- Email: [email protected]
Assay of sulfotransferases
ANALYTICAL
95, 82-86 (1979)
BIOCHEMISTRY
Assay of Sulfotransferasesl RONALD Section
D. SEKURA, on Enzymes
CAROL
J. MARCUS,~
and Cellular Biochemistry, Diseases, National Institutes
ELLEN
SUE LYON,
AND WILLIAM
National Institute of Arthritis, of Health, Bethesda, Maryland
Metabolism, 20014
B. JAKOBY and Digestive
Received October 13, 1978 Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3’-phosphoadenosine5’-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [%]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.
In attempting a systematic study of the sulfotransferases we have found limitations in the methods available for their assay. Our interest was focused on the activity of the sulfotransferases utilizing the hydroxyl functional group (Reaction [I]), a category encompassing such varied sulfate acceptors as simple alcohols, phenols, and sterols. ROH + 3’-phosphoadenosine5’-phosphosulfate + ROS03H + Adenosine 3’,5’-bisphosphate. [l] An existing method, effective in the nanomolar range, is based on the extraction from aqueous solution of the ion pair formed with methylene blue and any of several sulfate monoesters (1-3). We have used this system successfully for routine assay of phenol sulfotransferases with /3-naphthol as the acceptor substrate but greater sensitivity was
required for less effective substrates. Other approaches have included the separation of substrate from product by direct extraction with organic solvents (4,5) or by chromatography (5-8). We were concerned with the effectiveness of simple extraction methods when working with small amounts of product, particularly since certain of the sterols and their sulfates are tightly bound to the transferases as well as to other proteins. Chromatography is useful but time consuming for a large number of samples unless additional simplification can be achieved. In order to avoid some of these difficulties, we have designed two assay procedures for the low range of product formation and have applied them to crude enzyme preparations as well as to purified sulfotransferases. One method is based on the differential binding of radioactive sterols and their corresponding sulfate esters to filters. The second system, applicable to a far broader range of substrates, relies on the synthesis of radioactive products from [35S0,]PAPS3; subsequent thin layer chro-
* This paper is dedicated to the memory of Dr. Alvin Nason. * Present address: Laboratory of Experimental Pathology, NIAMDD, National Institutes of Health, Bethesda, Md. 20014. This work was carried out under an Intergovernmental Personnel agreement with The University of Tennessee, Center for the Health Sciences. 0003-2697i79/070082-05$02.00/O Copyright Q 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
3 Abbreviations used: PAPS, 3’-phosphoadenosine5’-phosphosulfate. 82
83
ASSAY OF SULFOTRANSFERASES
0
25
50
75
Fractm
FIG. 1. Elution pattern resulting from chromatography of [“%]PAPS on a 0.9 by IO-cm column of DEAEcellulose developed with triethylamine-carbonate. The pooled fractions are indicated by brackets.
matography of the assay mixture with a solvent that brings the product close to the solvent front, allows ready separation. MATERIALS
All sterols and some phenols were products of the Sigma Chemical Company and other phenols were from Eastman Organic Chemicals or from Aldrich Chemical Company. 14C- and 3H-labeled sterols were obtained from New England Nuclear. 3’-Phosphoadenosine-5’-phosphosulfate (PAPS) was prepared synthetically (9). [35S]PAPS was obtained from New England Nuclear and purified by chromatography on DEAE-cellulose. This step was necessary because commercially obtained [35S]PAPS is contaminated with radioactive material that migrates with product in the described chromatographic systems and thereby reduces the effectiveness of the procedure. The labeled nucleotide, 250 &I in 50% ethanol, was applied to a column (0.9 x 10 cm) of DE-52 (Whatman) that had been equilibrated with 50 mM triethylaminecarbonate at pH 7.6. The column was washed with the same buffer and eluted with a linear gradient (150 ml total volume) established between the starting buffer and 1 M triethylamine-carbonate at the same pH; fractions of 2 ml were collected. PAPS eluted sharply with a peak concentration in frac-
