98, 394-401


A Highly Sensitive and Rapid Fluorometric Assay for UDP-Glucuronyltransferase Using 3-Hydroxybenzo(a)pyrene as Substrate JASWANT SINGH AND FRIEDRICH Abil.


Gesellschaft fir StrahlenNeuherberglMiinchen, Received

und Um,veltjiwschung. Federal Republic February

J. WIEBEL lngolstiidter of Germany


I, D-8042

2, 1979

A rapid and highly sensitive fluorometric assay for UDP-glucuronyltransferase (EC has been devised using 3-hydroxybenzo(u)pyrene as substrate. The sensitivity of the procedure is based on (a) the high coefficient of fluorescence of the product, benzo(a)pyrene-3.glucuronide, and (b) the very low background which is obtained by an efficient differential extraction of substrate and product and their widely differing fluorescence characteristics in alkaline solution. As little as 5-10 pmol of product can be determined. The procedure involves essentially a single extraction and transfer step. The method may be applicable in measuring transferase activity in a few micrograms of tissue protein or of cultured cells as well as in the routine processing of large numbers of samples. Some of the properties of glucuronyltransferase activity directed toward 3-hydroxybenzo(a)pyrene are described such as kinetic constants and the sensitivity of the reaction to detergents and organic solvents.

Glucuronidation by microsomal transferases (EC is a major pathway for the detoxification of numerous xenobiotics. Because the metabolism of biologically active chemicals is increasingly studied in cells in culture or in extrahepatic tissues which may have a low capacity for glucuronidation and be available only in small quantities there is an apparent need for the development of highly sensitive assays of glucuronyltransferase activity. Presently, several assays for UDP-glucuronyltransferase activity are in use which are based on spectrocolorimetric (1,2), fluorometric (3,4.5), as well as radiometric (6,7) procedures and employ a large variety of substrates. Nemoto and Gelboin (7) have shown that 3-hydroxybenzo(a)pyrene (3-OH-BP)’ and other phenols of benzo(u)pyrene (BP) are

readily converted to glucuronides in the presence of UDP-glucuronic acid and hepatic microsomes. The determination of BPglucuronide formation involved the separation of tritium-labeled substrate and product by thin-layer chromatography and radiometric analysis (7). The following describes a fluorometric assay for the glucuronidation of 3-OH-BP which is very simple and improves the sensitivity by taking advantage of (a) the high fluorescence coefficient of the product, BP-3-glucuronide, and (b) the ease with which the substrate, 3-OH-BP, and the glucuronidated product can be separated by differential extraction and distinguished by their widely differing fluorescence characteristics. MATERIALS Chemiwls.

1 Abbreviations used: 3-OH-BP, 3-hydroxybenzo(rr)pyrene; BP. benzo(tr)pyrene: PB, phenobarbital. 0003.2697/79/140394-08$02.00/O Copyright All rights

