time course for the hydrolysis of various substrates . . 5. Effect of pH (and Buffers) on the enzyme activity . . . . 6. Effect of pH on the enzymic hydrolysis of various SUbstrateS O O O C O C O O O . O O O O O O D O O O O O 7. Effect of phenolphthalein glucuronide concentration on the enzyme activity . . . . . . . . . . . . . . . . 8. Effect of p-nitrophenyl glucuronide concentration on the enzyme activity . . . . . . . . . . . . . . . . . 9. Effect of pH on the Michaelis Constant for phenol- phthalein glucuronide . . . . . . . . . . . . . . . . 10. Sedimentation of the enzyme preparation at h2,0hO rpm for the determination of the sedimentation coefficient vi Page 33 38 to 113 AS A? h8 A9 51 56a PURIFICATION AND PROPERTIES OF A NEW B-D—GLUCURONIDASE I. INTRODUCTION All mammalian tissues and fluids contain group-specific enzymes known as B-glucuronidasesl. B-Glucuronidases have been obtained also from diverse sources other than mammalian tissues. They catalyze the hydrolysis of the B-D—glucopyranosiduronic acids of all types to the aglycons and D-glucuronic acid. The presence of D-glucuronic acid in hydrolyzates have been established by several ways (3), and it has been shown in experiments carried out in H2018 that hydrolytic cleavage ‘occurs at the glycosyl—O bond (5). Fishman and Green (6) have shown that these enzymes exhibit transferase activity also; thus, liver, bacterial and molluscan B-glucuronidases catalyze transfer of the E-D- glucuronosyl group from aryl and cycloalkyl B—D-glucuronides to ali- phatic alcohols and glycols but not to phenols or alicyclic alcohols. The function of these enzymes in the animal body is still a subject of speculation. Its wide distribution in the mammalian tissues and fluids, coupled with changes in activity that have been observed in various physiological and pathological processes, suggests that they may play important metabolic and physiological roles. The contention, held for some time, that B-glucuronidases were responsible for the synthesis of glucuronides in 3122 has been abandoned (6). The now established pathway for glucuronide synthesis employs uridine S-(D- glucosyluronic acid dihydrogenpyrophosphate) and the enzyme glucuronyl- transferase (7,8). The presence of B-glucuronidase activity in crude lFor recent reviews of the B-glcuronidase literature the reader should consult references 1-h. II' 'I l Ilil.ll|llllll Illirlll l.| lull; lie-Ill llr 2 testicular hyaluronidase preparations (9), and the action of this glucuronidase (or these glucuronidases) on oligosaccharides released by hyaluronidase from.hya1unmfic acid and chondroitin sulfate (10), suggest that they may play a role in the mucopolysaccharide catabolism. This role is also suggested by the changes in glucuronidase activity which have been observed in certain organs in reSponse to free hormones (in 2129), in common with other mammalian glycosidases that also act on degradation products of hyaluronic acid (11). Historical Background The first report of the decomposition of B-glucuronides by plant and bacterial preparations appeared in 19Gb (12). It was reported that raw emulsin from almonds and extracts from the kefir grains contained a splitting enzyme for conjugated gluCuronides. This finding was con- firmed in 1906 (13) and in 1907 (lb). In 1908 appeared the first re- port of decomposition of conjugated glucuronides by a mammalian prepara- tion (15); menthol glucuronide was hydrolyzed by a dog liver extract. Hamalainen (16) reported in 1910 that yeast extracts did not hydro— lyze borneol- and camphorglucuronic acids. Sera (17) reported in 191h that orcinol and phloroglucinol glucuronides were decomposed by mam- malian extracts; these glucuronides, as well as vanillin glucuronide, were not split by emulsin (18, 19). Later, Ishidate (20) showed that menthol and p-hydroxycamphorglucuronic acids could be hydrolyzed by means of emulsin if proper conditions of pH and temperature were maintained. In 1933, Helferich and Sparmberg (21) found that the hydrolysis of 1-menthol-B-D-glucuronide by almond emulsin in different stages of purification did not parallel that of l-menthol—B-D-gluCOSide. 3 It was assumed that a specific enzyme was present in emulsin for the cleavage of the glucuronides. Masamune (22), in 193h, commenced the systematic study of B—gluc— uronidases with the characterization of the enzyme from beef kidney. In that year, Oshima (23) reported the distribution of B-glucuronidase in the tissues of the dog and the ox, and later in 1936, the purification of the enzyme from ox—spleen (2h). The purification procedure consisted of autolysis followed by adsorption on Kaolin at acid pH and elution at alkaline pH. At the same time, Hofmann (25) studied the hydrolysis of 1-menthol and B-naphthol B-D-glucuronide by mammalian extracts while investigating the specificity of mammalian glycosidases; earlier in- consistencies were explained. In 1939, Fishman (26, 27) started study- ing the mammalian B-glucuronidase to determine whether the enzyme was concerned with the synthesis of estriol glucuronide in the female organ- ism. He reported a 137-fold purification of the enzyme from ox-spleen and investigated the action of the purified enzyme on borneol, menthol, and estriol B-glucuronides. The ox-spleen enzyme was again purified by Graham (78) in 19h6. A BlS-fold purification was obtained by a procedure which involved mincing in water, precipitation of proteins with acetone, and ammonium sulfate fractionation at three different pH values. The study of B—glucuronidases was greatly facilitated when a colorimetric method of assay which utilizes phenolphthalein mono-B—D- glucuronide as substrate was introduced in 19h6 (28). Mills (29), in 19h8, showed the presence in spleen extracts of two protein fractions having B-glucuronidase activity. The two fractions were separated and purified by ammonium sulfate fractionation. The two fractions exhibited h different pH optima at pH h.5 and pH 5.0. This was confirmed by Kerr §£_al. (30) in the liver and kidney of the mouse. Sarkar and Sumner (31) employed dioxane fractionation, calcium phosphate adsorption, and ammonium sulfate fractionation to purify the enzyme from ox—liver. A 6000-f01d purification was reported but it appears that the activity of 'the purified fraction was 31,000 Fishman Units per mg. of protein (37). Jarrige and Henry (32) found the digestive juice of the land snail, Helix pomatia, a very rich source of the enzyme and studied some of the properties of the enzyme. Locusts (33) and marine molluscs (3h) were shown to contain large amounts of the enzyme also. Mills 33 a1. (35), continuing with the investigation of the B- glucuronidase from ox-spleen,Ip£sented evidence in 1953 for the occurrence in this tissue of three glucuronidase fractions with pH optima at pH 3.h, pH h.5, and pH 5.2. This work has not been confirmed. In that same year, Smith and Mills (36) purified the enzyme from ox-liver by applying a procedure involving metallo-protein reactions, celite adsorption, and elution from celite with ammonium sulfate solu- tions of different concentrations. The purified preparation has a specific activity of 32,000. Then, Fishman et a1. (37), employing alkaline ammonium sulfate fractionation followed by anion exchange and methanol fractionation, obtained a preparation from calf liver with Specific activity of 107,000. This preparation was regarded as 85 per cent pure on the baéis of physical tests of homogeneity. Recently, a new approach was adopted for the purification of the mammalian enzyme (38). Instead of a tissue readily available, but with low enzyme activity, the tissue with the highest activity (female rat preputial gland), was chosen as the starting material. The gland, 5 which is very small, has an initial specific activity of about 18,000. After a simple fractionation procedure employing ammonium sulfate and ethanol, a preparation was obtained with a specific activity of b55,000. The preparation was colorless and was stable for at least 18 months when buffered at pH 5.0. Alfsen and Jayle (39) have reported the only crystalline B—glucur- onidase preparation. It was obtained from the land snail, Helix pomatia, and exhibited a specific activity of 120,000. The purification proced- ure employed ammonium sulfate fractionation followed by ethanol frac- tionation. The preparation was found homogeneous by electrophoresis and analytical ultracentrifugation. Later, Wakabayashi and Fishman (hO) described an improvement in the method of Alfsen and Jayle which gave a preparation free of sulfatase activity, although not of greater B-glu- curonidase activity. The method (heat denaturation) of freeing B-glucur- onidase from arylsulfatase (hl) yielded a preparation from the limpet, Patella vulgata, also almost free from arylsulfatase (h2). Still, a pure B-glucuronidase preparation needs to be obtained in sufficient amounts to permit a complete study of the enzyme properties. For this it is necessary to find a source readily available and from which the enzyme could be isolated by a simple procedure. Although these enzymes are widely distributed in mammals and molluscs very few highly purified preparations have been obtained. The best preparation of these enzymes is the one from the richest source, the rat preputial gland, but this source presents the inconvenience of being very small and requiring a delicate technique for its separation from other tis- sues. The viscaral hump of the limpet or the digestive tract of the snail appears to be the most promising sources for these enzymes since 6 these are the richest alternatives to the rat preputial gland (3). Pre- vious work in this laboratory (h3) suggested that the digestive tract of the aquatic snail, Ampullaria cupina, might be a good source for one of