tion 38 (Fig. 1). The major fractions were pooled and brought to dryness at 37°C with a rotary evaporator; the samples were dissolved in water and the procedure was repeated several additional times to remove traces of triethylamine (yield: 67%). PAPS was stored in aqueous solution at -20°C. Since most of the acceptor substrates are only minimally soluble in aqueous buffers, all were dissolved in acetone such that the final solvent concentration in the reaction mixture was 4 to 5%. Crude extracts of rat liver, prepared by homogenization in 3 vol of 10 mM Trischloride at pH 7.4 containing 0.25 M sucrose and 5 mM 2-mercaptoethanol, were centrifuged at 100,000 g for 90 min. The supernatant fluid served as an enzyme source as did homogeneous enzymes (9) from the same organ. Radioactivity was measured by adding the sample to 10 ml of Aquasol (New England Nuclear) with the use of a Model LS8100 scintillation spectrometer (Beckman). BINDING ASSAY
A number of steriod sulfates are separable from radioactive sterol substrates by a filter technique which uses to advantage the greater polarity of the sulfate esters. Sulfotransferase activity was measured in reaction mixtures of 50 ~1 total volume containing 0.25 M sodium acetate at pH 5.5, 50 PM radioactive sterol (about 10 &il pmol), 1.8 mM PAPS and 4% acetone. In preparing assay mixtures it is convenient to first evaporate the radioactive sterol mixed with unlabeled carrier to dryness and to dissolve the dry compound in acetone. When the resulting solution is added to the sodium acetate buffer and agitated with a Vortex mixer, a dispersion of the sterol is formed that is stable for several hours. Aliquots are then removed for individual assays, to which are added the other components; enzyme is added last to initiate the reaction. After 30 min at 37”C, the reaction vessel is placed in a boiling water
84
SEKURA
ET AL.
bath for 2 min. Upon cooling, 50 ~1 of 0.5 mM carrier sterol in absolute ethanol is added and mixed. A 20+1 aliquot of the reaction mixture is applied to the center of a 3-cm square of silica gel-impregnated glassfiber sheet (Gelman Instrument Co., ITLCSG). After the spot has dried, the square is placed on a Hoefer FH- 12 filtration apparatus. A circular disk, with the radioactive material in the middle, is easily cut from the square filter by placing the cylindrical steel sleeve onto the filter apparatus with a twisting motion. The resultant filter disk is washed Time lminl Protein lpll under slightly reduced pressure three times FIG. 2. Dependence of the binding assay and the with 3-ml portions of 25% dioxane in hexane (v/v). Under these conditions, sterol sulfates chromatographic assay on protein concentration and time of incubation. [WlDehydroepiandrosterone was remain bound to the filter whereas unesterthe substrate in the binding assay (0) with a crude liver ified sterol is removed by the solvent. extract (30 mg/mi). The chromatographic assay was The entire filter disk is placed in a scintilused with [%]PAPS and a partially purified enzyme (0.3 mg/ml) with butanol as acceptor (Cl) as well as lation vial and counted in 10 ml of Aquasol. Nonspecific binding is determined by in- with a homogeneous enzyme (6 &ml) and p-naphthol cluding a parallel assay lacking PAPS. The as acceptor (0). amount of radioactive product is calculated level of product recovery was estimated by by determining the fraction of the radioactivity in the isolated sulfate product com- allowing the reaction in a complete assay pared to that present in a sample not sub- mixture to proceed to apparent completion. By this means it was demonstrated that more jected to filtration. than 95% of the sterol substrate could be reEvaluation and Comments covered on the filter as the sulfate. In addition to dehydroepiandrosterone, the method With homogenates of rat liver as enzyme source and dehydroepiandrosterone as ac- has been used with As-androsten-38, 17pdiol, P-estradiol, and testosterone. ceptor, sulfate transfer to sterol was linear We have observed several factors that afwith protein concentrations as high as 2.5 fect the amount of background radioactivity mg/ml for a period of more than 40 min. binding to the filters. High blanks are found Linearity was observed in the formation of when high concentrations of protein are between 0.1 to 1 nmol of product (Fig. 2). present in the incubation mixture but can be We emphasize that the large amount of PAPS used here i.e., 1.8 mM, is essential compensated for by including an appropriate control without PAPS for each sample. Adsince lower concentrations result in deviation there is significant day to day from linearity. This observation appears to ditionally, variation in nonspecific binding to the filters reflect the rapid hydrolysis of PAPS by crude which amounts to 1 to 10% of the applied homogenates. When the assay is used with radioactivity. This variation is a function of partially purified or homogeneous preparaambient humidity and is predictable from tions of sterol sulfotransferases these difthe weather. Lower and more uniform levels ficulties are not encountered and the PAPS of nonspecific binding are attained by placconcentration may be reduced to 0.2 mM. after spotting The method is reasonably precise; five ing the filters, immediately duplicate samples led to a SD of +3%. The with sample, on a rack within a covered
