Sulfation of mono- and diaryl oximes by aryl sulfotransferase isozymes

Aryl sulfotransferases (3'-phosphoadenylsulfate:phenol sulfotransferase, EC 2.8.2.1) catalyze the sulfonation of a wide variety of hydroxyl-containing substrates, including numerous xenobiotics. The chemical diversity of aryl sulfotransferase substrates is in part attributable to the presence of multiple isozymes, each of which has broad substrate specificity. Of the aryl sulfotransferase isozymes in rat liver cytosol, two (designated isozymes I and II) have previously been shown to sulfonate phenolic compounds exclusively and, moreover, have very similar substrate specificity patterns. The recently reported unusually efficient, rapid isozyme I-catalyzed sulfonation of 9-fluorenone oxime (Mangold, J.B., Mangold, B.L.K. and Spina, A. (1986) Biochim. Biophys. Acta 874, 37-43) was therefore unexpected and suggested that aryl oximes may represent a useful class of model compounds to probe isozymic differences in substrate steric and electronic requirements. In the present study, several mono- and diaryl oximes have been prepared and tested as potential substrates for partially purified aryl sulfotransferases I and II from rat liver cytosol. The results indicate that steric factors, specifically planarity and hydroxyl group position, appear to be important requirements for enzyme-catalyzed sulfonation. In addition, although isozymes I and II had comparable activity with diaryl oximes, some striking differences in the ability of these two isozymes to sulfonate both substituted and unsubstituted monoaryl oximes were observed. This dissimilarity is consistent with distinct differences in the active sites of these isozymes.     

In vivo and in vitro oxidative regulation of rat aryl sulfotransferase IV (AST IV)

Sulfotransferase catalyzed sulfation is important in the regulation of different hormones and the metabolism of hydroxyl containing xenobiotics. In the present investigation, we examined the effects of hyperoxia on aryl sulfotransferase IV in rat lungs in vivo. The enzyme activity of aryl sulfotransferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.     

Assay of purified aryl sulfotransferase suitable for reactions yielding unstable sulfuric acid esters

An assay procedure for purified aryl sulfotransferase is described. The method utilizes isocratic paired-ion reverse-phase HPLC analysis of adenosine-3',5'-diphosphate formed in the reaction. Evaluation of the assay procedure was carried out with 1-naphthalene-methanol as a model substrate for purified rat hepatic aryl sulfotransferase IV. Kinetic constants for sulfation of 1-naphthalenemethanol determined by this method compared favorably with those determined using thin-layer chromatographic assays of 35S incorporation. These results indicate that the method will be suitable for determination of kinetic constants in sulfotransferase-catalyzed reactions where the product sulfuric acid ester may be chemically unstable.     

4-Hydroxytamoxifen sulfation metabolism

Tamoxifen (TAM) is an important chemotherapeutic agent for the treatment of breast cancer. It has also been shown to decrease breast cancer incidence in healthy women at high risk for the disease. The increased risk of endometrial cancer in women has raised concerns in the use of the drug. Tamoxifen has also been shown to be a potent hepatocarcinogen in rats. The oxidative metabolites of TAM include alpha-hydroxytamoxifen (alpha-OH-TAM) and 4-hydroxytamoxifen (4-OH-TAM). The studies on the sulfation of these metabolites are very limited. It has been reported that alpha-OH-TAM is a substrate for rat hydroxysteroid sulfotransferase a (STa). Our studies on the sulfation of 4-OH-TAM demonstrated that 4-hydroxytamoxifen can be sulfated by human liver and human intestinal cytosols. Human phenol-sulfating sulfotransferase and human estrogen sulfotransferase are the major enzymes for the sulfation of 4-OH-TAM. Human dopamine-sulfating sulfotransferase also has sulfation activity for 4-OH-TAM. In contrast, rat liver and intestine cytosols have no detectable sulfation activity for 4-OH-TAM. The results suggest that the alpha-OH-TAM sulfation pathway leads to bioactivation of TAM, and the 4-OH-TAM sulfation pathway leads to detoxification of TAM. This agrees with the fact that TAM is more toxic for rats than for human beings.     

Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.     

Benzylic alcohols as stereospecific substrates and inhibitors for aryl sulfotransferase

Aryl sulfotransferase IV catalyzes the 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent formation of sulfuric acid esters of benzylic alcohols. Since the benzylic carbon bearing the hydroxyl group can be asymmetric, the possibility of stereochemical control of substrate specificity of the sulfotransferase was investigated with benzylic alcohols. Benzylic alcohols of known stereochemistry were examined as potential substrates and inhibitors for the homogeneous enzyme purified from rat liver. For 1-phenylethanol, both the (+)-(R)- and (-)-(S)-enantiomers were substrates for the enzyme, and the kcat/Km value for the (-)-(S)-enantiomer was twice that of the (+)-(R)-enantiomer. The enzyme displayed an absolute stereospecificity with ephedrine and pseudoephedrine, and with 2-methyl-1-phenyl-1-propanol; that is, only (-)-(1R,2S)-ephedrine, (-)-(1R,2R)-pseudoephedrine, and (-)-(S)-2-methyl-1-phenyl-1-propanol were substrates for the sulfotransferase. In the case of 1,2,3,4-tetrahydro-1-naphthol, only the (-)-(R)-enantiomer was a substrate for the enzyme. Both (+)-(R)-2-methyl-1-phenyl-1-propanol and (+)-(S)-1,2,3,4-tetrahydro-1-naphthol were competitive inhibitors of the aryl sulfotransferase-catalyzed sulfation of 1-naphthalenemethanol. Thus, the configuration of the benzylic carbon bearing the hydroxyl group determined whether these benzylic alcohols were substrates or inhibitors of the rat hepatic aryl sulfotransferase IV. Furthermore, benzylic alcohols such as (+)-(S)-1,2,3,4-tetrahydro-1-naphthol represent a new class of inhibitors for the aryl sulfotransferase.     

Enzymatic sulfation of galactosyl- and lactosylceramides in cell lines derived from renal tubules.

1. The renal cell lines, JTC-12 and MDCK, not only synthesize galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate in vivo, but also contain enzymes that catalyze the transfer of sulfate to galactosylceramide and lactosylceramide in vitro. 2. Concentration of cations necessary for maximum sulfotransferase activity occurred at 40 mM Ca2+ with galactosylceramide and 15 mM Ca2+ with lactosylceramide as the substrate. Na+ was also found to stimulate the sulfation of galactosylceramide, but was slightly inhibitory for the sulfation of lactosylceramide. 3. The products of the in vitro assay mixture were characterized as galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate by a variety of TLC separations. 4. The apparent Km of JTC-12 cells for galactosylceramide was 17 microM, while that for lactosylceramide was 82 microM. The Km values of MDCK cells were comparable to those of JTC-12 cells. Competition studies suggested that galactosylceramide and lactosylceramide were sulfated by a single enzyme in both cell lines.     

In vivo expression and stoichiometric sulfation of the artificial protein sulfophilin, a polymer of tyrosine sulfation sites.

To gain insight into the structural requirements for tyrosine sulfation in vivo, we have constructed and expressed an artificial gene encoding a polypeptide substrate for tyrosylprotein sulfotransferase. This gene codes for a protein, referred to as sulfophilin, which consists of a 12-times repeated heptapeptide unit corresponding to the identified tyrosine sulfation site of chromogranin B (secretogranin I), Glu-Glu-Pro-Glu-Tyr-Gly-Glu. The gene was fused to the signal sequence of secretogranin II to direct the sulfophilin protein to the secretory pathway. Stable expression of the artificial gene in NIH 3T3 cells resulted in the secretion of sulfated sulfophilin. Analysis of the stoichiometry of sulfation revealed that each of the 12 tyrosyl residues in sulfophilin was sulfated. Remarkably, up to 50% of the total protein-bound tyrosine sulfate secreted by the cells was contained in sulfophilin. The results indicate that the structural information contained in the heptapeptide motif is sufficient for stoichiometric tyrosine sulfation to occur in the living cell.     