these enzymes. The purpose of the present work was to look further into the isolation of the enzyme from this source, improve the purifica- tion procedure, and study some of the properties of the purified enzyme. Occurrence The distribution of B—D-glucuronidases is general in mammalian tissues and body fluids. Highest in activity are the liver, kidney, spleen, epididimis, and cancer tissues. The distribution of these enzymes is probably general also in other vertebrates, as well as in insects and molluscs (a comprehensive list of known sources of these enzymes is found in reference 3). The digestive juices or digestive tracts of molluscs are specially rich sources of these enzymes. They occur sporadically in plants and are randomly distributed in bacterias. The distribution of these enzymes within the cell has been studied in homogenates of mouse and rat tissues (3). The enzyme in isotonic homogenates of mammalian tissues (hh) was sedimented with the mitochon- drial and microsomal fractions; little was present in the nuclear frac- tion or free in the cytoplasm. When mammalian tissues were homogenized in water (hS), more than half of the enzyme escaped into solution and the residue within the granules was completely accessible to substrate. Assay of Enzyme Activity There is a variety of sensitive and specific methods, employing different substrates, for the measurement of B—glucuronidase activity (for description of most convenient methods see reference 3). Most of 7 the methods of assay depend upon colorimetric determination of the amount of aglycon liberated from a specified concentration of substrate at a fixed pH value. The unit of enzyme activity has been almost uni- versally expressed as that which liberates 1 ug of aglycon or D-glu- curonic acid in 1 hour at 380. When the substrate used is phenolphtha- lein mono-B-D—glucuronide, the most commonly used, this unit is known as the Fishman Unit. When it is more convenient to measure the liber- ated glucuronic acid this can be effected by the method of Fishman and Green (h6) which is based on the Tollens naphthoresorcinol color re- action for uronic acids. Recently, p-nitrophenyl B-D-glucuronide was synthesized (h?) and it has been suggested as a better substrate than phenolphthalein mono-B—D-glucuronide (h8). Variation of the Activity with pH A list of the pH optima for hydrolysis of B-glucuronides by mam- malian and non—mammalian B-glucuronidase preparations has been compiled (3). Mean values for the optimum pH are h.5 and 5.2. Mammalian prep- arations might exhibit either or both depending on the source and purity of the enzyme, and on the conditions of the assay. Non—mammalian preparations of these enzymes display only single pH optima. The different enzyme preparations appear to fall in two groups: (a) the bacterial preparations with Optima near neutrality and (b) the remaining preparations with optima below pH 5.0. Mammalian and non—mammalian B-glucuronidase preparations have shown little change in the pH optima for glucuronides of different aglycons in agreement with the finding (h9) that the nature of the aglycon has little effect on the ionization of a glucuronide. However, 8 the pH optima for the hydrolysis of phenyl B—D-galacturonide by the rat preputial gland preparation was 3.9 while that for the phenyl glucuron- ide was h.5 (50). pH—Stability and the Effect of Temperature The mouse liver B-glucuronidase was found pH-stable between pH 5.0 and pH 7.0 at 0°C (51). The range of stability of the limpet B-glucuron— idase was greater by one pH unit on both the alkaline and acid sides of neutrality (51). A period of contact with the acid or base of one minute or one hour made no difference with this limpet preparation. However, raising the temperature to 370 narrowed the stability range. Another molluscan preparation has shown the same stability that the limpet preparation did (52). Rumen B-glucuronidase was stable only at the pH region of maximum activity (53). The ox—liver B-glucuronidase was stable to 30 minutes of heating at 500 (31). It was not stable above this temperature. The energy of activation for the B—glucuronidase of human urine was -l3,600 ca1./m01e (5h), and for the enzyme from.H. pomatia this value was -lh,200 (55). Mammalian preparations have been reported to double their activity for every 100 rise in temperature (2h, 31, 35). This is probably true for molluscan preparations also (56). Interfering Substances A variety of natural and synthetic substances have been shown to interfere with the B-glucuronidase activity (3, h). Heparin, chondroi- tin sulfate, and hyaluronic acid are weak, non-competitive inhibitors. Ionic resins inhibit the enzyme non—competitively also, and the inhibition 9 by detergents (alkyl sulfates) rapidly becomes irreversible, with complete inactivation of the enzyme. Highly purified mammalian B-glucuronidase preparations when highly diluted appear to lose activity, but they are activated by albumin, deoxyribonucleic acid, chitosan, heat inactivated glucuronidase, starch, suramin and certain diamines (36, 57). Albumin showed no effect on the position of the pH optima, but deoxyribonucleic acid shifted the pH optima to the alkaline side of the pH-activity curve (38, 36). Phthalic acid produced the same type of effect as deoxyribonucleic acid (36). An unidentified, non-dialyzable and thermostable agent which has the capacity of inhibiting the mammalian enzyme has been reported present in the blood plasma (58) and in aqueous rat liver suspensions (59). The effect of this inhibitor was not pH—dependent. Recently, a new unidentified agent which is also non-dialyzable and thermostable has been extracted from water-insoluble cell debris of a rat liver homogenate. It has the ability to activate calf liver B-glucuronidase (60). The effects of cations on the B-glucuronidase activity have been studied for mammalian and non-mammalian preparations (3, 61). Only cupric, silver and mercuric ions have been found strong inhibitors. The action of cupric ion was weak unless potentiated by a reducing agent such as L-ascorbic acid or sodium bisulfite (61, 62). These substances had no effect on the inhibition by silver and mercuric ions. It was observed that low cation concentrations caused slight activation of crude mouse and rat liver preparations (63). The inhibitory action of silver, cupric and mercuric ions on the rat preputial gland preparation appeared to be primarily competitive (61). IO Inhibition by p-chloromercuribenzoic acid has been reported for a bacterial preparation of these enzymes (6h) and for the rat preputial gland preparation (61). The inhibition of the bacterial enzyme was reversed by cysteine; that of the mammalian enzyme was competitive. Specificity and Inhibition The mammalian B—glucuronidase preparations investigated appear to catalyze the hydrolysis of all natural and synthetic B-D—glucopyranosid- uronic acids whether aliphatic or aromatic (3, h8, 5h). They also catalyze the hydrolysis of 1—0-acyl-B-D-g1ucuronic acids (65, 66), and of B-D-glucopyranosiduronic acid-l-phosphate (67). The comparative ability of mammalian, molluscan and bacterial enzyme preparations to hydrolyze steroid B—glucuronides has been studied recently (ho, 68, 69). There was no evidence for a clear superiority of any one of the enzyme preparations for all the steroid glucuronides studied. The mammalian enzyme has no action on a-D-glucopyranosiduronic acids or on B-D—glu- copyranosides. Probably, non-mammalian preparations exhibit a similar behaviour (3, 55). Whether these enzyme preparations can catalyze hydrolysis of B-D- glucuronides with a furanose ring is still unsettled, since substances having this structure have not been prepared. It is of interest, however, that Nakao at 31. (70) reported that p-aminobenzoyl—B-D- glucuronide has a furanose structure. This glucuronide is hydrolyzed by B-glucuronidase (66). The glycosides of D—glucofuranuronolactone are not attacked. B-glucuronidases from all sources often display marked inhibition in the presence of excess substrate. ll Hydrolysis of B-glucuronides by mammalian or non-mammalian enzyme preparations is powerfully and competitively inhibited by D-glucaro- l,h-lactone (71). The inhibition is pH-dependent (5h, 72); it decreases with increasing pH. D—glucaro-l,h-lactone is a highly specific inhib- itor for B-glucuronidases (72), and it was potent inhibitor of the transferase activity of these preparations also (6). Inhibition by solutions of D-glucaric acid was shown to be due to D—glucar0-1,h-lac- tone present in the solutions (71). D—Glucarolactone requires a free carboxylic acid group at the 6—position to be an effective inhibitor. D-glucaro-6,3-1actone and D-glucuronolactone do not appear to inhibit the glucuronidase prepar- ations (73). The affinity of D-glucaro-l,h-lactone was shown to be lOOO-times as great as that of its 6-methy1 and 6-ethyl esters (5h). Solutions of galactaric acid have been shown to inhibit B-glu- curonidase preparations of mammalian and molluscan origin markedly (35, 71, 72, 73). Solutions of D-glucuronic acid also inhibit the enzyme, although to a lesser extent (71). The inhibition of B-glucuronidase preparations by solutions of galactaric acid was considered an anomalous behaviour until it was found that mammalian and limpet enzyme preparations hydrolyze 8-D— galactopyranosiduronic acids (38, 50). The identity of B-glucuronidase and B-galacturonidase was suggested by the action of specific inhibi- tors and by the high activity of preparations from rat preputial gland and from P. vulgata toward both types of substrate. The two activities in P. vulgata preparations displayed identical pH-stability. The preparations from the limpet, unlike the mammalian preparations, hydrolyzed d-glucuronides also (50, 