Aryl Sulfate Research Articles

Quantification of 19‐nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry

Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-nortestosterone-17beta-sulphate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) had potential for the detection of 19-nortestosterone misuse in the male horse. The high concentrations of sulphate conjugates of the female sex hormones naturally present in male equine urine were overcome by selective hydrolysis of the aryl sulphates using glucuronidase from Helix pomatia; this was shown to have little or no activity for alkyl sulphates such as 19-nortestosterone-17beta-sulphate. The 'free' phenolic steroids were removed by solid-phase extraction (SPE) prior to LC/MS/MS analysis. The method also allowed for the quantification of the sulphate conjugate of boldenone, a further anabolic steroid endogenous in the male equine with potential for abuse in sports. The method was applied to the quantification of these analytes in a population of samples. This paper reports the results of that study along with the development and validation of the LC/MS/MS method. The results indicate that while 19-nortestosterone-17beta-sulphate is present at low levels as an endogenous substance in urine from 'normal' male horses, its use as an effective threshold substance may be viable.

Direct Evidence for ArOS Bond Cleavage upon Inactivation of Pseudomonas aeruginosa Arylsulfatase by Aryl Sulfamates

Pseudomonas aeruginosa arylsulfatase catalyses the cleavage of aryl sulfates and is an excellent model for human estrone sulfatase, which is implicated in hormone-dependent breast cancer. Aryl sulfamates are inactivators of sulfatases; however, little is known about their mechanism. We studied the inactivation of Pseudomonas aeruginosa arylsulfatase A by a range of aryl sulfamates, including the clinical agent 667COUMATE (STX64) used to inactivate estrone sulfatase. Inactivation was time dependent, irreversible, and active-site directed, consistent with a covalent modification at the active site. In terms of the kinetic parameters of inactivation k(inact) and K(i), K(i) values are in the micromolar to nanomolar range, and the inactivation half-life is less than 30 s. A Brønsted plot of k(inact)/K(i) has a steep slope (beta(lg) = -1.1), which implies that the transition state for the first irreversible chemical step of inactivation involves a high degree of charge transfer and cleavage of the ArO-S bond. Detection of the released phenol and titration of the residual activity showed the stoichiometry of inactivation to be in the range 3-6, with the greatest values found for the most effective inactivators. Thus, multiple sulfamoylation events appear to occur during the inactivation process. These data provide valuable insight into the mechanism of sulfatase inactivation by sulfamates.

Vibrational spectroscopic study of sulphated silk proteins

Degummed Bombyx mori ( B. m.) silk fibroin fabric and mutant naked pupa cocoons (Nd-s) consisting of almost pure silk sericin were treated with chlorosulphonic acid in pyridine and investigated by FT-IR and FT-Raman spectroscopies. Untreated silk fibroin and sericin displayed typical spectral features due to characteristic amino acid composition and molecular conformation (prevailing β-sheet with a less ordered structure in sericin). Upon sulphation, the degree of molecular disorder increased in both proteins and new bands appeared. The IR bands at 1049 and 1014 cm −1 were attributed to vibrations of sulphate salts and that at 1385 cm −1 to the ν asSO 2 mode of organic covalent sulphates. In the 1300–1180 cm −1 range various contributions of alkyl and aryl sulphate salts, sulphonamides, sulphoamines and organic covalent sulphates, fell. Fibroin covalently bound sulphate groups through the hydroxyl groups of tyrosine and serine, while sericin through the hydroxyl groups of serine, since the δOH vibrations at 1399 cm −1 in IR and at 1408 cm −1 in Raman disappeared almost completely. Finally, the increase of the I 850/ I 830 intensity ratio of Raman tyrosine doublet in fibroin suggested a change towards a more exposed state of tyrosine residues, in good agreement with the more disordered conformation taken upon sulphation.

Symbioimine and Neosymbioimine, Amphoteric Iminium Metabolites from the Symbiotic Marine Dinoflagellate Symbiodinium sp.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Symbioimine and neosymbioimine, amphoteric iminium metabolites from the symbiotic marine dinoflagellate Symbiodinium sp.