ASSAY OF SULFOTRANSFERASES
8.5
80 PM [35S]PAPS (170 Ql~mol), and the appropriate alcohol at a concentration of 5 mM. After addition of enzyme, the reaction is allowed to proceed for 20 min at room temperature and is stopped with 50 ~1 of CHROMATOGRAPHIC ASSAY acetone. Aliquots (5 ~1) of reaction mixtures are Measurement of sulfotransferase activity applied to a thin-layer strip (1 x 10 cm) of by chromatographic assay is more versatile cellulose on a plastic backing (Eastman No. and its special recommendation is that blank 6064). Once the samples are dry, the chrovalues are close to zero, a situation of parmatographic strips are developed with nticular importance in the determination of propanol:ammonia:water (6:3: 1) at 4°C for kinetic constants with poor substrates. The phenol and at room temperature for sterol method has been useful for a large number sulfates. For the alcohol sulfates, a lo- to of phenols,4 sterols5 and aliphatic alcohols6 15-~1 aliquot is applied to a strip of silica although optimum incubation conditions coated gel (Eastman No. 13 181) and dediffer because of separate requirements of veloped with 2-propanol:chloroform:meththe enzymes catalyzing the reactions. anol:water (10: 10:5:2) at room temperature. Sulfate transfer to phenols is assayed in a After the solvent has moved about 8 cm, total volume of 50 ~1 containing 0.25 M sothe strips are removed and the solvent front dium phosphate at pH 6.5, 5 mM 2-mercapis marked. For the phenol system, a 2-cm toethanol, 0.1 mM [35S]PAPS (130 &i/ section is cut below and including the solvent Fmol), the appropriate phenol at a concenfront and placed directly into a scintillation tration of 0.5 mM, and 5% acetone. The revial containing 10 ml of Aquasol; a 1.2-cm action is initiated by addition of enzyme and, after 10 min at 37°C is terminated by addi- section is cut for the alcohol and sterol sulfates. tion of 13 ~1 of 2 M acetic acid. With sterols as acceptor, the incubation Evaluation and Comments mixture in a total volume of 50 ~1 includes 0.25 M sodium acetate at pH 5.5, 50 PM With [“‘SIPAPS that has been purified by sterol, 60 PM [35S]PAPS (270 ~Cil~mol), and chromatography on DEAE-cellulose, there 4% acetone. After addition of enzyme and is essentially no radioactive contaminant incubation at 37” for 30 min, the reaction is that migrates with the sulfate esters thereby terminated by placing the covered reaction enabling the determination of low levels of vessels into a boiling water bath for 2 min. product. The limits of the assay are delineFor aliphatic alcohols, transferase activated in Fig. 2 for sulfotransferases active with ity is measured in a total volume of 100 ~1 P-naphthol and with butanol. It will be evicontaining 0.1 M Tris-chloride at pH 7.5, dent that linearity is achieved both as a function of time and of protein concentration ’ a- and fl-naphthol, o-, m-. andp-chlorophenol, 2.4within the conditions described. Although dinitrophenol, acetaminophenol, o-, m-. and p-nitronot shown, linearity over a similar time phenol, m- andp-cresol. phenol, 5-hydroxytryptamine, course and range of protein concentration 5-hydroxyindole, hydroquinone, p-methoxyphenol, phydroxybiphenyl. m-methylphenol. was attained with dehydroepiandrosterone. j Androsterone, dehydroepiandrosterone, h”-androRecovery of product was apparently comsten-3P, 17,&diol: J5-pregnen-3p-ol-20-one, estradiol. plete since the results of the chromatocorticosterone, hydrocortisone, and testosterone. graphic and the methylene blue (2) assays 6 I-Butanol. I-heptanol, and 2-heptanol; the method were identical when performed on the same is not applicable to the sulfates of methanol, ethanol, and propanol which migrate at a lower R,. sample over a wide range of product contrough containing warm water. The filters are allowed to remain in the humid atmosphere for 10 min and are then washed with solvent as described above.