7h). The enzyme responsible to 12 the hydrolysis of d-glucuronides was distinguished from B-glucuronidase since it displayed different pH and heat stability and was not inhibited by g1ucaro-l,h—1actone or galactarolactone. B-D-glucuronidase activity has been found present in crude, testi- cular hyaluronidase preparations (9). It is believed that hyaluronic acid is composed predominantly of alternate, B-linked, D-glucuronic acid and N-acetyl—D-glucosamines residues (75); chondroitin sulfate is a similar type of polymer in which the amino sugar is N-acetyl—D- galactosamine. Testicular hyaluronidase degrades hyaluronic acid and chondroitin sulfate to oligosaccharides from which, as has been shown (9, 10), B-glucuronidases and B-N-acetylhexosaminidase split off alter- nately D—glucuronic acid and hexosamine from their non-reducing ends. Considering this behaviour, the enzyme may be regarded an exo-B-D— glucuronidase. Recently, it has been reported that the hyaluronidase from the medicinal leech is an endo-B-D-glucuronidase (75, 76). This enzyme hydrolyzes hyaluronic acid to aligosaccharides which have the uronic acid moiety on the free reducing end. More recently, this enzyme has been purified by Yuki and Fishman (77). The specificity of the enzyme forhyaluronic acid appears to be unique and exclusive for the endo—B- glucuronide linkage. Simple glucuronides were not hydrolyzed. Crude extracts contained the exo-B-D—glucuronidase but not the purified fraction. Other properties also indicated that this enzyme is not identical to the exoglucuronidases. II. EXPERIMENTAL 1. Apparatus Spectrophotometer. —— Absorbance measurements in the visible range were carried out in the Beckman Model B Spectrophotometer. The cell compartment was replaced with a test tube compartment. Centrifuges. -— The International Centrifuge Model HR-l was used for preparative purposes. The Spinco Model E Analytical Ultracentri- fuge was used to study the sedimentation properties of a purified enzyme preparation. Columns for Fractional Elution of Proteins. -— Columns fitted with fritted glass discs (coarse) or glass-wool plugs and packed with the desired resin were employed in the purification of B-glucuronidase. Zone ElectrOphoresis.—— Experiments on paper (Whatman #1) were carried out in the Paper Electrophoresis Apparatus Type L. K. B. 3276 (Ivan Sorvall, Inc.). The experiments on oxoid cellulose-acetate strips (Consolidated Lab., Inc.) were carried out in the Shandon Elec- trophoretic Cell (Shandon Scientific Co., London). Standardized Test Tubes. -—-Soft—g1ass test tubes were standard- ized by comparing the absorbance reading of an alkaline phenolphthalein solution when transferred from one tube to another. The roundness of the tubes was checked by rotating the tubes in the instrument. 13 lb 2. Materials Snails. -— Aquatic snails, Ampullaria cupina, were obtained from the Streamland Aquarium, Florida. Protein. —— Bovine serum albumin, 3x crystallized, was purchased from Pentex, Inc. Resins. ——-Sephadex G-75 (Lot To 8h92 M) and diethylaminoethyl— sephadex-A50 (Lot To 787h M) were purchased from Pharmacia (Sweden). Diethylaminoethyl—cellulose (Lot 107h18, 0.78 meq. per 9; Lot 500391, 0.62 meq. per g) and diethylaminoethyl-Solk-Floc (Lot 10hl69, 0.50 meq. per g) were obtained from California Corporation for Biochemical Re- search. Chemicals. - p-Chloromercuribenzoate (sodium salt, Lot 102735) was obtained from California Corporation for Biochemical Research. N-Ethyl maleimide (Lot c2282) was obtained from Mann Research Lab. Reagents. -— (a) Folin-Ciocalteu phenol reagent was obtained from the Hartman—Leddon Co. (b) Folin-Ciocalteu alkaline reagent: 73 g of anhydrous sodium carbonate in 1927 m1. of water; 0.6 g of cupric sulfate pentahydrate in l9.h m1. of water; and 1.05 g of NaKtartrate tetrahydrate in 18.9 m1.of water. The sodium carbonate solution is made first and filtered. The cupric sulfate and tartrate solutions are mixed and added to the carbonate solution. (c) 0.h% aqueous naphthoresorcinol solution: The naphthoresor- cinol is pulverized and suspended in water. The suspension is shaken 15 for ten minutes in an amber colored mixing-cylinder. The filtered solution is kept away from light. A fresh solution is prepared every day. Buffers. —— (a) 0.1M phosphate buffer, pH 7.0 -- 28.8 g of sodium monohydrogenphosphate (anhydrous) and 8.16 g of sodium dihydrogen phos- phate monohydrate were dissolved in 1500 ml. of water, the pH was ad- justed if necessary and then it was diluted to 2000 ml. (b) 0.1M acetate buffer, pH h.5 —- 8.2 g of sodium acetate and 7.73 ml. of glacial acetic acid were put in 700 m1. of water, the pH was adjusted if necessary and then it was diluted to 1000 m1. (0) 0.2M glycine—NaOH buffer, pH 10.h -— 15 g of glycine and 11.7 g of sodium chloride were dissolved in 700 m1. of water. The pH was adjusted to 10.h with 10% NaOH solution and the mixture was diluted to 1000 ml. (d) Carbonate buffer, pH 10.1 —- 8.h grams of anhydrous sodium bicarbonate and 36 g of anhydrous sodium carbonate are dissolved in a liter of water. (e) 0.1M phthalate buffers ofvarious pH values (37°C) -— 50 ml. aliquots of 0.2M Kththalate solution were adjusted to the desired pH values with 1M HCl or 1M NaOH solutions at 37° and diluted to 100 ml. (f) 0.1M acetate and citrate-phosphate buffers of various pH values -- The buffers were prepared as described in Methods in Enzymol- ogy, Vol. 1, pages 138-h6. Each buffer solution was checked in the pH meter and if necessary, adjusted to the desired pH with dilute acid or base solutions. 16 Substrates. -— Phenolphthalein mono-B-D-glucuronide (Lot 51 B-690), borneol-B-D-glucuronide (Lot 111 B-801), l-menthol—B-D-glucuronide (Lot 36—h0), and pregnanediol-B—D-glucuronide were purchased from.Sigma Chemical Co. 3. Preparation of_pgNitrgphenyl-B—D-Glucuronide (h7) The method of Kato et al. was slightly modified for this prepara- tion: 8.0 g of methyl(tri-O—acetyl-o-D-glucopyranosyl bromide)—uronate (79) and lh.0 g of p—nitrophenol were dissolved in 100 ml. of acetoni- trile. h.0 g of silver oxide were added and the suspension was shaken overnight. The reaction mixture was filtered. The filtrate was col— lected in 100 m1. of chloroform. The silver salts were extracted with 50 ml. of chloroform and the extract was combined with the chloroform solution containing the bulk of the product. The chloroform solution was extracted three times with water, then three times with 2M KOH solu— tion, and again with water. The mixture was dried over drierite and evaporated under diminished pressure. The residue was crystallized twice from isopropyl alcohol. Yield of methyl(p-nitrophenyl-tri-0- acetyl~B-D—gluc0pyranosid)-uronate: 50%. m.p. 150-529 Methy1(o—nitrophehy1-tri-0-acety1-B-D-glucopyranosid)—uronate was obtained by this procedure from 10 g of methyl (tri-O-acetyl-d-D-gluco- pyranosyl bromide)—uronate. The product was crystallized from acetone. The yield was 70%. m.p. 175-760. 7.32 g of methyl(p-nitrophenyl-tri-0-acetyl-B-D-glucopyranosid)- uronate were dissolved in 50 m1. of 0.1M sodium methoxide solution by stirring with a swirling motion. The solution was allowed to stand at room temperature for 20 hours. The methanol was evaporated under 17 diminished pressure. The residue was taken up in hO m1. of 0.h3N barium hydroxide solution and allowed to stand at room temperature for one hour. Then, cation exchange resin (IR-120) was added and the suspension was shaken until a clear solution of pH about 2.5 was obtained. The suspen— sion was filtered and the filtrate was evaporated under diminished pressure. The solid residue was dissolved in ethyl acetate which pre- viously had been shaken with water in a separatory funnel. Ether was added to the solution until it became permanently cloudy, the flask was stoppered, and allowed to stand at room temperature. After one to three days the compound crystallized. The product (p-nitrophenyl-B-D—glucur- onide) was collected on a filter and dried in a vaccum dessicator over , phosphorus pentoxide. m.p. 95°. Specific rotation at 23°C, D line of Na, was equal to -112° (water, 0.2836). This product corresponds to the monohydrate compound.. Repeated attempts to obtain o-nitr0phenyl—B-D-glucuronide by this procedure were unsuccessful. h. Determination of Protein Folin-Ciocalreu Phenol Reaction (80). -— An aliquot of the protein solution is placed in a soft-glass test tube and the volume is brought to 1.0 ml. with water. 5.0 m1. of the alkaline reagent are added to the tube and this is incubated at 37° for 20 minutes. The phenol rea- gent is diluted (1:2) and 0.5 m1. are added to the tube; the contents are mixed immediately. The absorbance at 560 mu is read after 30 min- utes of standing at room temperature. A bovine serum albumin solution (1 mg. per ml.) was used to prepare standard curves (0.D. at 660 mu versus ug of protein). 