0 1979 by Academic Preys, Inc. of reproductmn in any term reserved

salt), 394



UDP-Glucuronic acid (sodium X-100, D-saccharic acid-l ,4-






lactone were obtained from Sigma Chemtransferred to a tube fitting the sample icals, and Brij-58 from Atlas Chemie. p- holder of the fluorometer and made alkaline Glucuronidase (bovine liver, 243 Fishman by addition of 0.1 ml of 5 N NaOH. The units/mg) was from Serva. All other chemresulting turbid solution was centrifuged or icals used were of analytical grade. 3-OH-BP allowed to clear by standing for a few minwas obtained from the Carcinogenesis utes before fluorometric determination. The Standard Reference Compound Bank, NCI, relative fluorescence intensity was then read Bethesda, Maryland. at 378-nm excitation and 425-nm emission Pwpurariotl of tnictwsotnes. Male Wistar for glucuronide conjugates formed using an rats (150-200 g body weight) were treated Aminco Bowmann fluorometer. The specific with phenobarbital (PB) supplied in the enzyme activity is expressed as nanomoles drinking water as a 0.1% solution for 12 of BP-3-glucuronide formedlminimg prodays. PB-Treated and untreated animals tein. The fluorescence coefficient of BP-3were sacrificed by cervical dislocation, glucuronide was determined as described and microsomes were prepared as de- under Results and Discussion. scribed by Hesse and Wolff (8). In the Rrpt-otlucihiliry . All assays were perfollowing, microsomes from PB-treated or formed in duplicates or triplicates. The countreated rats are referred to as PB and efficient of variation was generally less than control microsomes, respectively. 5% The volumes given above can readily Ccl1 c.ultrlr.r.s. 4-H-H-E cells derived from be reduced, e.g.. by half without loss of Reuber H-35 hepatoma were generously sensitivity or reproducibility. supplied by Dr. B. Thompson, Bethesda, Sllfety prec.rrufiott.s. 3-OH-BP has to be Maryland. The cells were cultured in handled with caution since it has been Dulbecco’s modified Eagle’s medium supfound to be cytotoxic (10). weakly mutagenic plemented with 10% fetal calf serum, 100 in Saltnotzello ryplzittluriuttl ( 11,12), and units of penicillin. and 100 pg of streptovery weakly if at all tumorigenic (13,14). mycin, harvested as previously described The danger of exposure can be minimized (9) and stored at -80°C. They were by using disposable materials, i.e., tubes utilized for the transferase assay without and tips of automatic pipetting devices. further homogenization. Gllrcurotzidrtsr treclttnetlt. An aliquot Etz:ytnc crsstry. The entire procedlure was containing approximately 2 nmol of BP-3performed under subdued light. The standglucuronide and 0.08 nmol of 3-OH-BP was ard reaction mixture contained in 0.4 ml: extracted with ch1oroform:methanol. The 100 mM Tris-HCI buffer, pH 7.6: 5 mM MgCI,. 3 mM UDP-glucuronic acid, 50 aqueous methanol phase was dried under a p.g protein, and 50 pM 3-OH-BP added in stream of nitrogen and the residual material 20 ~1 methanol to give a final 5% (v/v). The was dissolved in ammonium acetate buffer, 0.5 M, pH 4.6. Duplicate samples were substrate was added after the enzyme prepincubated in the presence of p-glucuronidase aration. The reaction was started by placing (200 Fishman units) and 5 mM saccharic the samples in a water bath at 37°C. Incubaacid- 1.4~lactone. The aliquots were removed tion time was 3 min if not stated otherwise. After incubation under mild shaking the at various time intervals and l-ml portions with 6 ml of chloroform: reaction was terminated by addition of 6 ml were extracted was then of chloroform:methanol (2: I, v/v) and 0.6 methanol (2:l). The fluorescence read in aqueous and organic phases as deml of water. The mixture was vigorously scribed above. shaken and centrifuged at low speed for faster separation of the phases. An aliquot ProFeiti detrrt?iitiutiotz. Protein was meas(1 ml) of the aqueous methanol phase was ured by the method of Lowry et ~1. (15).






Figure 1 shows the fluorescence spectra of 3-OH-BP and the product(s) formed in the presence of UDP-glucuronic acid and hepatic microsomes which closely resemble those of BP-3-glucuronide described by Nemoto et al. (16) and Baird rt ~1. (17). The similarity of the maximum excitation and emission wavelengths of 3-OH-BP and BP-3-glucuronide precludes their quantitative fluorometric determination without prior separation or selective change of fluorescence properties. Both approaches proved to be feasible. Of the various solvents and solvent mixtures tested (diethylether, ethylacetate.

FIG. 1. Fluorescence spectra of 3-OH-BP and of the aqueous soluble product of UDP-glucuronic aciddependent 3-OH-BP metabolism. (- - -) Fluorescence in the water:methanol phase after incubation of 50 PM 3-OH-BP, 400 pg microsomal protein, 6 mM UDP-glucuronic acid, 0.0125% Brij-58 for 60 min and extraction of the incubation mixture with chloroform:methanol (for other conditions see Materials and Methods). More than 90% of the substrate 3.OH-BP was utilized. (-1 Fluorescence of 3.OH-BP in the water:methanol phase. Conditions were as described above except that UDP-glucuronic acid was omitted from the incubation mixture. The amount of substrate remained unchanged over the incubation period. (. ‘1 Fluorescence of 3-OH-BP in the water: methanol phase after addition of 5 N NaOH (0.1 ml/ml). Excitation spectra t/eff sit/e) were recorded at an emission wavelength of (- - -) 425 nm. (-) 438 nm. and (. .) 520 nm. Emission spectra (right side) were recorded at an excitation wavelength of (- - -) 378 nm. (---) 375 nm. and (. ) 392 nm.


acetone:hexane (1:3), propanol:hexane (1:3), chloroform, chloroform:methanol) a chloroform:methanol mixture of 2: I gave the best differential extraction of substrate and product. Extraction of 1 part of the aqueous reaction mixture with 6 parts of chloroform: methanol (2: 1) removed more than 98% of the substrate into the organic solvent phase. Under these conditions about 90% of the product remained in the water:methanol phase. Following the extraction step, the fluorescence at 378-nm excitation/425-nm emission was linear with the concentration of BP-3-glucuronide in the reaction mixture in the range of 5 nM to 5 PM. Extraction with chloroform:methanol proved also to be advantageous in that most of the protein appears in the intermediate layer of organic and aqueous phases. Addition of various amounts of microsomal protein to reaction mixtures containing a given amount of BP-3-glucuronide did not alter the fluorescence intensity in the aqueous phase after extraction.