86
SEKURA ET AL.
centration. At the level of 100 pmol formed in an incubation mixture, the SD was 22%.
ily applicable to the determination of monoester sulfates in the range of 50 to 400 pmol. REFERENCES
DISCUSSION In the course of a year of experience with the two assay procedures, we have found them dependable and simple to perform. The binding assay is particularly rapid allowing 100 samples to be processed per day but requires a radioactive compound as acceptor and is limited in its sensitivity by appreciable binding of substrate itself to the filter. Although less rapid, the chromatographic assay results in clean separation of substrate and products and may be increased in sensitivity by use of lower dilutions with carrier PAPS. Both assay systems are read-
Roy, A. B. (1956)Biochem. J. 62,41-50. Nose, Y., and Lipmann, F. (1958) J. Biol. Chem. 233, 1348-1351. Banerjee, R. K., and Roy, A. B. (1966) Mol. Pharmacol. 2, 56-66. Singer, S. S., Giera, D., Johnson, J., and Sylvester, S. (1976) Endocrinology 98, 963-974. 5. Rozhin, J., Huo, A., Zemlicka, J., and Brooks, S. C. (1977) J. Biol. Chem. 252, 7214-7220. 6. Spencer, B. (1960) Biochem. J. 77, 294-304. 7. Mattock, P., and Jones, J. G. (1970) Biochem. J. 116, 797-803. 8.
McEvoy, F. A., and Carroll, J. (1971)Biochem.
J.
123, 904-906. 9.
Sekura, R. D., and Jakoby, W. B. (1979) J. Biol. Chem. 254, in press.
95, 82-86 (1979)
BIOCHEMISTRY
Assay of Sulfotransferasesl RONALD Section
D. SEKURA, on Enzymes
CAROL
J. MARCUS,~
and Cellular Biochemistry, Diseases, National Institutes
ELLEN
SUE LYON,
AND WILLIAM
National Institute of Arthritis, of Health, Bethesda, Maryland
Metabolism, 20014
B. JAKOBY and Digestive
Received October 13, 1978 Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3’-phosphoadenosine5’-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [%]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.
In attempting a systematic study of the sulfotransferases we have found limitations in the methods available for their assay. Our interest was focused on the activity of the sulfotransferases utilizing the hydroxyl functional group (Reaction [I]), a category encompassing such varied sulfate acceptors as simple alcohols, phenols, and sterols. ROH + 3’-phosphoadenosine5’-phosphosulfate + ROS03H + Adenosine 3’,5’-bisphosphate. [l] An existing method, effective in the nanomolar range, is based on the extraction from aqueous solution of the ion pair formed with methylene blue and any of several sulfate monoesters (1-3). We have used this system successfully for routine assay of phenol sulfotransferases with /3-naphthol as the acceptor substrate but greater sensitivity was
required for less effective substrates. Other approaches have included the separation of substrate from product by direct extraction with organic solvents (4,5) or by chromatography (5-8). We were concerned with the effectiveness of simple extraction methods when working with small amounts of product, particularly since certain of the sterols and their sulfates are tightly bound to the transferases as well as to other proteins. Chromatography is useful but time consuming for a large number of samples unless additional simplification can be achieved. In order to avoid some of these difficulties, we have designed two assay procedures for the low range of product formation and have applied them to crude enzyme preparations as well as to purified sulfotransferases. One method is based on the differential binding of radioactive sterols and their corresponding sulfate esters to filters. The second system, applicable to a far broader range of substrates, relies on the synthesis of radioactive products from [35S0,]PAPS3; subsequent thin layer chro-
* This paper is dedicated to the memory of Dr. Alvin Nason. * Present address: Laboratory of Experimental Pathology, NIAMDD, National Institutes of Health, Bethesda, Md. 20014. This work was carried out under an Intergovernmental Personnel agreement with The University of Tennessee, Center for the Health Sciences. 0003-2697i79/070082-05$02.00/O Copyright Q 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
3 Abbreviations used: PAPS, 3’-phosphoadenosine5’-phosphosulfate. 82
83
ASSAY OF SULFOTRANSFERASES
0
25
50
75
Fractm
FIG. 1. Elution pattern resulting from chromatography of [“%]PAPS on a 0.9 by IO-cm column of DEAEcellulose developed with triethylamine-carbonate. The pooled fractions are indicated by brackets.