Two amphoteric iminium metabolites, symbioimine ( 1) and neosymbioimine ( 2), were isolated from a cultivated symbiotic marine dinoflagellate Symbiodinium sp. Compounds 1 and 2 have a characteristic 6,6,6-tricyclic iminium ring structure and an aryl sulfate moiety. The plausible biogenetic pathway of 1 and 2 can be explained by an intramolecular Diels–Alder reaction followed by imine cyclization. Symbioimine ( 1) inhibited the differentiation of RAW264 cells into osteoclasts (EC 50 = 44 μM), and significantly inhibited cyclooxygenase-2 activity at 10 μM. Thus, symbioimine is a potent anti-resorptive and anti-inflammatory drug.

Do electrostatic interactions with positively charged active site groups tighten the transition state for enzymatic phosphoryl transfer?

The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline phosphatase (AP) using linear free-energy relationships (LFERs). We determined k(cat)/K(M) for a series of aryl sulfate ester monoanions to obtain the Brønsted coefficient, beta(lg), and compared the value to that obtained previously for a series of aryl phosphorothioate ester dianion substrates. Despite the difference in substrate charge, the observed Brønsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis (-0.76 +/- 0.14 and -0.77 +/- 0.10, respectively) are strikingly similar, with steric effects being responsible for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition states in solution. These observations suggest an apparent equivalency of the transition states for phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active site electrostatic interactions on charge distribution in the transition state.

Synthesis and Protection of Aryl Sulfates Using the 2,2,2‐Trichloroethyl Moiety.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Symbioimine exhibiting inhibitory effect of osteoclast differentiation, from the symbiotic marine dinoflagellate Symbiodinium sp.

An amphoteric iminium metabolite, symbioimine (1), was isolated from a cultivated symbiotic marine dinoflagellate Symbiodinium sp. Its structure was deduced by spectroscopic analysis and X-ray crystallographic analysis. Symbioimine (1) has a characteristic 6,6,6-tricyclic iminium ring structure and an aryl sulfate moiety. The plausible biogenetic pathway of 1 can be explained by an intramolecular Diels-Alder reaction followed by imine cyclization. Symbioimine (1) inhibited the differentiation of RAW264 cells into osteoclasts, whereas its cell viability was not affected. Thus, symbioimine (1) is an antiresorptive drug candidate for the prevention and treatment of osteoporosis in postmenopausal women.

Scavenger properties of cultivated pig liver endothelial cells

BackgroundThe liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC.ResultsEndocytosis of five 125I-labelled molecules: collagen α-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of α-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells.ConclusionPig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC.

Synthesis and protection of aryl sulfates using the 2,2,2-trichloroethyl moiety.

[reaction: see text] The 2,2,2-trichloroethyl (TCE) group was utilized as the first protecting group for aryl sulfates. Aryl sulfates, protected with the TCE group, were prepared in high yield by reacting phenols with chlorosulfuric acid TCE ester. Deprotection was accomplished using Pd/C-ammonium formate or with Zn-ammonium formate to give aryl sulfate monoesters in high yield. This approach to aryl sulfate synthesis was successfully applied to the construction of estrone sulfate derivatives, which could not be prepared by previous methodologies.

Amperometric Ion Detection in Capillary Zone Electrophoresis by Ion Transfer across a Liquid−Liquid Microinterface

Ion transfer across the electrically charged interface of two immiscible electrolytes has been employed for the amperometric detection of ions in capillary zone electrophoresis (CE). The method is inherently selective to hydrophobic ions, which are characterized by a low standard Gibbs energy of partition between the aqueous CE buffer solution and 2-nitrophenyl n-octyl ether employed as organic phase. The applicability of this detection scheme for nonredox ions is demonstrated by means of the cations choline and acetylcholine and by some alkyl and aryl sulfate and sulfonate anions. The response of the detector is sufficiently fast; no additional peak broadening has been observed. Plate numbers of 100 000(170 000 m -1 ) were determined for the quickly migrating choline cation. It is shown that the use of buffer modifiers such as organic solvent additives, cyclodextrins, and polyelectrolytes is compatible with the detector within certain boundaries. The limit of detection was ∼ 1 μM for the ions tested.

The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-313.

Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded by ssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa.

Thermospray mass spectrometry and tandem mass spectrometry of polar, urinary metabolites and metabolic conjugates.