In order to suppress the fluorescence arising from residual substrate in the aqueous phase after extraction which may interfere with the determination of low amounts of products, the aqueous phase was made alkaline by addition of 0.1 ml of 5 N NaOH. This causes the excitation and emission peaks of the substrate, 3-OH-BP. to shift from 375 to 392 nm and from 435 to 520 nm, respectively (Fig. l), but affects neither the fluorescence maxima nor intensity of the product BP-3-glucuronide. In the presence of alkali the product was stable for several hours. The fluorescence of the substrate in alkaline solution at the wavelengths of interest (378-nm excitation/425-nm emission) drops to less than 1% of its value in neutral water:methanol solution. Thus. by the combination of differential extraction





and alkali treatment, the fluorescence of the substrate can be reduced to about 0.102% of its initial value, i.e., at a substrate concentration of 20 nmolireaction mixture the background fluorescence corresponds to less than 5 pmol. Maximum suppression Iof substrate-dependent fluorescence requiires at least a final 0.05 N NaOH: above this value the normality of the solution is not critical. From the foregoing it is apparent that an even simpler assay procedure may be employed when the amounts of product formed comprise at least 5- 10% of the substrate, a condition which is usually met using microsomes from rodent liver. Mere addition of alkali to the reaction mixture suppresses the fluorescence due to thle substrate to 1.O- I .5% and allows the fluorescence of the product to be read directly in the reaction vessel. However, fluorescence of the product in alkaline aqueous solution is also decreased by 50% without concomitant change in the fluorescence characteristics. This reduction in fluorescence intensity does not occur in the ,water: methanol phase obtained after chloroform: methanol extraction as noted above.

Figure 2a shows the disappearance of choloroform:methanol extractable fluorescent material due to 3-OH-BP and the concomitant formation of fluorescent material in the aqueous phase, presumably the BP-3-glucuronide. The sum of the fluorescent units consumed and appearing during UDP-glucuronic acid-dependent metabolism was the same at all incubation times provided corrections were made for the different degree of fluorescence of both product and substrate in organic and aqueous phases (1.0 and 1.4, respectively) and for the fraction of product extracted in1.o the organic phase amounting to about 10%. These results sugget that the coefficient of fluorescence of the substrate and the prod-


FIG. 2. Appearance and disappearance of fluorescence during the formation and hydrolysis of BP-3glucuronide. (a) Microsome-mediated formation of BP-3-glucuronide (W from 3-OH-BP (3). The incubation mixture contained in a total volume of 1.0 ml: 400 fig microsomal protein. 6 rnM UDP-glucuronic acid. and 0.0125% Brij-58. At various times aliquots were removed in duplicate from the reaction mixture and upon addition of 0.9 ml H,O were processed for determination of fluorescence as described under Materials and Methods. (b) Hydrolysis of BP-3.glucuronide by P-glucuronidase: disappearance of glucuronide (W) and appearance of 3-OH-BP (0). P-Glucuronidase treatment as described under Materials and Methods.

uct may be very similar if not identical. This is confirmed by reverting the enzyme rethe aqueousaction, i.e., by subjecting soluble product to hydrolysis by ,&glucuronidase (Fig. 2b). Within a few minutes the aqueous-soluble material was quantitatively converted into organic-soluble material which by fluorescence spectra was identified as 3-OH-BP. Again, the sum of the fluorescence due to substrate and product remained unchanged during the reaction (Fig. 2b). The fluorescence of the product did not decrease when p-glucuronidase hydrolysis was carried out in the presence of its inhibitor saccharic acid-l .4-lactone. The results indicate that BP-3-glucuronide is the only water-soluble fluorescent metabolite formed from 3OH-BP in the presence of microsomes and UDP-glucuronic acid. BP-Phenols have been shown to be substrate for microsomal monooxygenases (18) as well as for soluble sulfotransferases (19.20). However, taking into account a contamination of the microsomal prepara-




tion with the soluble transferase. the lack of their cofactors in the reaction mixture most likely prevents the metabolism of 3OH-BP by these alternative routes. The fact that no water-soluble fluorescent products are detectable in the absence of UDPglucuronic acid does not exclude the formation of other derivatives of 3-OH-BP than the glucuronide but it shows that even if they are formed in substantial amounts they do not interfere with the quantitation of the BP-3-glucuronide.