matography of the assay mixture with a solvent that brings the product close to the solvent front, allows ready separation. MATERIALS
All sterols and some phenols were products of the Sigma Chemical Company and other phenols were from Eastman Organic Chemicals or from Aldrich Chemical Company. 14C- and 3H-labeled sterols were obtained from New England Nuclear. 3’-Phosphoadenosine-5’-phosphosulfate (PAPS) was prepared synthetically (9). [35S]PAPS was obtained from New England Nuclear and purified by chromatography on DEAE-cellulose. This step was necessary because commercially obtained [35S]PAPS is contaminated with radioactive material that migrates with product in the described chromatographic systems and thereby reduces the effectiveness of the procedure. The labeled nucleotide, 250 &I in 50% ethanol, was applied to a column (0.9 x 10 cm) of DE-52 (Whatman) that had been equilibrated with 50 mM triethylaminecarbonate at pH 7.6. The column was washed with the same buffer and eluted with a linear gradient (150 ml total volume) established between the starting buffer and 1 M triethylamine-carbonate at the same pH; fractions of 2 ml were collected. PAPS eluted sharply with a peak concentration in frac-
tion 38 (Fig. 1). The major fractions were pooled and brought to dryness at 37°C with a rotary evaporator; the samples were dissolved in water and the procedure was repeated several additional times to remove traces of triethylamine (yield: 67%). PAPS was stored in aqueous solution at -20°C. Since most of the acceptor substrates are only minimally soluble in aqueous buffers, all were dissolved in acetone such that the final solvent concentration in the reaction mixture was 4 to 5%. Crude extracts of rat liver, prepared by homogenization in 3 vol of 10 mM Trischloride at pH 7.4 containing 0.25 M sucrose and 5 mM 2-mercaptoethanol, were centrifuged at 100,000 g for 90 min. The supernatant fluid served as an enzyme source as did homogeneous enzymes (9) from the same organ. Radioactivity was measured by adding the sample to 10 ml of Aquasol (New England Nuclear) with the use of a Model LS8100 scintillation spectrometer (Beckman). BINDING ASSAY
A number of steriod sulfates are separable from radioactive sterol substrates by a filter technique which uses to advantage the greater polarity of the sulfate esters. Sulfotransferase activity was measured in reaction mixtures of 50 ~1 total volume containing 0.25 M sodium acetate at pH 5.5, 50 PM radioactive sterol (about 10 &il pmol), 1.8 mM PAPS and 4% acetone. In preparing assay mixtures it is convenient to first evaporate the radioactive sterol mixed with unlabeled carrier to dryness and to dissolve the dry compound in acetone. When the resulting solution is added to the sodium acetate buffer and agitated with a Vortex mixer, a dispersion of the sterol is formed that is stable for several hours. Aliquots are then removed for individual assays, to which are added the other components; enzyme is added last to initiate the reaction. After 30 min at 37”C, the reaction vessel is placed in a boiling water
84
SEKURA
ET AL.
