Nature of o-phthalaldehyde reaction with pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase (FAS) was inactivated irreversibly by stoichiometric concentration of o-phthalaldehyde exhibiting a bimolecular kinetic process. FAS-o-phthalaldehyde adduct gave a characteristic absorption maxima at 337 nm. Moreover this derivative showed fluorescence emission maxima at 412 nm when excited at 337 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The inactivation is caused by the reaction of the phosphopantetheine -SH group since it is protected by either acetyl- or malonyl-CoA. The enzyme incubated with iodoacetamide followed by o-phthalaldehyde showed no change in fluorescence intensity but decrease in intensity was found in the treatment of 2,4,6-trinitrobenzenesulphonic acid (TNBS), a lysine specific reagent with the enzyme prior to o-phthalaldehyde addition. As o-phthalaldehyde did not inhibit enoyl-CoA reductase activity, so nonessential lysine is involved in the o-phthalaldehyde reaction. Double inhibition experiments showed that 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a thiol specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 moles of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation.     

Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a ''half-of-the-sites'' mechanism.

Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)     

A method for the estimation of thiol esters.

1. A method is described for the estimation of thiol ester groups. The thiol ester is converted into the corresponding thiol by reaction with ammonia; the thiol is then titrated amperometrically with mercuric chloride. 2. The method may be used in the presence of SH and S.S groups. The SH groups are titrated at pH3 in the presence of excess of chloride; under these conditions thiol esters do not react with mercuric chloride. Thiol ester plus thiol is then estimated by titration after reaction with ammonia. Finally, titration after reaction with ammonia and sulphite gives the thiol ester plus thiol plus disulphide. 3. The procedure has been applied to glyceraldehyde phosphate dehydrogenase. The enzyme was found to contain 15-16 SH groups/mol. and no S.S groups. After reaction with acetyl phosphate 1.8-3.5 thiol ester groups were detected, the number depending on the conditions of acetylation. In the absence of bound NAD, the number of thiol ester groups formed was 1.8/mol., although a value of 2.9 labile acetyl groups/mol. was given by the method of Lipmann & Tuttle (1945). The presence of thiol ester groups in the S-(d-3-phosphoglyceryl)-enzyme was also demonstrated.     

The differential reaction of histamine and N tau-methylhistamine with Pauly's diazo reagent: application to assay of histamine N-methyltransferase activity

A simple nonradioisotopic fluorescent method for assay of histamine N-methyltransferase (HMT) activity was developed. After termination of the HMT reaction, the remaining excess substrate, histamine, was degraded by Pauly 's diazo reagent, whereas the product, N tau-methylhistamine (N- MeHA ), was not degraded by the reagent. Then the mixture was applied to high-performance liquid chromatography under conditions in which N- MeHA was not separated from histamine, and N- MeHA was measured fluorometrically by condensation with o-phthalaldehyde. The method would be convenient for measurement of HMT activity during enzyme purification.     

DNA strand scission by hemin-thiolate complexes as models of cytochrome P-450

Hemin-thiolate complexes, as chemical models for cytochrome P-450 monooxygenases, have been shown to cause strand scission of DNA. Circular super-coiled DNA was degraded to open-circular and linear forms by these complexes in 30 min at pH 7.8 under aerobic conditions, the degradation depending on the structure of the thiol ligand and the ratio of thiol ligand to hemin concentration. The relationship between the structure of the thiol ligand and DNA strand cleaving activity was examined. Complete cleavage of DNA was observed by complexes containing TGE and ME at 400-600 moles excess of thiol ligand to hemin, those containing Cys, CysMe, and CysEt at 50-200 moles excess, and those containing MPG, GSH, penta- and nona-peptides at 5-20 moles excess. Complexes containing NACys and MEA caused no cleavage of DNA. Inhibition experiments suggested the involvement of active oxygen species in the cleavage.     

Improving the stability of thiol–maleimide bioconjugates via the formation of a thiazine structure

Linker stability is critically important for the efficacy and safety of peptide and protein conjugates used for biological applications. One common conjugation strategy, thiol–maleimide coupling, generates a succinimidyl thioether linker with limited stability under physiological conditions. We have shown in previous work that when a peptide with an N-terminal cysteine is conjugated to a maleimide reagent, a thiazine structure is formed via a chemical rearrangement. Our preliminary work indicated that the thiazine linker has favorable stability. Here, we report the evaluation of a thiazine linker as an alternative to the widely used succinimidyl thioether linker for thiol–maleimide bioconjugation. The stability of the thiazine conjugate in comparison to the thioether conjugate was assessed across a broad pH range. Additionally, the propensity for retro-Michael reaction and cross-reactivity with other thiols was evaluated by treating conjugates in the presence of glutathione. The studies indicated that the thiazine linker degrades markedly slower than the thioether conjugate. In addition, the thiazine linker is over 20 times less susceptible to glutathione adduct formation. The NMR study of the thiazine structure confirmed that the formation of the thiazine linker is a stereoselective process that yields a single diastereomer. In summary, we propose the use of the thiazine linker obtained by conjugation of maleimide-containing reagents with peptides or proteins presenting an N-terminal cysteine as a novel approach for bioconjugation. The advantages of this approach are the formation of a linker with a well-defined stereochemical configuration, increased stability at physiological pH, and a strongly reduced propensity for thiol exchange.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde

Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of Copper-Catalyzed Oxidation of Glutathione

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal *OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and *OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.     