Dependency of’ Transferase Actil!ity Time and Protein Concentration




01 02 OL i3OH~Benro~olp”rene,~MI





The glucuronidation of 3-OH-BP by microsomes or preparations of cultured cells was linear with time of incubation for at least 30 min (Fig. 3a). Linearity of the reaction increased to 60 min when protein concentrations as low as 20 &reaction mixture were used. At microsomal protein concentration of 200 pg, the reaction was linear up to 20 min (data not shown). Enzyme activity was also proportionate to the amounts of protein used in the range of 20 to 80 wg after 30 min of incubation (Fig. 3a). These results were obtained in the absence of detergents from the reaction mixture. In

FIG. 3. Glucuronidation of 3-OH-BP as a function of time and protein concentration. Microsomal protein: 20 pg, (0): 40 pg. (0); 80 pg, (a); and 50 kg protein of 4-H-H-E cells. (0). Other conditions as under Materials and Methods. (a) In the absence of detergents; (b) in the presence of 0.005 and 0.0075% of Brij-58 at 20 (0) and 40 ~g (0) of microsomal protein, respectively.

FIG. 4. BP-3-Glucuronide formation as a function of 3-OH-BP or UDP-glucuronic acid concentration. The reaction was carried out with PB-microsomes (20 pg) in the presence of 0.0125% of Brij-58. Other conditions as described under Materials and Methods. (a) Double-reciprocal plot of 3-OH-BP concentration vs glucuronide formation. UDP-Glucuronic acid concentration was 3 mM. Methanol was added as solvent for the substrate to a final concentration of 5%. (b) Double-reciprocal plot to UDP-glucuronic acid concentration YS glucuronide formation. 3-OH-BP concentration was 50 /rM.

the presence of the detergent Brij-58, at concentrations which cause maximal activation of the transferase (see below), the rate of reaction decreased rapidly depending on the concentration of protein used (Fig. 3b): Enzyme activity was linear for about 5 min with 20 pg protein/reaction mixture. At 40 pg protein/reaction mixture the rate of reaction declined even earlier. Triton X- 100 at a concentration of 0.0125%~ had qualitatively similar effects (data not shown). Early loss of linearity was also observed by Bock and White (5) on the glucuronidation of lnaphthol by microsomes in the presence of 0.05% detergent. The reason for the loss of






linearity in the presence of the detergents is not known. Dependency on Substrrrte and UDP-Glucuronic Acid Concentrat,ion

The activity of microsomal glucuronyltransferase at various concentrations of 3OH-BP and of UDP-glucuronic acid is shown in Lineweaver-Burk plots in Fig. 4. The curve obtained with 3-OH-BP (Fig. 4a) is linear in the range of 1 to 50 PM and corresponds to an apparent K,,, of 10 PM. This value is considerably lower than those reported for other substrates (21,22), a fact which might be attributable to the high lipophilicity of the 3-OH-BP and hence its likely accumulation in a lipophilic environment of the enzyme within the microsomal membrane (23). For the standard assay a substrate concentration of :5O PM was adopted. V,,, of the reaction vvas 20 nmoliminimg protein (Fig. 4a). V,,,, values of the same order of magnitude have been reported for other substrates of the transferase such as 2-aminophenol, I-naphthol, 4nitrophenol, or 4-methylumbelliferone (5,31). In contrast to the substrate, the reciprocal plot for the cosubstrate UDP-glucuronic acid is not linear (Fig. 4b), in agreement

FIG. 5. Effect of detergents on the glucuronlidation of 3-OH-BP. The reaction mixture contained 20 pg of microsomal protein. Other conditions as described under Materials and Methods. PB microsomes: control microsomes: - - -. Brij-58: 0. Triton -. X-100: 0. The detergents did not interfere with the fluorometric determinations.