18 5. Assay of Enzyme Activity (a) Phenolphthalein mono-B-D—glucuronide. —— The B-D-glucuronidase activity was assayed with this substrate by the method of Fishman et a1. (28) slightly modified. Each determination was run in duplicate with a single control. Controls on the spontaneous hydrolysis of the substrate always showed that the amount of hydrolysis occurring under the condi- tions of the tests was undetectable. The assay mixtures consisting of 0.5 ml. of 0.1M acetate buffer, pH h.5, 0.5 m1. of 0.0015M substrate solution, and 0.5 m1. of enzyme dilution were incubated at 37° for exact periods of time (usually 30 minutes). Bovine serum albumin (100 ug per ml.) was added to the dilutions of highly purified enzyme prepar- ations; this was not necessary with crude preparations. The reaction was stopped by the addition of 5.0 m1. of 0.2M glycine buffer, pH 10.h, and the optical density was read at 5h0 mu with the Beckman Model B spectrophotometer. For the determination of the initial velocity of reaction by this procedure the incubations were done for only several minutes (counted after the first minute in the water bath), and a com- plete assay mixture, incubated for one minute, was used as the blank for the reading of the optical density. In the present work the B—D-glucuronidase activity is expressed in phenolphthalein units. One such unit was defined as the activity which liberates one microgram of phenolphthalein per hour in acetate buffer, pH h.5, at a temperature of 37°, and a 0.0005M substrate concen- tration. This unit is not identical to the Fishman Unit since it is not defined for the pH of optimum enzyme activity. Although the enzyme is more active at pH values below h.5 (Figure 5), the assay of the 19 activity at pH h.5 was convenient because the enzyme has great stability at this pH (Table v11). (b) p-Nitrophenyl-B-D—Glucuronide. -—-The assay procedure used for the phenolphthalein glucuronide was applied to this substrate, but the optical density was read at hOO mu. (c) l-Menthol-, d-Borneol—, and Pregnanediol-B-D—Glucuronides. —— 0.5 m1. of the substrate solution, 1.0 m1. of 0.1M phthalate buffer of the desired pH, and 0.5 m1. of enzyme dilution were mixed. 0.5 ml. of boiled enzyme were added to the controls. The mixtures were incubated (at 37°) for exact periods of time at the end of which the tubes were immersed in boiling water for one minute. The extent of hydrolysis was determined employing the method of Fishman and Green (h6) for determina- tion of free and conjugated glucuronic acid. The method depends on carrying out the naphthoresorcinol reaction before and after the oxida- tion of the free glucuronic acid by hypoiodite to saccharic acid at pH 10.1. The difference in values obtained for glucuronic acid before and after the oxidation procedure gives a measure of the unconjugated glucuronic acid. The assayemixtures were diluted with water so that the concentra- tion of total glucuronic acid (free plus conjugated) was not more than 20 ug per m1. Aliquots of 5.0 ml. were put in 50 m1. erlenmeyer flasks containing 2.05 ml. of carbonate buffer, pH 10.1. 1.5 m1. of 0.1N iodine solution were added, shaken gently, and the flasks were stoppered and allowed to stand in the dark for 30 minutes. At the end of this, 0.15 ml. of 1.0M sodium bisulfite solution were added, the flasks agi- tated, and an addition made of 0.3 m1. of 6N sulfuric acid. Any 20 ‘residual iodine coloration was removed by one additional drop of bisul- fite solution. The flasks were shaken to remove the excess carbon di- oxide from the solution. This mixture yielded the value for glucuronide glucuronic acid. To obtain the figure for total glucuronic acid, another 5.0 m1. of solution was pipetted into a solution containing iodine, bisulfite, and sulfuric acid prepared in the amounts and sequence as before. Four ml. aliquots (in duplicate) were then pipetted into pyrex test tubes (capacity about 50 ml.). To each were added 2.0 ml. of 0.h% naphthoresorcinol solution and 2.0 m1. of 18N sulfuric acid. The contents of the tubes were mixed well, and the tubes, unstoppered, were placed in a boiling water bath for one hour. The tubes, still in the rack, were immersed in cold water. After cooling, 10 ml. of 95% alcohol were added to each tube, the tubes were shaken to dissolve the pigment, and 8 m1. of toluene were added. Cork stoppers were inserted and the tubes were vigorously shaken 100 times to extract the violet pigment into the toluene phase. The aqueous layer was removed by suction with a tube drawn out to a capillary attached to an aspirator and collecting bottle. The toluene extracts were then transferred into standardized soft-glass test tubes. After allowing the extracts to stand in the dark (5 minutes) to permit them to clear, the optical density of each tube was measured at 565 mu (0 optical density with a reagent blank). The reagent blank should not read below 85% transmittance (0 optical density with a tol- uene blank). Calibration Curve: 5.0 ml. of solutions that contain 1.25, 2.5, 5.0, 10, and 16 ug of glucuronic acid per m1., respectively, are pipetted 21 into erlenmeyer flasks that already contain the previously stated amounts of buffer, iodine, bisulfite, and acid (final volume, 9.0 ml.). Four m1. aliquots of the mixtures (in duplicate) are then pipetted for the naphthoresorcinol reaction. The concentration of glucuronic acid in the h.0 m1. aliquots is plotted against optical density to yield a straight line. 6. Purification of the Enzyme Step 1: Extraction.-—- The shells of the snails (killed in batches of 150 to 200), Ampullaria cupina, were removed with scissors. The foot, the reSpiratory system and reproductive tract were cut and discarded. The remainder of the snails, consisting primarily of the hepato-pancreas, intestines and crop, was put in cold 20 per cent saturated ammonium sulfate solution (300 ml.). The cold suspension was homogenized in a waring-blender (1 minute). The homogenate was centrifuged in a refrig- erated International Centrifuge Model HR-l at 11,000 rpm (all preparative centrifugations were done in this machine). After 15 minutes of centri- fugation, the sedimented material was extracted with additional ammonium sulfate solution. The two extracts combined made about 500 ml. of a brown mixture (fractional). The insoluble residue was discarded without further treatment. Step 2: Heat denaturation. -— Wakabayashi and Fishman (hl) 0b— tained the B-D-glucuronidase from Helix pomatia free of sulfatase activity by heat denaturation. By the same procedure, our extract was heated to 70-7h° and kept at that temperature for four minutes. Then, the suspension was cooled in an ice bath. When cold, then suspension was centrifuged at 11,000 rpm for 20 minutes. The sedimented precipitate 22 was extracted once with 20 per cent saturated ammonium slufate solution and then discarded (fraction II). The combined supernatants made about 550 m1. (fraction III). Step 3: Ammonium slufate precipitation. —— Fraction III was ad- justed to 55 per cent salt saturation with solid ammonium sulfate. The suspension was stirred for 30 minutes and then centrifuged at 11,000 rpm for 20 minutes. The supernatant (fraction IV) was discarded. The pre— cipitate (fraction V) was dissolved in 150 m1. of water and the mixture allowed to stand in the cold overnight. The material that sedimented was removed by centrifugation at 16,000 rpm for 30 minutes. The solid (fraction VI) was discarded. The supernatant was dialysed in 20 per cent saturated ammonium slufate solution in the cold room (2 days, 3 changes of salt solution). If more material sedimented, it was removed by centrifugation and discarded. Step h: Fractional precipitation with ammonium sulfate. -— The dialysed mixture (fraction VII) was adjusted to 38 per cent salt satur- ation with solid ammonium sulfate and stirred for 30 minutes. The pre- cipitated protein (fraction VIII) was removed by centrifugation (16,000 rpm) and discarded. The supernatant (fraction IX) was further adjusted to 50 per cent saturation, stirred for 30 minutes and centrifuged at 16,000 rpm for 20 minutes. The supernatant (fraction X) was discarded. The precipitated protein (fraction XI) wasdiluted to 25 m1. and put to dialyse in 0.005M phosphate buffer, pH 7.0. Step 5: Fractional elution from DEAR-cellulose. -— The diethyl- aminoethyl—cellulose (22 g) was suspended in 0.005M phosphate buffer, pH 7.0, stirred several minutes and filtered by suction. The slow sedimenting particles of the resin were discarded by suspending the 23 resin in the buffer, allowing the fast sedimenting particles to settle and decanting the supernatant fluid with the slow sedimenting particles; this was repeated several times. The remaining slurry was put in a filtering flask and the entrained air removed in the water pump. The slurry was packed to a height of about 30 cm. (into a glass cylinder 2.5 x 37 cm.). The column was put in the cold room and 0.005M phos- phate buffer was allowed to flow through it overnight from a 500 ml. capacity separatory funnel used as solvent reservoir. The crude enzyme preparation (usually between 250 and h00 mg. of protein) was placed in the column. Then, the column was washed with 500 m1. of 0.005M phos- phate buffer, pH 7.0, at a rate of 1 ml. per minute and collecting fractions of 10 to 15 ml. When the washing elution was completed, the buffer was changed to 0.01M phosphate buffer, pH 7.0, and the column was eluted with 250 m1. of the buffer. The fractions containing the bulk of the activity were combined (usually made 25 to hO ml.). Step 6: Ammonium sulfate precipitation. -— The DEAE-cellulose eluate was adjusted to 60 per cent salt saturation with ammonium sul- fate, stirred for 30 minutes, and centrifuged at 17,000 rpm. The supernatant was discarded. The precipitate was dissolved in a few ml. of water and centrifuged at 17,000 rpm for 15 minutes. The insoluble matter was discarded. The supernatant was adjusted to 60 per cent salt saturation with ammonium sulfate and stored in the refrigerator. 