Development of a simple homogeneous assay to screen for inhibitors of N-acetylglucosamine-6-sulfotransferases

L-selectin, a leukocyte adhesion molecule, plays a central role in lymphocyte homing to secondary lymphoid tissue and to certain sites of inflammation. Carbohydrate sulfation was implicated in this process, when it was demonstrated that carbohydrate sulfotransferase-mediated sulfation of N-acetylglucosamine (GlcNAc) within sialyl Lewis X of cognate endothelial ligands for L-selectin was an essential modification for L-selectin binding. The recently identified GlcNAc-6-sulfotransferases GlcNAc6ST-1 and -2, which facilitate GlcNAc sulfation by catalyzing the transfer of a sulfonyl group from 3(')-phosphoadenosine 5(')-phosphosulfate (PAPS) to the 6-hydroxy group of the acceptor GlcNAc moiety, contribute to the biosynthesis of the 6-sulfosialyl Lewis X motif. Due to their pivotal role in L-selectin ligand biosynthesis, this enzyme class has recently emerged as an important and relatively unexplored class of potential targets for anti-inflammatory therapy. However, no inhibitors have been reported to date and screening for lead inhibitors has been hampered by the lack of simple assay formats suitable for high-throughput screening. Here, we report the development of a simple homogeneous in vitro sulfotransferase assay using a newly synthesized biotinylated glycoside as a substrate. The assay is based on GlcNAc6ST-2-mediated [35S]sulfate transfer from [35S]PAPS to the biotinylated glycoside and subsequent detection using streptavidin-coated SPA beads. K(m) values with partially purified GlcNAc6ST-2 for PAPS and the biotinylated glycoside were estimated to be 8.4 and 34.5 microM, respectively. The sulfotransferase reaction could be inhibited by 3('),5(')-ADP with an IC(50) of 2.1 microM. The assay can be operated in 384-well format; is characterized by a high signal-to-noise ratio, low variation, and excellent Z factors; and is highly suitable for high-throughput screening.     

Sulfation in vitro of mucus glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid

Enzymatic sulfation of mucus glycoprotein by rat submandibular salivary gland and the effect of prostaglandin and acetylsalicylic acid on this process were investigated in vitro. The sulfotransferase enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein has been located in the detergent extracts of Golgi-rich membrane fraction of the gland. Optimum enzyme activity was obtained at pH 6.8 with 0.5% Triton X-100, 25 mM NaF and 4 mM MgCl2, using the desulfated glycoprotein. The enzyme was also capable of sulfation of the intact mucus glycoprotein, but the acceptor capacity of such glycoprotein was 68% lower. The apparent Km of the submandibular gland sulfotransferase for salivary mucus glycoprotein was 11.1 microM. The 35S-labeled glycoprotein product of the enzyme reaction gave in CsCl density gradient a 35S-labeled peak which coincided with that of the glycoprotein. This glycoprotein upon reductive beta-elimination yielded several acidic 35S-labeled oligosaccharide alditols which accounted for 75% of the 35S-labeled glycoprotein label. Based on the analytical data, the two most abundant oligosaccharides were identified as sulfated tri- and pentasaccharides. The submandibular gland sulfotransferase activity was stimulated by 16,16-dimethyl prostaglandin E2 and inhibited by acetylsalicylic acid. The rate of enhancement of the glycoprotein sulfation was proportional to the concentration of prostaglandin up to 2.10(-5) M, at which point a 31% increase in sulfation was attained. The inhibition of the glycoprotein sulfation by acetylsalicylic acid was proportional to the drug concentration up to 2.5.10(-4) M at which concentration a 48% reduction in the sulfotransferase activity occurred. The apparent Ki value for sulfation of salivary mucus glycoprotein in presence of acetylsalicylic acid was 58.9 microM. The results suggest that prostaglandins may play a role in salivary mucin sulfation and that this process is sensitive to such nonsteroidal anti-inflammatory agents as acetylsalicylic acid.     