bath for 2 min. Upon cooling, 50 ~1 of 0.5 mM carrier sterol in absolute ethanol is added and mixed. A 20+1 aliquot of the reaction mixture is applied to the center of a 3-cm square of silica gel-impregnated glassfiber sheet (Gelman Instrument Co., ITLCSG). After the spot has dried, the square is placed on a Hoefer FH- 12 filtration apparatus. A circular disk, with the radioactive material in the middle, is easily cut from the square filter by placing the cylindrical steel sleeve onto the filter apparatus with a twisting motion. The resultant filter disk is washed Time lminl Protein lpll under slightly reduced pressure three times FIG. 2. Dependence of the binding assay and the with 3-ml portions of 25% dioxane in hexane (v/v). Under these conditions, sterol sulfates chromatographic assay on protein concentration and time of incubation. [WlDehydroepiandrosterone was remain bound to the filter whereas unesterthe substrate in the binding assay (0) with a crude liver ified sterol is removed by the solvent. extract (30 mg/mi). The chromatographic assay was The entire filter disk is placed in a scintilused with [%]PAPS and a partially purified enzyme (0.3 mg/ml) with butanol as acceptor (Cl) as well as lation vial and counted in 10 ml of Aquasol. Nonspecific binding is determined by in- with a homogeneous enzyme (6 &ml) and p-naphthol cluding a parallel assay lacking PAPS. The as acceptor (0). amount of radioactive product is calculated level of product recovery was estimated by by determining the fraction of the radioactivity in the isolated sulfate product com- allowing the reaction in a complete assay pared to that present in a sample not sub- mixture to proceed to apparent completion. By this means it was demonstrated that more jected to filtration. than 95% of the sterol substrate could be reEvaluation and Comments covered on the filter as the sulfate. In addition to dehydroepiandrosterone, the method With homogenates of rat liver as enzyme source and dehydroepiandrosterone as ac- has been used with As-androsten-38, 17pdiol, P-estradiol, and testosterone. ceptor, sulfate transfer to sterol was linear We have observed several factors that afwith protein concentrations as high as 2.5 fect the amount of background radioactivity mg/ml for a period of more than 40 min. binding to the filters. High blanks are found Linearity was observed in the formation of when high concentrations of protein are between 0.1 to 1 nmol of product (Fig. 2). present in the incubation mixture but can be We emphasize that the large amount of PAPS used here i.e., 1.8 mM, is essential compensated for by including an appropriate control without PAPS for each sample. Adsince lower concentrations result in deviation there is significant day to day from linearity. This observation appears to ditionally, variation in nonspecific binding to the filters reflect the rapid hydrolysis of PAPS by crude which amounts to 1 to 10% of the applied homogenates. When the assay is used with radioactivity. This variation is a function of partially purified or homogeneous preparaambient humidity and is predictable from tions of sterol sulfotransferases these difthe weather. Lower and more uniform levels ficulties are not encountered and the PAPS of nonspecific binding are attained by placconcentration may be reduced to 0.2 mM. after spotting The method is reasonably precise; five ing the filters, immediately duplicate samples led to a SD of +3%. The with sample, on a rack within a covered
ASSAY OF SULFOTRANSFERASES
8.5
80 PM [35S]PAPS (170 Ql~mol), and the appropriate alcohol at a concentration of 5 mM. After addition of enzyme, the reaction is allowed to proceed for 20 min at room temperature and is stopped with 50 ~1 of CHROMATOGRAPHIC ASSAY acetone. Aliquots (5 ~1) of reaction mixtures are Measurement of sulfotransferase activity applied to a thin-layer strip (1 x 10 cm) of by chromatographic assay is more versatile cellulose on a plastic backing (Eastman No. and its special recommendation is that blank 6064). Once the samples are dry, the chrovalues are close to zero, a situation of parmatographic strips are developed with nticular importance in the determination of propanol:ammonia:water (6:3: 1) at 4°C for kinetic constants with poor substrates. The phenol and at room temperature for sterol method has been useful for a large number sulfates. For the alcohol