7. Zone Electrophoresis The homogeneity of the most purified enzyme fraction (specific activity 168,000) was tested by paper electrophoresis (81, 82). The Whatman #1 filter paper strips (2.5 x L7 cm.) were saturated with the desired buffer by passing them through the buffer; each strip was 2h blotted with another dry strip to remove excess buffer. Twenty to 30 ul. of the enzyme solution (1.16 mg per ml.) were applied on the paper either directly with a small pipette or the sample was first transferred from the pipette to the sample applicator (SA 3276). Either way of application produced a narrow band of the sample on the mid- point of the strip. The strips were placed in the apparatus (L.K.B. 3276) located in the cold room and a field of 270 volts (10 we.) was applied for the desired period of time. Experiments were carried out at pH 3.0 (0.1M glycine-RC1) and pH 3.8 (0.1M acetate) for h.5 hours; pH h.5 (0.1M acetate) for 9.5 hours; pH 7.5 (0.1M phosphate) for 9.0 hours; pH 9.0 (0.1M veronal) for 12 and 2h hours; and pH 10.0 (0.1M phosphate-borate) for 9.0 hours. The experiments on cellulose—acetate matrix were run for h5 minutes at pH 8.6 (0.05M veronal) and pH 8.6- 9.1 (discontinuous buffer) under a field of 275 volts (5 ma.). Paper strips containing a sample of 1% starch solution were used to establish the extent of migration of solvent in the L.K.B. apparatus. When the electrophoretic run was complete, the paper strips were hung to dry in the air. When dry, the strips were immersed in a brom- phenol blue solution (1 g of the indicator per liter of ethanol satur- ated with mercuric chloride). The strips were washed with 1% acetic acid solution and the dye was fixed by immersing in an acetate buffer (50 ml. of glacial acetic acid and h.0 g of sodium acetate in 1 liter of water) and drying the strips in an oven at 120°. The cellulose-acetate strips were stained with light green SF dye and fixed with dilute trichloroacetic acid. 25 8. Analytical Ultracentrifugation TwO purified enzyme preparations were combined and adjusted to 60 per cent salt saturation with ammonium sulfate. The cloudy sus- pension was centrifuged at 16,000 rpm and the supernatant was discarded. The precipitate was taken in 0.5 m1. of water and the mixture was centrifuged again at 16,000 rpm to sediment the insoluble matter. The supernatant was decanted into a cellophane tubing. The insoluble mat- ter was suspended in 0.3 m1. of water, the suspension was centrifuged, and the clear supernatant was added to the cellophane tubing. Then, the enzyme preparation was dialysed in 0.1M sodium chloride solution (2 liters) for 6 hours. The resulting enzyme solution contained 5.93 mg of protein per m1. (specific activity 153,000). This enzyme preparation was examined in the Spinco Model E analyt- ical centrifuge (83). The sedimentation velocity experiment was run at h2,0h0 rpm, and photographs of the sedimenting boundaries were taken at intervals of 8 minutes. For the determination of the molecular weight of the major component by the Archibald method, the enzyme preparation was subjected to 10,589 rpm and photographs of the resulting boundary were taken at intervals of 16 minutes (0 = 750). The enzyme solution was transferred to the synthetic boundary type cell, the synthetic boundary was photographed, and then, it was subjected to 59,780 rpm (the sedimentation pattern photographed at intervals of 8 minutes). Measurements on the sedimentation patterns were carried out with a microcomparator. 9. Stability of Enzyme vs. pH One ml. of enzyme solution (5,h00 phenolphthalein units per m1., 26 Spec. act. 108,000) was diluted to 5.0 ml. with water. Two tenths m1. (of this dilution were added to 0.2 ml. of the appropriate buffer: pH 2.2 and 3.0 (0.1M glycine-HCl); pH 3.8, h.3, h.9, and 5.7 (0.1M acetate); pH 8.1 and 9.1 (0.1M triS-HCl); and pH 10.h (0.1M glycine—NaOH). The mixtures were allowed to stand at room temperature (23°) for four hours at the end of which the pH of the mixtures was readjusted to pH 3.8 by adding 3.6 m1. of 0.1M acetate buffer. Five tenths m1. of phenol- phthalein glucuronide solution were added to 1.0 m1. aliquots of the resulting mixtures, and the hydrolysis was allowed to proceed for 30 minutes at 37°. The determinations were carried out also with an enzyme dilution to which bovine serum albumin was added (100 pg per ml.) 10. Effect of Temperature An assay mixture consisting of 0.5 m1. of 0.1M acetate buffer, pH h.5; 0.5 ml. of 0.0015M phenolphthalein glucuronide solution; 0.h m1. of water; and 0.1 ml. of enzyme dilution (0.006 mg of protein per m1. specific activity 80,000) was incubated for 30 minutes at tempera- .9 tures of 10°, 25°, and 37°. The assay mixture was incubated for only 5 minutes at h7°. 11. Inhibition by Cations A purified enzyme solution (0.76 mg per m1., Spec. act. 112,000) was diluted 1:250 for the experiments. No albumin was added to this dilution. The assay mixtures consisted of 0.3 ml. of serial dilutions of 0.01M cation solution; 0.9 ml. of 0,1M.acetate buffer, pH h.5; 0.2 m1. of the enzyme dilution; and 0.1 m1. of 0.0075M phenolphthalein glu- curonide solution. The mixtures were incubated at 370 for 30 minutes. 27 The effect of silver, cupric, mercuric, calcium, magnesium, manganese and zinc ions was investigated. 12. Inhibition by Sulfhydryl-Group Reagents (a) N-Ethyl maleimide. —— A 0.02M solution of this inhibitor was prepared in 0.1M acetate buffer, pH h.5. Aliquots of 0, 0.075, 0.30, and 0.75 ml. of this solution were placed in test tubes and the volumes were brought to 1.2 ml. with additional acetate buffer. Two tenths m1. of the enzyme dilution (3 ug of protein per m1., Spec. act. 112,000) were added to each test tube and the tubes were allowed to stand at room temperature for 90 minutes. Then, 0.1 m1. of 0.0075M phenolphtha— lein glucuronide solution was added and the mixtures were incubated for 30 minutes at 37°. (b) p-Hydroxy mercurihenzoate. -—.A 0.0001M solution of this substance was prepared in 0.1M acetate buffer, pH h.5. Aliquots of 0, 0.15, 0.30, 0.75, and 1.2 m1. of this solution were placed in test tubes and the volumes adjusted to 1.2 ml. with additional acetate buffer. Two tenths ml. of the enzyme dilution (same as in a) were put in each test tube and the mixtures were allowed to stand at room temperature for 30 minutes. Then, 0.1 m1. of 0.0075M phenolphthalein glucuronide solution was added and the mixtures were incubated for 30 minutes at 37°. To check the effect of this agent at pH 8.1, a 0.0001M solution was prepared in 0.1M tris-HCl buffer, pH 8.1. Two tenths m1. of this solution were added to 0.2 ml. of enzyme dilution (1,080 units per m1., spec. act. 108,000) and the mixtures were allowed to stand 30 minutes at room temperature. At the end of the 30 minutes, 3.6 m1. of 0.1M 28 acetate buffer, pH 3.8, were added to each test tube. One ml. aliquots of the resulting mixtures, combined with 0.5 ml. of 0.0015M phenolphtha- lein glucuronide solution, were incubated for 30 minutes at 37°. III. RESULTS AND DISCUSSION 1. Purification of the Enzyme Initially, the snails were killed and the digestive tract was con— verted to acetone-powder. The acetone-powder retained full activity of the enzyme and this could be extracted by suspending the powder in 20 per cent saturated ammonium sulfate. The preparation of acetone- powder was discontinued because during the fractional elution of the enzyme from DEAE—cellulose two consecutive fractions were obtained with identical Specific activity; this did not happen when the digestive tracts of the snails were homogenized in dilute ammonium sulfate solu- tion. Otherwise, steps 1-5 of the procedure, when starting with ace- tone-powder, yielded the'same degree of purification as when starting with the homogenate of fresh tissues. It was found convenient, in order to work with a cleaner extract, always to centrifuge the homogenate, remove the black insoluble matter, and then heat the extract to 70-7h°. Nevertheless, heating of the homogenate gave the same results as far as increasing the specific activity was concerned. The heat denaturation step always yielded very good recovery of the glucuronidase activity (Table I). The ex- tract, after the heat denaturation step, retained a deep red—brown color. The bulk of the colored material was removed in Step 3 when the proteins were precipitated with ammonium sulfate and the colored supernatant was discarded. 29 30 Table I. Effect of heating the enzyme extract at 70-7h° Number of Spec. Act. of Extract Per cent Snails Killed Before Heating After Heating Activity 200* 300 600 ’ 87 81 360 1,030 85 150 N30 1,100 89 115 5h0 1,260 91 lho too 933 91 200 220 580 81 200 220 590 80 200 200 618 7h 185 220 700 86 105** 200 730 99 500% 2A2 750 95 .‘L I\ Enzyme was extracted from the acetone—powder of snails. 7%The denatured proteins were centrifuged out and extracted once be—— fore discarded. Application of steps 1-h of the procedure gave about 12-f01d purification of the material in the first extract (Table II). The values obtained with one of the batches of snails suggest that the snails in different batches may show a great difference in their enzyme content. The purification of the enzyme from acetone-powder was included in the table for comparison. The crude enzyme preparations destroyed the ce110phane tubing during pervaporation and during dialysis in phosphate buffer, pH 7.0. However, it was found safe to dialyse these preparations in ammonium [I'llllllllllllllllllllil'l fl Illl‘lrll ll.‘ ll 31 Table II. Extent of enzyme purification given by steps 1-h. Snails in Batch mg Prot. spec. Act. Fold-Purif. 7:71372; 200* 3110 11,500 15 69 h86 1,h78 3,150 7.6 63 785 2,365 2,610 12 53 105 625 2,560 13 75 500 1,000 2,h82 11 57 7Enzyme was extracted from the acetone-powder of snails. Table III. Fractional elution of the enzyme from DEAE-resins. Resin7 Fraction Placed in Column DEAE-Eluate mg Prot. Spec. Act. Spec. Act. Per cent Act. h g of (a) 130 2,600 71,000 23 22 g of (b) 620 3,200 110,000 16 22 g of (b) h50 2,600 91,000 12 22 g of (b) h50 2,360 91,000 17 22 g of (b) — h50 2,570 87,000 12 22 g of (c) 570 2,870 h7,000** 23 22 g of (c) 312 2,560 60,000** 32 22 g of (c) 500 2,h80 73,000 13 22 g of (c) 250 2,b80 100,000 21 22 g of (c) 250 2,h80 95,000 2h Resins: (a) DEAE-Sephadex A50 (3.9 meq/g); (b) DEAE-cellulose (0.78 meq/g); (c) DEAR-cellulose (062 meq/g). The enzyme was eluted during the washing of the column with 0.005M buffer. 