This paper describes the characterization of glucuronide and sulfuric acid conjugates and alkyl phosphates by thermospray tandem mass spectrometry (MS/MS). Primary thermospray mass spectra were generated using ammonium acetate buffer and filament-on ionization with both positive and negative ion detection. In positive ion mode molecular weight information was obtained for glucuronic, phosphoric and other weak acids. Under these conditions, however, spectra were not obtained for the sulfate adducts or phosphate salts. Negative ion thermospray mass spectrometry was more versatile, providing spectra of all metabolic structures examined. Positive and negative ion mass spectra provided complementary information for glucuronic acids. Collisionally activated dissociation (CAD) mass spectra of the glucuronide [M + NH4]+ or [M - H]- ions exhibited characteristic glucuronide 'fingerprints' as well as prominent aglycone ions. The aryl sulfates were hydrolyzed to the corresponding phenols under buffer/thermospray conditions and for these analytes CAD mass spectra were obtained from [phenate]- or [phenol + acetate]- parent ions. The M- of dimethyl thiophosphate underwent sequential loss of alkyl and alkoxy radicals and formaldehyde with collisional activation. MS/MS greatly enhances the power of the thermospray interface by providing fragmentation information useful both in the identification of unknowns and for improved mass spectrometry specificity.

Arylsulfatases: Colorimetric and fluorometric assays

This chapter discusses the colorimetric and fluorometric assays of arylsulfatases. The assay of arylsulfatases is simple in principle, and any of the reaction products may be determined. The general method is simple and requires only that the reaction mixture, after incubation with the enzyme, be made alkaline so that the liberated phenol can be determined, as the phenoxide, either spectrophotometrically or fluorometrically. With crude enzyme preparations deproteinization with, for example, ethanol or trichloroacetic acid may be necessary but any acid treatment should be as mild and as brief as possible because all aryl sulfates are acid-labile. In some cases, the spectra of the phenol and its sulfate ester differ sufficiently at the pH of arylsulfatase activity for the formation of the former to be followed directly so that continuous spec trophotometric assays are possible. Unhydrolyzed substrate can be determined spectrophotometrically. The lysosomal sulfatases A and B have pH optima at about 5, and acetate buffers, or formate buffers at lower pH, are generally suitable for their assay. These enzymes are inhibited by phosphate. The microsomal sulfatase C of mammalian tissues, and the arylsulfatases of mollusks and of microorganisms, have pH optima in the neutral or slightly alkaline region so that imidazole or Tris buffers may be used in their assay. Arylsulfatases may be quite sensitive to changes in the salt or buffer concentration of the reaction mixture so that careful control of the assay conditions is essential. Sulfatases A are hysteretic enzymes that undergo a substrate-induced inactivation with a half-time of a few minutes. The chapter also discusses the substrates for the arylsulfatases.

Electrophilic catalysis of sulphate (–SO3–) group transfer: hydrolysis of salicyl sulphates assisted by intramolecular hydrogen bonding

The hydrolysis of substituted salicyl sulphates has been measured over a range of pH values at 70 °C to obtain kinetic parameters for hydronium ion (KH) and carboxylic acid (Kp) catalysis. A Jaffe treatment of the carboxy-catalysis parameter for a range of nuclear substituents yields ρphenol 1.51; ρcarboxy 0 indicates that the carboxy-function does not ionise on going from the ground-to the transition-state, consistent with hydrogen bonding rather than catalysis through proton transfer. The change in ‘effective’ charge on the phenol oxygen on going from the ground-to the transition-state confirms less build up of negative charge than in the uncatalysed hydrolysis of aryl sulphates.

A method for the sulfation of sugars, employing a stable, aryl sulfate intermediate

A method for the synthesis of sugar sulfates is described which, unlike the methods in general use, involves incorporation of the sulfate function in the form of a protected organosulfate. For example, the reaction of phenyl chlorosulfate with 1,2:5,6-di- O-isopropylidene-α- d-glucofuranose afforded the 3-(phenyl sulfate) of the latter in high yield. Deprotection, to obtain the 3-sulfate derivative, was readily effected by catalytic hydrogenolysis. As the (phenyl sulfate) substituent is relatively stable under a variety of conditions, it would be expected to survive an array of chemical transformations made on esterified sugars, or derivatives. Also, it is more compatible with general synthetic and purification procedures than an ionic sulfate group. For these reasons, the (phenyl sulfate), or analogous organosulfate, substituent should be particularly well suited to the synthesis of complex sulfates, including those of higher saccharides.