“\ ‘V 0.05 01 0.2

kc-r-----., 0 0005 001 BRIJ -58


FIG. 6. Activation of glucuronyltransferase various concentrations of Brij-58: Dependency on amount of protein. Reaction mixtures contained (0). 80 ((I), or 200 Kg (0) of microsomal protein microsomes). Transferase activities are expressed percentage of controls, i.e., of the activities in absence of detergent.

by the 20 (PB as the

with the observation of Winsnes and Rugstad (21). The reasons for the nonlinearity are not clear. From the slope of the higher UDP-glucuronic acid concentrations a K,u value of 0.17 mM was estimated which agrees with the observation of others (22,24).

Detergents are known to cause a severalfold activation of glucuronyltransferase activity (3,25,26). As shown in Fig. 5, Triton X-100 and Brij-58 have a biphasic effect on the glucuronidation of 3-OH-BP. In the presence of low concentrations of the detergents (approx. 0.0125%) transferase activity is strongly activated. The degree of activation rapidly decreases above 0.0125% Triton X-100 to turn into inhibition at 0.05% of the detergent. For Brij-58 the fall in enzyme activity with increasing concentrations is less precipitate and reaches the nonactivation level only at a concentration of 0.2% before inhibition sets in. The detergent concentration which causes maximum activation of3-OH-BPglucuronidation is strongly dependent on the amount of protein used in the assay (Fig. 6): With increas-




ing amounts of microsomal protein ranging from 20 to 200 &reaction mixture, the optimal concentration of Brij-58 increases from 0.005 to 0.0125%, respectively, to yield an approximate sevenfold activation independently of the protein concentration. The results point out that great care has to be taken to stay within the proper range of detergent concentrations under the particular experimental conditions. Acti\,ation


pg/reaction mixture). However, in freshly collected 4-H-B-E cells or in these cells after 2 h of freezing at -8o”C, methanol did not cause an activation at concentrations ranging from 0.25 to 7.5%. Likewise, at a concentration of microsomal protein used under standard conditions (approx 50 pg/reaction mixture) methanol at 2.5-8.75s was ineffective in activating the transferase (data not shown).

oj’ Gl~r~uron~ltr~~nsferuse

Methanol used as solvent for the substrate 3-OH-BP conceivably activates glucuronyltransferase activity as has been shown for other solvents (27). We thus examined the effect of increasing concentrations of methanol on the glucuronidation of 3-OH-BP in microsomes (Fig. 7) or preparations of cultured cells in vitro. Methanol concentrations above 2.5% (v/v) increasingly activated the rate of glucuronidation to reach a plateau of 50% activation at about 5% (v/v) in microsomal preparations (8

FIG. 7. Effect of methanol on glucuronidation of 3.OH-BP. The substrate, 3-OH-BP, was added in 1 ~1 of methanol to a final 2.5 x IO-” M. Amount of microsomal protein was 8.0 pg. Other conditions as described under Materials and Methods. 100% of controls corresponds to the activity at the lowest methanol concentration (1 #0.4 ml reaction mixture) and represents the mean of six determinations. The width between the dashed lines gives the range of the controls. Points represent the mean and range of duplicate determinations.



The assay for glucuronyltransferase activity described here offers a combination of high sensitivity and great simplicity. The sensitivity is afforded by two features: (a) The BP-glucuronide formed has a high coefficient of fluorescence which, e.g., exceeds that of I-naphthol, another substrate in a fluorometric assay (5), by a factor of 30. (b) The “background” fluorescence due to the substrate can virtually be eliminated by differential extraction and by selectively shifting the fluorescence maxima of the substrate to higher wavelengths than those of the product. The assay procedure requires only one simple extraction and transfer step which avoids the more lengthy and cumbersome radiometric analysis of :‘HOH-BP using thin-layer chromatography (7). It has been well established that at least two forms of glucuronyltransferases exist which can be distinguished by their specific range of substrates and inducers (22,28,30), their physicochemical properties (29), and their developmental characteristics (31). Experiments using 3-OH-BP in comparison with 1-naphthol and morphine as substrates showed? that 3-OH-BP belongs to the substrate group represented by I-naphthol (29,30), the “late fetal” group according to Wishart (31). In view of its high sensitivity, the present assay appears to be well suited for determining the activity of UDPglucuronyltransferases directed toward the 2 Schwarz, servations.










latter substrate group in tissues with low enzyme levels or in minute tissue samples. Likewise, simplicity and brevity recommend this assay for the routine processing of large numbers of biological probes.

13. 14.

ACKNOWLEDGMENTS We thank Dr. L. R. Schwarz and Dr. A. Pawlak for their helpful advice. and Dr. H. Greim for his critical reading of the manuscript. The expert secretarial assistance of Ms. J. Byers and the technical assistance of P. Bannach are gratefully acknowledged.