32 sulfate solutions. This effect was attributed to the presence of cellu- lose activity in the crude preparations. When it was necessary to dialyse crude fractions in 0.005M phosphate buffer, the ce110phane tubings were changed at intervals of about two hours. The fractional elution of the enzyme from DEAE-cellulose gave about 35—fold purification of the crude fractions placed in the column (Table III). Usually, very little of the glucuronidase was eluted with the 0.005M buffer, but when the columns were overloaded the bulk of the activity was eluted during the washing-elution. The elution with 0.01M buffer gave a protein peak coinciding with a sharp peak of enzyme activity (Figure 1). The fractions corresponding to both sides of the peak were worth saving since they contained considerable amounts of the enzyme, although of less purity than the fractions corresponding to the top of the peak. Re—elution of these fractions from DEAE—cellu- lose usually yielded eluates of high specific activity. DEAE-cellulose (0.78 meq/g) was the resin most effective for the purification of the enzyme; DEAE-cellulose (0.62 meq/g) was effective when the column was charged with small crude enzyme fractions. DEAE-Solk Floc and DEAE-Seph- adex A50 were even less effective, and Sephadex G-75 gave no purifica- tion of the crude fractions at all. Precipitation of the DEAE-eluates with ammonium sulfate, besides concentrating the proteins, always led to an increase in the purity of the enzyme preparation (Table IV). The increase in specific activity given by Step 6 could also be obtained by placing the eluates in a DEAE—cellulose column for a second elution. However, the ammonium sul— fate precipitation gave a much better recovery of the activity. Prepar- ations with Specific activity higher than 110,000 were not affected by the ammonium sulfate precipitation. 33 . ' Mg of Prot. per ml O - N U «b O I r r I. r r ‘ 9 O 0 an or =- tn 6 'fi _.- I 2 a- i l o o s 2 3; .°. 3 £- 0! .. o O . o O a... E. -|. 3 Cor .- CT .‘° -<-—- O E. 9 30" ‘7 C7 (D :r. q 0 .J C) " *— a 34 - N l I: 07 o 7 0. 1 l ‘1 l I, I A a) "’ -' N to N 0 o No , Units per ml x-IO‘3 Figure 1. Fractional elution of B-Deglucuronidase from DEAE-cellu» lose (0.78 meq per g). .A fraction of 620 mg of protein (apec. Act. 3,200) was placed in the column. 3h Table IV. Effect of precipitating the DEAE-eluates with ammonium sulfate DEAE—Eluate Water-Soluble Precipitate Per cent mg Prot. Spec. Act. mg Prot. Spec. Act. Activity 9.90 55,000 h.90 100,000 90 8.10 86,000 5.35 122,000 9h 15.8 h2,000 10.1 57,000 88 7.56 5A,000 5.00 77,000 9S l.h6 l2h,000 1.28 123,000 90 22.2 113,000 18.7 11h,000 85 6.9h 92,000 h.50 118,000 83 Table V summarizes a typical purification procedure. The whole procedure gave a 500-fold purification of the material in the first extract. The purified preparation was colorless and amorphous. It was free of arylsulfatase activity as evidenced by the fact that 9 ug of protein from the enzyme preparation did not cause hydrolysis of nitrochatecol sulfate and p-nitrophenol sulfate after one hour at 37° and pH 5.5 (8h). The preparation was probably free of cellulase activ- ity also. I An attempt to crystallize the enzyme was made by subjecting a combination of various purified preparations (18.7 mg prot. in 5.5 ml.; Spec. act. 11h,000) to slowly increasing ammonium sulfate concentra- tion. By this slow increasing of salt concentration, the preparation required less than 35 per cent saturation to precipitate. The protein separated as a white, floculent precipitate. The suspension was cen- trifuged at 7,000 rpm to sediment the precipitate. It appeared amorphous under the microscope, but it exhibited the same specific activity as 35 Table V. Purification of B-D-glucuronidase from the aquatic snail, Ampullaria cgpina. Step Fract. mg Prot. Enz. Act. Spec. Act. YiZId ASE? l I 18,800 h,52h,000 2h2 100 2 III 5,720 h,298,000 750 95 3 VII 2,0h0 3,908,000 1,920 86 h ‘ XI‘ 1,000 2,h82,000 2,h80 57 5 DEAE-eluate 6.9h 639,000 92,000 1h.l 6 Sol. ppt. h.50 530,000 118,000 11.7 500 snails (Mainly small) 36 did the preparation placed in the cellophane tubing. The supernatant showed a specific activity of only 37,000. The ratio of the absorb- ance at 280 mu to the absorbance at 260 mu for the precipitated protein dissolved in water (50 ug/ml.) was 1.25. This was a rather low value for a protein and suggested the presence of contaminating materials in the preparation. The precipitate was redissolved in water and centri- fuged to remove the insoluble material. For another four times, the enzyme preparation was subjected to increasing salt concentrations. The rate of addition of the salt solution was made slower every time; the last addition running for nearly two weeks. Every time a precip- itate appeared, samples were observed in the microscope and they were found to be amorphous. The precipitates were redissolved by dialysis in distilled water and any insoluble material in the preparation was removed by centrifugation. These five precipitations did not cause the crystallization of the enzyme, but they improved the purity of file enzyme since after the treatment the specific activity of the prepara- tion was 168,000. The activity of this individual preparation was higher than any previously reported for non-mammalian preparations (3, 39, hO) and second only to the rat preputial gland preparation (38). In phthalate buffer, pH 3.1, the activity of this preparation was h20,000 Fishman Units per mg. of protein (Figure 5). 2. Test of Homogeneity (Zone Electr0phoresis) The most active fraction obtained (specific activity 168,000) was found electrophoretically homogeneous on paper and on cellulose-acetate. The enzyme was not stable at pH values below h.5, but it was observed that the migration was toward the cathode. The enzyme appeared to have 37 the isoelectric point near pH h.5. The migration on paper as a Single spot, at different pH values, was as shown in Table VI. Table VI. Paper electrophoresis in L.K.B. apparatus. Distance Moved Buffer, pH Time (hrs.) Toward Anode 0.1M acetate, pH h.5 9.5 0.0 cm. 0.1M phosphate, pH 7.5 9.0 5.8 cm. 0.1M veronal, pH 9.0 12.0 5.6 cm. 0.1M veronal, pH 9.0 2h.0 9.0 cm. 0.1M phos.-borate, pH 10 20.0 7.5 cm. 3. Analytical Ultracentrifugation Since the Archibald method of molecular weight determination as well as the determination of the sedimentation coefficient do not re— quire a homogeneous preparation, the enzyme solution with specific activity of 153,000 was sedimented in the analytical ultracentrifuge with the purpose of approximating these physical properties and also establishing the relative composition of the preparation. When the preparation was subjected to h2,0h0 or 59,780 rpm, it gave a sharp, symmetrical peak which was preceded and followed by very small peaks. Figure 2 shows the synthetic boundary (zero time) and the sedimentation pattern obtained at 59,780 rpm. The sedimentation coefficient of the major peak (8203w) was calculated to be 11 S. The value of the coeffic- ient did not change with time for the entire run (72 minutes at h2,0h0 rpm). The area of the sedimentation pattern, corrected for radial dilution (85), for the major peak at top speed was accounted for as .menspofim wepscfia mm Adv one «mepaafia 4N AUW nwopnafia 0H “0V ameuscfis w Anv mesa» open an unmucvon emponpahm Amv .Aenn oww mmv poomm mop pm «mammupaoomupab Hmoprqmd< one a“ ooonmmfi ho aUH>Hpom ofimgoumm no“: :oHpmpmmenm eesuco am no oofipmpaeafiuom .m shaman 39 80 per cent of the synthetic boundary at zero time and at top speed (initial area). This was indication that the major component represented 80 per cent of the material in the enzyme preparation. This result suggests that the limiting value of the specific activity of the enzyme is 190,000 at pH h.5 (acetatebuffer). During the approach to equilib- rium experiment the preparation gave a boundary pattern which after h8 minutes began to look like a peak (Figure 3). Measurements for the ‘computation of the molecular weight were taken from the 32, 6h, and 112-minute photographs. Using the density of the sodium chloride solu- tion (1.0025) and assuming a value for the specific volume of the pro- tein (0.7h CC/g), the molecular weight was calculated to be h07,000 i 10,000. A molecular weight as high as this supports the proposal that the inactivation of purified mammalian glucuronidase preparations on dilution is due to dissociation into inactive components (57). h. Stability as Function of pH Table VII. Effect of pH on the stability of the enzyme. __ Per cent Recovety pH 0B3.Abq%L KDugBSJflhfim. 2.2 0 7 0 3.0 3 h? 3.8 8 86 h.9 31 92 5.7 A? 89 8.1 55 76 9.1 58 8h 10.h 55 80 hO .zumuason vaponpahm Amy new AHV «megapofia amepncfie me Anv new amopscaa NHH Amv «mouzawa om Amy amopscfis Om .Emp mam E E 3 3 on «mopscfia 40 “UV «mooscfia m4 Auv «mopvcfia mm Anv .mopsnme 0H Amy OH pm Aoooqmmfi >HH>Hoom uHmHoeamv coHpmumnoem cashew opp mo cofipmucosfipom .m shaman b1 These results Show that the enzyme from A. pppipg resembles prep- arations from.P. vulgata (51) and L. littorea (52) in stability when albumin is added to the dilutions. The activating effect by albumin is probably attributable to a stabilizing action. P. Bernfeld pp EI’ (57) studied the inactivation of mammalian preparations on dilution and explained the phenomena as dissociation of the enzymic protein into inactive components. They explained the activation by albumin, deoxy- ribonucleic acid and other agents as a prevention of such dissociation. At 37°, the A. cupina enzyme was stable only at pH values above h.5. 