The structural basis of the anomalous kinetics of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K i values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of 35S-labeled substrate at pH values near the pH optimum (pH 5–6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.

Subunit affinity chromatography and its application to the isolation of aryl sulfatase A enzymes

The monomeric form of rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) was covalently coupled to CNBr-activated Sepharose and the catalytic properties of the covalently coupled monomer subunit were examined. The immobilized subunit showed one pH optimum near pH 5.6 which appears to be the characteristic pH optimum of the monomer. The enzyme-Sepharose complex exhibited the characteristic anomalous kinetic behavior at pH 5.5 but there was no turnover-induced inactivation of the immobilized enzyme at pH 4.5. The covalently coupled subunit column was examined for its ability to act as a subunit affinity chromatography medium. It was found that dissolved aryl sulfatase A was removed from solution at pH 4.5 and pH 5.0, I = 0.2, and became associated with the affinity column of Sepharose-aryl sulfatase A. The retained subunit of the enzyme could subsequently be quantitatively eluted with 0.2 m Tris-HCl, pH 7.5. Extraneous protein such as bovine serum albumin did not measureably affect the rate or equilibrium for association of the enzyme to the covalently bound subunit. The extent of binding of the enzyme to the affinity column was found to be strongly dependent on the time of equilibration and on the pH. About 90% of the enzyme was retained after 24 h at pH 5.0, I = 0.2. Under otherwise comparable conditions, use of Sepharose-6MB resulted in slightly faster association than did Sepharose-4B. Under the experimental conditions employed, the total capacity of the affinity column was approx 50% of the total aryl sulfatase A coupled to the Sepharose. The rabbit liver subunit column also permits the purification of several other aryl sulfatase A enzymes. Thus, the subunit affinity column provides a simple, convenient, and rapid procedure for the isolation of most mammalian aryl sulfatase A enzymes as well as for studying inter- and intraspecific subunit association interactions.

Sulphatase A: an arylsulphatase and a glycosulphatase.

Sulphatase A was first described as an arylsulphatase but was subsequently shown to have cerebroside sulphatase activity, bydrolysing galactose 3-sulphate residues in certain lipids. A characteristic feature of the arylsulphatase activity is the substrate-induced inactivation of the enzyme which occurs during the catalytic reaction. Present views of the course of this modification are considered. A similar modification of the enzyme does not occur during the cerebroside sulphatase reaction and the fall in velocity noted during most such assays can be explained by disappearance of substrate and accumulation of sulphate. Possible reasons for the difference between the two types of activity are considered. Sulphatase A also hydrolyses hexose sulphates at rates comparable to those of aryl sulphates. The specificity of sulphatase A towards its natural substrates, sulpholipids, is considered in the light of these findings.

The monomer-dimer association of rabbit liver aryl sulfatase A and its relationship to the anomalous kinetics

The polymerization of aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) has been studied by frontal gel chromatography on Sephadex G-200 and Bio-Gel A-5m under various conditions of pH, ionic strength, and temperature. The aryl sulfatase A molecule exists as a monomer and as a dimer at pH 7.5 and pH 4.5, respectively. The extent of dissociation is markedly pH-, protein concentration-, and ionic strength-dependent. Only a small effect of temperature was observed. The enthalpy change (Δ H o ) for the dissociation was −2.5 ± 1 kcal/mol at pH 5.5–5.6, and the entropy change for dissociation of the enzyme dimer to two monomeric units was −47 cal mol −1 deg −1. Sulfate ion has little effect on the extent of dissociation of the enzyme at pH 5.6. The present studies suggest that the dissociation of rabbit liver aryl sulfatase A is regulated by the ionization of amino acid residues whose apparent p K is between pH 5 and 6. The driving force for the association of the subunits of the enzyme is primarily ionic and/or ionic/hydrogen bond formation. The small enthalpy change and the fact that dissociation is strongly favored by an increase in the ionic strength suggest that hydrophobic interactions play only a minor role in stabilizing the dimeric quaternary structure relative to the monomeric state. The monomeric form of the enzyme exhibits the anomalous kinetics often observed with sulfatase A but the dimer does not show anomalous kinetics. Since aryl sulfatase A is probably in the dimeric form in the lysosome, the anomalous kinetics of the enzyme are unlikely to be of physiological importance in the intact lysosome.