Modulation of testicular galactolipid sulfotransferase activity in vitro by ATP

Testicular galactolipid sulfotransferase activity is an early marker of differentiation during mammalian spermatogenesis. The enzyme will catalyze the sulfation of galactosylglycerol in the 3' position of the galactose moiety at 37 degrees C in vitro. However, sulfotransferase activity was found to be completely lost on preincubation of the solubilized enzyme preparation at 37 degrees C. This loss of activity was completely prevented by inclusion of ATP and Triton in the preincubation step. This protective effect was synergistic, pH dependent and correlated with an inhibition of endogenous phosphatase activity. These results are interpreted to suggest that the galactolipid sulfotransferase may be regulated by a phosphorylation mechanism.     

Tyrosine-ester sulfotransferase from rat liver: bacterial expression and identification.

A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and transformed into Escherichia coli. The protein, expressed in large amounts, was not aryl sulfotransferase (EC 2.8.2.1) but rather tyrosine-ester sulfotransferase (EC 2.8.2.9), a sulfotransferase also active with phenols but having a much wider substrate range that includes hydroxylamines and esters of tyrosine. The recombinant tyrosine-ester sulfotransferase was identified by its unique substrate spectrum, by comparison with three peptides that were sequenced from homogeneous tyrosine-ester sulfotransferase isolated directly from rat liver, and by the specificity of antibody raised to the rat liver enzyme. Two isoforms were obtained, each of which was difficult to solubilize upon sonication of E. coli. Both forms were solubilized with a solution of polyols (glycerol and sucrose) and subsequently purified to homogeneity.     

Enzymatic sulfation of glycochenodeoxycholic acid by tissue fractions from adult hamsters

Using a radiometric assay with glycochenodeoxycholic acid as substrate, bile acid:3'-phosphoadenosine-5'-phosphosulfate sulfotransferase activity was found in 105,000 g supernatant fractions of liver, proximal intestine, and adrenal gland homogenates from adult hamsters. Optimum conditions for measurement of the hepatic enzyme were determined. In both male and female animals sulfation only occurred at the 7 alpha-position. Saturation analysis with glycohenodeoxycholic acid revealed that the higher activity observed in fractions from female compared to male hamsters was due to a 4-fold lower apparent Km (79 muM vs. 317 muM) for this bile acid in the females. The sulfation of glycohenodeoxycholic acid was competitively inhibited by glycolithocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid. The data are consistent with the concept that sulfation of many, if not all, bile acids can occur in vivo.     

Role of surfactants in optimizing fluorene assimilation and intermediate formation by Rhodococcus rhodochrous VKM B-2469

Biodegradation of fluorene by Rhodococcus rhodochrous VKM B-2469 was investigated and optimized by adding non-ionic surfactants to the liquid media. The utilization of 1-1.5% Tween 60 or 1% Triton X100 allowed to solubilize 1 mM fluorene over 150 times more than in water medium (from 9-11 microM to above 1.5 mM at 28 degrees C). We observed that Tween 60 was useful to enhance the fluorene biodegradation rates further supporting R. rhodochrous VKM B-2469 growth as an additional carbon source and to decrease fluorene toxicity for bacterial cells whereas Triton X100 resulted to be toxic for this strain. An additional enzyme induction step before starting the bioconversion process and the increase of incubation temperature during fluorene bioconversion led to further improvements in rates of fluorene utilization and formation of its intermediates. In the optimized conditions 1 mM fluorene was degraded completely within 24h of incubation. Some intermediates in fluorene degradation built up during the process reaching maxima of 31% for 9-hydroxyfluorene, 2.1% for 9-fluorenone and 1.9% for 2-hydroxy-9-fluorenone (starting from 1 mM substrate). In the presence of Tween 60 the appearance and following conversion of 2-hydroxy-9-fluorenone was observed for R. rhodochrous VKM B-2469 revealing the existence of a new pathway of 9-fluorenone bioconversion.     

Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases.

Sulfation is an important pathway in the metabolism of thyroid hormones. Sulfated iodothyronines are elevated in nonthyroidal illnesses and in the normal human fetal circulation. We assayed and characterized COS-1 cell expressed recombinant human liver dehydroepiandrosterone sulfotransferase (DHEA ST or SULT2A1) and estrogen sulfotransferase (EST or SULT1E1) activities for the first time with triiodothyronine (T(3)) as the substrate. Several biochemical properties that included apparent K(m) values, thermal stabilities, and responses to the inhibitors 2, 6-dichloro-4-nitrophenol and NaCl were tested. SULT2A1, a member of the hydroxysteroid sulfotransferase family, used 3,3'-T(2) more readily than T(3) and 3,5-T(2) as substrates, but had the lowest apparent K(m) value for T(3) of any reported human SULT. SULT1E1, a member of the phenol sulfotransferase family, used 3,3'-T(2) and rT(3) more readily than T(3), and also displayed the greatest specificity for T(4) among human SULTs. SULT2A1 may contribute more to iodothyronine sulfation than previously suspected. Potential roles of both steroid sulfotransferases in the enhanced sulfation of nonthyroidal illnesses and fetal development invite further investigation.     

Enzymes responsible for synthesis of corneal keratan sulfate glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.     

Tyrosine sulfation of statherin

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.     

Analysis of the substrate specificity of tyrosylprotein sulfotransferase using synthetic peptides

Tyrosylprotein sulfotransferase (TPST) catalyzes the sulfation of proteins at tyrosine residues. We have analyzed the substrate specificity of TPST from bovine adrenal medulla with a novel assay, using synthetic peptides as substrates. The peptides were modeled after the known, or putative, tyrosine sulfation sites of the cholecystokinin precursor, chromogranin B (secretogranin I) and vitronectin, as well as the tyrosine phosphorylation sites of alpha-tubulin and pp60src. Varying the sequence of these peptides, we found that (i) the apparent Km of peptides with multiple tyrosine sulfation sites decreased exponentially with the number of sites; (ii) acidic amino acids were the major determinant for tyrosine sulfation, acidic amino acids adjacent to the tyrosine being more important than distant ones; (iii) a carboxyl terminally located tyrosine residue may be sulfated. Moreover, TPST catalyzed the sulfation of a peptide corresponding to the tyrosine autophosphorylation site of pp60v-src (Tyr-416) but not of a peptide corresponding to the non-autophosphorylation site of pp60c-src (Tyr-527). These results experimentally define structural determinants for the substrate specificity of TPST and show that this enzyme and certain autophosphorylating tyrosine kinases have overlapping substrate specificities in vitro.     

A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV.

We have developed a continuous spectrophotometric coupled-enzyme assay for sulfotransferase activity. This assay is based on the regeneration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from the desulfated 3'-phosphoadenosine-5'-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-IV) is expressed, resolved to the pure beta-form during purification, and utilized for the regeneration. The activity of betaAST-IV to catalyze the synthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capillary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. betaAST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod factor sulfotransferase is verified with an enzyme concentration study and substrate-specificity assays of N-chitoses. The potential applications of this assay include rapid kinetic determinations for carbohydrate and protein sulfotransferases, high-throughput screening of potential sulfotransferase substrates and inhibitors, and biomedical screening of blood samples and other tissues for specific sulfotransferase enzyme activity and substrate concentration.