5. Effect of Enzyme Concentration The hydrolysis of phenolphthalein glucuronide varied linearly with the amount of enzyme present in the assay mixture (Table VIII). Table VIII. Effect of enzyme concentration ug Phenolphthalein U9 E. Protu/mlxture Prod. per hour 0.30 22 0.h8 36 0.60 uh 0.78 57 0.90 66 1.20 88 Assays at pH h.5 (actate). B.S.A1bumin in E. dilution: 100 ug/ml. Specific activity of enzyme prep.: 80,000. 6. Time Course for the Hydrolysis of various Substrates The enzymic hydrolysis of phenolphthalein—, 1-menthy1, pregnane— diol, and p-nitrophenyl B—D- glucuronides as function of time is shown h2 in Figure h. Without apparent reason, the enzyme preparation failed to hydrolyse the glycosidic bond of borneol B-D—glucuronide. It was ob- served, however, that when this substrate was put in solution efferves— cent occurred; this did not happen with any of the other substrates. The enzyme dilution was sufficiently strong for complete hydrolysis of phenolphthalein glucuronide in less than h5 minutes. After 90 minutes, the hydrolysis of pregnanediol glucuronide was nearly complete. Menthyl glucuronide was 61 per cent and p-nitrophenyl glucuronide 78 per cent hydrolysed after two hours of incubation. The hydrolysis of pregnane- diol and phenolphthalein glucuronides appeared to be linear with time to nearly completion of reaction, but not the hydrolysis of the other two substrates. The little specificity exhibited by the present glu- curonidase preparation towards the aglycon part of the substrate, is the usual behaviour of B-glucuronidases regardless of the source. 7. Effect of Temperature on the Enzyme Activity Table IX. Effect of temperature on the B-glucuronidase activity. Temperature (°C) Velocity of Hydrolysis 10 0.29 25 1.6 37 h.h AB 9.b velocity in u moles of S. split per min. per mg of protein. From these results it was calculated that the energy of activation for the enzymic hydrolysis of phenolphthalein glucuronide is -16,000 cal. AB ‘l 45 l ' _ O 'umoles Subst. Decomp, . 19' I o o O O O’ . l o ' so 60' _ 90 :20 Minutes Figure h. Tlme course for the hydrolysis of several substrates. All‘ _ 'determinations were done with 0.1M phthalate buffer. An enzyme preparation with Spec. act. of 138,000 was diluted 'to 1.23 ug of protein per m1. and 0.5 m1 of this dilution used for the determinations. (a) 0.0005M phenolphthalein glucuronide at pH 3.6; (b) 0.0005M pregnanediol glucuronide at pH b.53 (c) 0.0033M 1-menthy1 glucuronide at pH h.2; and (d) 0.0029M p—nitrophehyl glucuronide at pH 3.9. Ah per mole. This result is in the range of other values reported for mammalian (35, 5h) or non-mammalian preparations (55). 8. Effect of pH (and Buffers) on the Enzyme Activity Previous preparations of the B-glucuronidases from other sources have been always reported to have an optimum pH for activity (3, h). Figure 5 shows the effect of pH on the velocity of hydrolysis, catalyzed by the enzyme from.A. cupina, of phenolphthalein glucuronide in acetate, citrate-phosphate, and phthalate buffers. The enzyme exhibits a sig— moid pH—velocity curve in which the activity approaches a maximum with decreasing pH. The activity was affected by the nature of the buffer- ing ions, being equal in acetate and citrate-phosphate buffers but nearly twice in phthalate buffer at very acidic pH values. At pH values above 5.0 the activity appeared to be equal in acetate and phthalate buffers; it was rather smaller in citrate-phosphate buffer. When the action of an enzyme is studied, it is always assumed that the enzyme combines with its substrate to form an unstable complex which then may break down to products. If the maximum celocity varies with pH, this indicates that the enzyme-substrate complex ionizes or that its breakdown is subjected to acid or base catalysis. If a bell- shaped pH-velocity curve is obtained experimentally, it is assumed that there are two groups in the enzymatic site which have a total effect on the kinetics (86). The bell-Shaped pH-velocity curve is considered to be a combination of the dissociation curves of the groups which may be of either the acidic or the basic types. If the pK values of the groups are sufficiently apart, these can be read from the inflection points on both Sides of the bell-shaped curve. 115 I l l l l I . i 0 l ' 20" r - C 0 I6—- _ - w . . . l2r- o .. V O O O 3 . x o 0 . o 8- .. 7. 1 . x 4!- . x .- O L l L l l 1 0 3.0 as 4.0 4.5 5.0 ,. 5.5 DH Figure 5. Effect of pH (and buffers) on the enzyme activity. 0.0005M phenolphthalein glucuronide hydrolyzed by the enzyme prepar- ation with Specific-activity of 168,000. 0' , with 0.1M phthalate buffer; X,7with 0.1M acetate buffer; and O , with 0.1M citrate-phpSphate buffer. A6 The velocity of the enzyme catalyzed hydrolysis of the phenol- phthalein, l-menthyl, and p-nitrophehyl B—D-glucuronides was determined at various pH values in phthalate buffer. The results, as shown in Figure 6, were in agreement with the fact that the nature of the aglycon has little effect on the ionization of the glucuronides (A9). The pH— velocity curve for the hydrolysis of glucuronides exhibits the shape of the dissociation curve for a group with an approximate pK of h.7. These results suggest that a carboxyl group of the enzyme is involved in the catalytic process that Splits the glucuronosyl-0 bond. 9. Effect of Substrate Concentration The initial velocity of the hydrolytic reaction was determined for various concentrations of phenolphthalein glucuronide and p-nitro- phenyl glucuronide. The velocity was determined from the amount of aglycon liberated in l to 3 minutes of incubation at 37°. The rate of hydrolysis of p-nitrophenyl glucuronide was maximum at 0.003M concentra- tion of the substrate. For phenolphthalein glucuronide, the rate was maximum at 0.0005M substrate concentration, and it was inhibited by higher concentrations. The determinations were made in acetate and phthalate buffers for the phenolphthalein glucuronide and in phthalate buffer only for p-nitrophenyl glucuronide. The results were analysed by the graphical method of Lineweaver and Burk (87). The effect of phenolphthalein glucuronide concentra- tion on the enzyme activity is shown in Figure 7; the Michaelis constant was calculated to be h.6 uM at pH 3.h (phthalate). In acetate buffer, at pH 3.6, the value was 11 uM. Figure 8 shows that effect of p-nitro— phenyl glucuronide concentration on the enzyme activity at pH 3.6 (phthalate). The Michaelis constant was calculated to be 60 uM. A7 T 28 24" 20- 3.5 4.0 4.5 5.0 5. 5 6.0 p H 1.. Figure 6. Effect 'of pH on the enzymic fwdrolysis of various substrates. A11 determinations were done with 0.1M phthalate buffer. The enzyme preparation with Specific activity of 138,000 was di- luted to 2.5 Hg 01‘ protein per ml. 0 ', 0.0033M l-menthyl glucuronide;~ O , 0.0029Mp-nitropheny1 glucuronide; and G , 0 .OOOhM pheno lphtha 1e in g lucuroni de . 118 Figure 7. Effect of phenolphthalein glucuronide concentration on the enzyme preparation with specific activity of 168,000 was diluted to 0.58 ug of protein per. m1. K was calculated to be 11.6 M. m ' . A9 1.0- 7 0‘ 0.8- _ 0.6 - ' ' 7 “’9 )C m] > 0.4» . ' 7 Figure 8. Effect of p-nitrophehyl glucuronide concentration on the enzyme activity at pH 3.6 (phthalate). Enzyme preparation with Specific activity of 138,000 diluted to 2.5 pg of protein per m1. Km was calculated to be 60 uM. 0.2 - . , . - 3.8 x 106 50 Dixon (88) has studied the influence of pH on the affinities of enzymes for their substrates. It was found that a plot of me (nega- tive logarithm of the Michaelis constant) versus pH would give valuable information of three kinds: (a) on the nature of the enzyme—substrate link, deduced mainly from the slope of the curve; (b) on the nature of the substrate—binding groups of the enzyme, deduced from the pH values as determined from the position of the dis- continuities of the curve; caution needs to be exercised here (89); (c) on the nature of the activation process, deduced from the ion- izations of the enzyme-substrate complex. It was of interest to find out how much information could provide the effect of pH on the Michaelis constant for the glucuronidase catalyzed hydrolysis of phenolphthalein and p—nitrophenyl glucuronides. The variation of enzyme activity with substrate concentration at various pH values was determined for phenolphthalein and p-nitr0phehyl glucuronides. The Michaelis constant for phenolphthalein glucuronide varied with pH (phthalate buffer) from h.6 uM at pH values below h.0 to 17 uM at pH values above 5.0, and the variation was not linear. In acetate buffer, the variation was from 11 uM to 20 uM. However, the constant for p—nitrophenyl glucuronide was independent of the pH. Plots of me versus pH are shown in Figure 9. The bending in the curve about pH h.0 (concave downward) would be due to either a group in the free enzyme or a group in the substrate. Since this bending did not appear with p-nitrophenyl glucuronide as the substrate, it is concluded that it is due to a group in the substrate (probably the carboxyl group in the phenolphthalein moiety). The fact that the Km for p-nitrophenyl 51 5.3 - 4.9 - 4.7 b L l l l l l I Figure 9. .3.0 . 3.5 _ 4.0 4.5 5.0 5.5 pH} Effect of pH on the K for the B—D—glucuronidase catalyzed hydrolysis of pheho'lpl'ithalein glucuronide. ,Enzyme prepara- tion with Specific activity of 168,000. 0 , determinations made in phthalate buffer; 0 , determinations made in acetate buffer. 52 glucuronide does not depend on the pH would be explained either by a non-ionic interaction of substrate and enzyme or by an ionic one. AS- suming that the binding is by ionic interaction, the groups involved probably would be an ammonium group in the enzyme and the carboxyl group of the substrate. Interaction between these groups should be pH-independent in the pH—region studied. It is possible that when phenolphthalein glucuronide is the substrate, a secondary interaction of the aglycon with the enzyme causes the Km to be pH-dependent. Linear variation of me with pH (pH 3.0 to 5.5), with slope equal to -2.h, was observed for the enzyme preparation from the limpet Cellana tramoserica (56). Phenolphthalein glucuronide was the sub- strate. 