sulfates, a lo- to of phenols,4 sterols5 and aliphatic alcohols6 15-~1 aliquot is applied to a strip of silica although optimum incubation conditions coated gel (Eastman No. 13 181) and dediffer because of separate requirements of veloped with 2-propanol:chloroform:meththe enzymes catalyzing the reactions. anol:water (10: 10:5:2) at room temperature. Sulfate transfer to phenols is assayed in a After the solvent has moved about 8 cm, total volume of 50 ~1 containing 0.25 M sothe strips are removed and the solvent front dium phosphate at pH 6.5, 5 mM 2-mercapis marked. For the phenol system, a 2-cm toethanol, 0.1 mM [35S]PAPS (130 &i/ section is cut below and including the solvent Fmol), the appropriate phenol at a concenfront and placed directly into a scintillation tration of 0.5 mM, and 5% acetone. The revial containing 10 ml of Aquasol; a 1.2-cm action is initiated by addition of enzyme and, after 10 min at 37°C is terminated by addi- section is cut for the alcohol and sterol sulfates. tion of 13 ~1 of 2 M acetic acid. With sterols as acceptor, the incubation Evaluation and Comments mixture in a total volume of 50 ~1 includes 0.25 M sodium acetate at pH 5.5, 50 PM With [“‘SIPAPS that has been purified by sterol, 60 PM [35S]PAPS (270 ~Cil~mol), and chromatography on DEAE-cellulose, there 4% acetone. After addition of enzyme and is essentially no radioactive contaminant incubation at 37” for 30 min, the reaction is that migrates with the sulfate esters thereby terminated by placing the covered reaction enabling the determination of low levels of vessels into a boiling water bath for 2 min. product. The limits of the assay are delineFor aliphatic alcohols, transferase activated in Fig. 2 for sulfotransferases active with ity is measured in a total volume of 100 ~1 P-naphthol and with butanol. It will be evicontaining 0.1 M Tris-chloride at pH 7.5, dent that linearity is achieved both as a function of time and of protein concentration ’ a- and fl-naphthol, o-, m-. andp-chlorophenol, 2.4within the conditions described. Although dinitrophenol, acetaminophenol, o-, m-. and p-nitronot shown, linearity over a similar time phenol, m- andp-cresol. phenol, 5-hydroxytryptamine, course and range of protein concentration 5-hydroxyindole, hydroquinone, p-methoxyphenol, phydroxybiphenyl. m-methylphenol. was attained with dehydroepiandrosterone. j Androsterone, dehydroepiandrosterone, h”-androRecovery of product was apparently comsten-3P, 17,&diol: J5-pregnen-3p-ol-20-one, estradiol. plete since the results of the chromatocorticosterone, hydrocortisone, and testosterone. graphic and the methylene blue (2) assays 6 I-Butanol. I-heptanol, and 2-heptanol; the method were identical when performed on the same is not applicable to the sulfates of methanol, ethanol, and propanol which migrate at a lower R,. sample over a wide range of product contrough containing warm water. The filters are allowed to remain in the humid atmosphere for 10 min and are then washed with solvent as described above.
86
SEKURA ET AL.
centration. At the level of 100 pmol formed in an incubation mixture, the SD was 22%.
ily applicable to the determination of monoester sulfates in the range of 50 to 400 pmol. REFERENCES
DISCUSSION In the course of a year of experience with the two assay procedures, we have found them dependable and simple to perform. The binding assay is particularly rapid allowing 100 samples to be processed per day but requires a radioactive compound as acceptor and is limited in its sensitivity by appreciable binding of substrate itself to the filter. Although less rapid, the chromatographic assay results in clean separation of substrate and products and may be increased in sensitivity by use of lower dilutions with carrier PAPS. Both assay systems are read-
Roy, A. B. (1956)Biochem. J. 62,41-50. Nose, Y., and Lipmann, F. (1958) J. Biol. Chem. 233, 1348-1351. Banerjee, R. K., and Roy, A. B. (1966) Mol. Pharmacol. 2, 56-66. Singer, S. S., Giera, D., Johnson, J., and Sylvester, S. (1976) Endocrinology 98, 963-974. 5. Rozhin, J., Huo, A., Zemlicka, J., and Brooks, S. C. (1977) J. Biol. Chem. 252, 7214-7220. 6. Spencer, B. (1960) Biochem. J. 77, 294-304. 7. Mattock, P., and Jones, J. G. (1970) Biochem. J. 116, 797-803. 8.
McEvoy, F. A., and Carroll, J. (1971)Biochem.
J.
123, 904-906. 9.
Sekura, R. D., and Jakoby, W. B. (1979) J. Biol. Chem. 254, in press.