16. 17.

REFERENCES I. Dutton. G. J. (1966) in Glucuronic Acid (Dutton, G. J.. ed.). pp. 186-299. Academic Press. New York. 2. Van Roy, F. P., and Heirwegh. K. P. M. (1968) Biochetn. .I. 107, 507-518. 3. Winsnes. A. (1969) Eiochim. Biophy.7. Acttr 191,279-291. 4. Mulder, G. J. (1975)AnnI. Biochrnz. 64. 3510-359. 5. Bock, K. W.. and White, I. N. H. (1974) Eur. J. Biochrrn. 46, 451-459. 6. Lucier. G. W., McDaniel, 0. S., and Malthews, H. B. (1971) Arch. Biochrm. Biophy:N. 145, 520-530. 7. Nemoto. N.. and Gelboin. H. V. (1976) Biocham. Phurmtrcd. 25, 1221- 1226. 8. Hesse, S.. and Wolff. T. (1977) Bioc,henl. Phcrrnruc~d. 26, 2043-2047. 9. Wiebel. F. J., Matthews, E. J., and Gelboin, H. V. (1972) .I. Bid. Chew/. 247, 4711-4717. 10. Celboin, H. V.. Huberman, E.. and Sachs, L. (1969) Prrw. ,Yur. Ac,ud. Sci. f Wush.) 64, 11881194. 11. Glatt. H. R.. and Oesch, F. (1976) Muttrtion Re.c. 36, 379-384. I?. Wislocki. P. G.. Wood. A. W.. Chang. R. L., Levin, W., Yagi. H.. Hernandez. 0.. Dansette.

18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29.

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P. M., Jerina. D. M., and Conney, A. H. (1976) Cuncer Rrs. 36, 3350-3357. Cook, J. W., and Schoental, R. (1952) Brit. J. Cunc,er 6, 400-406. Wislocki, P. G.. Chang, R. L., Wood, A. W., Levin, W., Yagi, H., Hernandez. 0.. Mah, H. D., Dansette, P. M., Jerina. D. M., and Conney, A. H. (1977) Cancer Rrs. 37, ?6082611. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L.. and Randall. R. J. (1951) J. Bid. Chem. 193, 265-275. Nemoto. N., Hirakawa. T., and Takayama, S. (1978) Cham. Bid. Inrrrcrcr. 22, I- 14. Baird, W. M., Chern, C. J.. and Diamond, L. (19’77) Cunc,er Rrs. 37, 3190-3197. Wiebel. F. J. (1975) Arch. Biochetn. Biophys. 168, 609-621. Cohen, G. M.. Moore, B. P., and Bridges. J. W. (1977) Biochem. Phurnmcnl. 26, 551-553. Nemoto, N., and Takayama, S. (1977) Bioc-hem. Phurmuc~ol. 26, 679-684. Winsnes, A., and Rugstad, H. E. (1973) Ac,fu Pharmucd. Toxicol. 33, 161- 176. Bock, K. W.. Frohling, W.. Remmer, H., and Rexer, B. (1973) Biochim. Biophys. Acts 327. 46-56. Zakim. D., and Vessey, D. A. ( 1977) J. Bid. Chrm. 252, 7534-7537. Gregory, D. H.. II. and Strickland, R. D. (1973) Biwhim. Binpllys. Ac,tu 327, 36-45. Lueders. K. K., and Kuff, E. L. (1967) Arch. Bio~~hrm. Biophys. 120, 198-203. Mulder, G. J. (1970) Biochcm. J. 117, 319-324. Gorski, J. P., and Kasper, C. B. (1977) J. Bid. Chrm. 252, 1336-1343. Lucier. G. W.. and McDaniel, 0. S. (1977) J. Sfrroiti Biochrm. 8, X67-872. Bock. K. W., von Clausbruch. U. C., Josting. D.. and Ottenwalder. H. (1977) Biochrnl. Phur1,7(1< 4. 26, 1097- 1 100. Wishart. G. J. (1978) Bir>chem. .I. 174, 671-672. Wishart, G. J. (1978) Biwhrm. J. 174. 485-489.

A highly sensitive and rapid fluorometric assay for UDP-glucuronyltransferase using 3-hydroxybenzo (a) pyrene as substrate.

ANALYTICAL BIOCHEMISTRY 98, 394-401 (1979) A Highly Sensitive and Rapid Fluorometric Assay for UDP-Glucuronyltransferase Using 3-Hydroxybenzo(a)py...
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