10. Inhibition The effects of Silver, cupric and mercuric ions on the enzyme activity is shown in Table X. Mercuric ion was inhibitory at all con— centrations while cupric and silver ions required concentrations higher than 2 x 1074M for effective inhibition. Low cupric and silver ions concentrations caused slight activation. A Similar behaviour was re- ported for a mammalian preparation (63). Calcium, magnesium, mangan- ese, and zinc ions had no effect on the enzyme activity. Dialysis in 0.01M versene solution caused no inhibition. The sulfhydryl group reagents N-ethyl maleimide and p-chloromer- curibenzoate failed to inhibit the enzyme; N-ethyl maleimide at pH h.5 and p—chloromercuribenzoate at pH h.5 and pH 8.0. These results appear to rule out the possibility of a sulfhydryl group being connected with the enzyme activity. 53 Table X. Inhibition hy heavy cations. Ion Conc. (M) NgrfigfIzonpercfigggcper mlosgivgil. O 306 306 306 2 x 10-7 29b 323 323 2 x 10‘6 257 323 350 2 x 10‘5 59 323 370 2 x 10-4 6 258 276 IV. SUMMARY 1. A ndw B-D-glucuronidase of high activity has been prepared from the digestive tract of the aquatic snail, Ampullaria cupina, by a Simple procedure. This procedure involved (1) extraction with 20% saturated ammonium sulfate., (2) heat denaturation, (3) precipitation of proteins with 55% saturated ammonium sulfate, (h) fractional pre- cipitation with ammonium sulfate, (5) fractional elution from DEAE— cellulose, and (6) ammonium sulfate precipitation. The procedure gave a 500-fold purification of the material in the first extract. The final product was colorless and amorphous. It was free of arylsulfa- tase activity. The specific activity of the product was 120,000. 2. After several reprecipitations with ammonium sulfate, intended to crystallize the enzyme, the preparation was still amorphous but its specific activity increased by h0%. This highly active fraction was electrophoretically homogeneous. 3. Data obtained in the analytical ultracentrifuge at top speed indicated that the limiting value for the specific activity of the enzyme is 190,000. The sedimentation coefficient of the enzyme was calculated to be 11 S and the molecular weight as h07,000. h. The diluted purified enzyme was stable between pH h.0 and pH 10.h if albumin was added to the enzyme dilutions. 5. The enzyme activity increased 2.3-fold for every ten degrees increase in temperature. 6. The velocity of hydrolysis, at pH 3.5 (phthalate), of pregnane- diol, phenolphthalein, l-menthyl, and p—nitropehnyl glucuronides was 5h 55 7.5, 17, 30 and 30 u moles of substrate decomposed per min. per mg of protein respectively when they were hydrolyzed by a preparation with a specific activity of 138,000. 7. The enzyme exhibited a sigmoid pH—activity curve with in- flection point about pH h.7. The activity of the enzyme in acetate buffer and citrate-phosphate buffer was about 55% of the activity in phthalate buffer. 8. The Michaelis constant for phenolphthalein glucuronide showed variation with pH; the constant for p-nitrophenyl glucuronide was pH independent. 9. The enzyme was not inhibited by sulfhydryl group reagents. Mercuric, Silver and cupric ions inhibited the enzyme activity, but only mercuric ion did it markedly. V. APPENDIX 1. Calculations for the Analytical Ultracentrifugation (a) Calculation of the sedimentation coefficient. The sedimen— tation experiment ran for 72 minutes at 52,050 rpm. Photographs of the sedimentation pattern were taken every eight minutes (0 = 700). 2.303 XM/Xm = —— log —._._._ wZ S t w2 = 19.381379 x 106 XM = distance from axis of rotation to maximum ordinate of sedimentation pattern Xm = distance from axis of rotation to meniscus Distance from axis of rotation to reference hole in the rotor = 120.289. Data and Results: Time (Sec.) DM Dm XM XMfi/Xm S 960 10.112 7.562 130.502 1.02075 11.015 . 1,550 11.538 7.555 131.728 1.0311 10.972 1,920 12.806 7 562 133.096 1.0518 11.008 2,500 15.185 7.553 135.575 1.0526 11.023 2,880 15.565 7.560 135.855 1.0635 11.008 3,360 16.972 7.565 137.262 1.0755 11.035 3,850 18.392 7.562 138.682 1.0855 11.023 5,320 19.835 7.562 150.125 1.0969 11.015 D - distance from reference hole to maximum ordinate of the sedimenta- tion pattern. Dm - distance from reference hole to meniscus. Xm - 120.289 + 7.561 = 127.75. 56 56a .mooscas «a Aha use .ao lav .om are .m: Asa .oa Awe .nm Act .an on .oa on .e Ana .a one we cofipmcfiauopou on» mom a wmv pm cexmp mnmmumoponm .pcefiufimmeoo soundpcesficem ozonm: pm Compmpmaopm weaves on» he coHpmoeoEHoom AH .oH nausea (b) ' concentration (synthetic boundary at low speed; 0 = 75°). 57 Calculation of the molecular weight. _ dx 7° 7 2.103 EZYn Determination of initial Data: n Rn(cm) Yn(cm) n Rn(cm) Yn(cm) 1 1.25 0.0570 18 1.52 1.3280 2 1.26 0.0550 19 1.53 1.2532 3 1.27 0.0705 20 1.55 1.1065 5 1.28 0.1015 21 1.55 0.9512 5 1.29 0.1395 22 1.56 0.8200 6 1.30 0.1938 23 1.57 0.6678 7 1.31 0.2588 25 1.58 0.5238 8 1.32 0.3580 25 1.59 0.5118 9 1.33 0.5592 26 1.50 0.3360 10 1.35 0.5625 27 1.51 0.2590 11 1.35 0.6850 28 1.52 0.1990 12 1.36 0.7878 29 1.53 0.1356 13 1.37 0.9050 30 1.55 0.0860 15 1.38 1.0152 31 1.55 0.0620 15 1.39 1.1060 32 1.56 0.0510 16 1.50 1.2118 33 1.57 0.0320 17 1.51 1.3290 35 1.58 0.0186 2Y0 = 17.5256 _ (0.01) = 0.08333 M = RT (dc/'dx)m W (l—Vf’)w2 xmcm w2 at 10,589 rev./min. = 9==1.0025 and v = 0.75 co/g T = 2930 m m 1.229615 X 106 58 Data at meniscus for 32 minutes centrifugation (10,589 rpm; 6 = 75°) (F = 2.103; V = 0.75 cc/g; T = 2930; w2 = 1.229615 x 106; e = 1.0025) n Rn(cm) Yn(cm) Xn an 0 0.732 0.8616 12.719 161.773 1 0.752 0.8602 12.729 162.027 2 0.752 0.8572 12.739 162.282 3 0.762 0.8572 12.759 162.539 5 0.772 0.8566 12.759 162.792 5 0.782 0.8516 12.769 163.057 6 0.792 0.8388 12.779 163.303 7 0.802 0.8058 12.789 163.559 8 0.812 0.7606 12.799 163.815 9 0.822 0.6850 12.809 165.070 _10 0.832 0.5916 12.819 165.327 11 0.852 0.5038 12.829 165.583 12 0.852 0.5308 12.839 165.850 13 0.862 0.3662 12.859 165.097 15 0.872 0.3105 12.859 , 165.355 15 0.882 0.2610 12.869 165.611 16 0.892 0.2195 12.879 165.869 17 0.902 0.1886 12.889 166.126 18 0.912 0.1598 12.899 166.385 19 0.922 0.1196 12.909 166.652 20 1 0.932 0.0975 12.919 166.901 21 0.952 0.0790 12.929 167.159 22 0.952 0.0628 12.939 167.518 23 0.962 0.0568 12.959 167.677 25 0.972 0.0376 12.959 167.936 25 0.982 0.0280 12.969 168.195 26 0.992 0.0150 12.979 168.555 2; ann — 1921.0253 dx = c - X 2Y cm 0 (Xn2)(2.103) Z; n n = 0.08333 - (0°01)(1921'0253) = 0.02686 (161.7737?2.103) _ 0.8616 Mw ”76,7143 T0.02686)(6.058) ll 507,120 59 (c) Approximate composition of the enzyme solution. Concentration was corrected for radial dilution with: = 2 c0 FX(Xr/Xm) AZ XYJ. Data for concentration at zero time (synthetic boundary at 59,780 rpm; zero time). 6 = 700 z Y J 5::5 :6.262 570 0.536 Z Z 575 1.255 fir = $23 55 580 3.932 m ' 585 8.922 F = 0.02536 590 13.962 X 595 16.895 c0 = 12.815 600 11.180 605 5.796 610 2.560 615 0.926 620 0.300 Data for concentration after 25 minutes centrifugation at top speed. z Yj 290 0 312 :E?; = 52.180 0. 735 1 $30 00 = 10.091 705 3.812 710 6.552 715 .8.758 720 10.038 725 8.500 730 5.688 735 3.295 750 1.735 755 0.728 750 0.350 6O 2. variation of Enzyme Activity with Substrate Concentration at various pH values (a) For phenolphthalein glucuronide in acetate buffer. Assay mixture: 0.5 ml. of 0.1M acetate buffer; 0.3 ml. of water; 0.2 ml. of enzyme dilution; and 0.5 ml. of phenolphthalein glucuronide dilution to give the desired final 5. concentration. Enzyme preparation with spec. act. of 168,000 was diluted to 1.56 X 10- mg of prot. per m1. Results: v of hydrolysis at (S) x 105M conc. of substrate pH 1.58 3.15 9.56 23.7 57.3 118 3.6 7.1 9.5 11 11 ll 3.8 7.1 8.9 11 ll .11 5.0 6.0 8.3 9.5 10 11 5.2 5.5 7.8 8.9 9.5 11 5.5 5.8 7.1 7.7 8.9 9.5 5.6 6.0 7.1 7.7 8.3 8.9 5.8 5.2 6.5 6.7 7.1 7.9 5.0 5.0 5.2 5.9 6.3 7.1 5.2 3.2 5.5 5.7 5.3 6.0 5.5 2.7 3.5 5.1 5.7 5.1 5.6 2.1 2.8 3.3 3.8 5.0 V = u moles substrate decomposed per min. per mg of prot. (b) For phenolphthalein glucuronide in phthalate buffer. Assay mixture: 0.5 m1. of 0.1M phthalate buffer; 0.5 m1. of the enzyme dilution; and 0.5 ml. of substrate dilution. Enzyme prep. with spec. act. of l68,000 was diluted to 5.8 x 10_4 mg of prot. per ml. Results: v of hydrogysis at (S) x 105M conc. of substrate pH 1.89 3.95 7.88 15.8 57.3 3.1 18 2O 21 22 21 3.5 17 19 2O 21 20 3.6 17 18 2O 2O 19 3.8 16 17 18 19 19 5.0 l5 l7 l8 l8 19 5.3 11 13 15 15 15 5.6 6.0 6.5 8.5 9.1 9.6 5.0 5.3 5.1 5.7 6.5 7.1 5.5 2.0 2.8 3.2 3.6 5.1 61 (c) For p-nitrophenyl glucuronide in phthalate buffer. Assay mixture: 0.5 ml. of 0.1M phthalate buffer, 0.5 ml. of p-nitro- phenyl glucuronide dilution; and 0.5 ml. of the enzyme dilution. Enzyme preparation with spec. act. of 138,000 was diluted to 1.23 x 10-3 mg of prot. per ml. Results: ‘ v of hydrolysis at (S) x 104M conc. of substrate pH 0.50 2.50 10.0 29.0 50.0 3.0 - - - 30 - 3.3 - 25 28 31 31 3.6 17 23 26 29 29 3.9 17 22 25 28 29 5.2 15 19 22 27 30 5.5 10 . 15 16 21 25 5.8 7.0 10 12 15 18 5.2 5.2 5.8 6.5 8.3 10 6.0 1.0 2.1 2.3 3.5 5.2 (l) (2) (3) (5) (5) (6) (7) (8) (9) (10) (ll) (12) (13) (15) (15) (16) (17) (18) (19) (20) G. W. G. :35: :<*< . Hamalginen, Skand. Arch. Physiol., 23, 297 (1910); c. 5., 5 REFERENCES A. Levvy, Vitamins and Hormones, 15, 267 (1956). H. Fishman, Advances in Enzymol., 16, 361 (1955). A. Levvy and C. A. Marsh, Advances in Carbohydrate Chem., 15, 381 (1959). . A. Levvy and C. A. Marsh in "The Enzymes", P. Boyer, H. Lardy and K. Myrback, eds., Academic Press, New York, 1960, Vol. IV, P. 397. . 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