Ammonium 4-chloro-7-sulfobenzofurazan: a new fluorigenic thiol-specific reagent

This paper describes the synthesis and study of a new fluorigenic, thiol-specific reagent, ammonium 4-chloro-7-sulfobenzofurazan, which is readily prepared by sulfonation of 4-chlorobenzofurazan. Evaluation of ammonium 4-chloro-7-sulfobenzofurazan was undertaken using glutathione as a model thiol peptide and bovine serum albumin and jackbean urease as thiol-containing proteins. Thiol specificity of this reagent was established using various amino acids and peptides including l-cysteine, l-lysine, l-histidine, l-tyrosine, glutathione, and oxidized glutathione. Nitrogen and sulfur derivatives of ethanediol and acetic acid were also investigated. Optimum conditions for the thiol labeling reaction have been investigated using the parameters of pH, buffer type, time, temperature, and relative concentrations. Under appropriate conditions the fluorescence produced by the reaction of ammonium 4-chloro-7-sulfobenzofurazan is linearly related to thiol concentration. The fluorescence intensity of 7-sulfobenzofurazan thiol derivatives are considerably greater than the corresponding 7-nitrobenzofurazan derivatives in both aqueous and aprotic solvents, rendering this reagent highly sensitive. Our preliminary experiments with proteins labeled with ammonium 4-chloro-7-sulfobenzofurazan have shown the probe to be removable by thiolysis with excess 2-mercaptoethanol.     

Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges.

A convenient, practical route to the synthesis of disulfide-bridged oligonucleotides has been developed. Aliphatic linkers with terminal thiol groups have been attached to the phosphodiester backbones of partially or fully complementary oligonucleotide sequences and oxidized to yield covalently closed oligonucleotides with disulfide bridges. This procedure has been used to prepare a duplex with disulfide bridges at both ends and stem-loop sequences with single disulfide bridges. Oxidation of a self-complementary duplex possessing terminal thiol groups produced both hairpin and duplex structures with disulfide bridges, the relative proportions of each being dependent upon the reaction conditions. These bridged hairpin and duplex structures were shown to be interconvertible by reduction and re-oxidation. The melting profiles of disulfide-bridged oligonucleotides were compared with the same sequences without bridges and with sequences possessing triethylene glycol bridges, and in all cases the introduction of disulfide bridges resulted in a considerable increase in thermal stability. EcoRI endonuclease was capable of cleaving a disulfide-bridged duplex possessing a recognition site for this enzyme, thus supporting a lack of distortion of the recognition site. The disulfide bridges could be cleaved using a large excess of DTT to regenerate the corresponding sulfhydryl compounds. A study of the serum stabilities of disulfide-bridged oligonucleotides showed that the bridged duplexes were much more stable than their unmodified counterparts, whereas the rate of degradation of the stem-loop structures was more dependent upon the size of the loop than the presence or absence of the disulfide bridge. In summary, we have described a novel methodology, employing commercially available reagents, for the stabilization of oligonucleotide duplexes or stem-loop structures by disulfide bridge formation.     

Identification of the amino acid residues essential for the activity and the interconversion of the molecular forms of biliverdin reductase

Biliverdin reductase (molecular form 1, EC 1.3.1.24, bilirubin:NAD(P)+ oxidoreductase) carries three thiol residues. Only one of them could be alkylated when a ratio N-ethylmaleimide (NEM)/mol enzyme's SH = 90 was used. The alkylation of this thiol group inhibited the conversion of molecular form 1 to its dimer, molecular form 3; however, it did not inhibit the enzymatic activity. At a ratio of NEM/enzyme's SH = 300, two thiol residues were alkylated and the activity of the enzyme was totally inhibited. The third thiol group could not be alkylated either by NEM or by iodoacetamide. Biliverdin as well as the co-substrate NADPH protected the thiol residue essential for the enzymatic activity from alkylation. Spectroscopic evidence was obtained that this thiol group binds covalently to the C-10 of biliverdin to form a rubinoid adduct. The presence of a lysine residue, which is also essential for the enzymatic activity, could be inferred from the fact that by reduction of the Schiff base formed by the enzyme with pyridoxal phosphate the catalytic activity was irreversibly abolished. The location of a lysine residue in the vicinity of the thiol group involved in the catalytic activity was evident when the enzyme was treated with o-phthalaldehyde. The inactivation of the enzymatic activity was coincident with the formation of the fluorescent isoindole derivative which originates when the thiol and epsilon-NH2 groups are located about 3 A apart. The presence of a positively charged ammonium ion in the vicinity of the NADPH binding site was inferred from the shifts in the UVmax of NADPH from 340 nm to 327 nm and of 3-acetyl NADPH from 360 nm to 348 nm when the pyridine nucleotides bind to the reductase. The involvement of arginine residues in the enzymatic activity was established by inhibition of the latter after reaction with butanedione. This inhibition was totally protected by NADPH but not by biliverdin. The similarity of the structural features of biliverdin reductase with those of several dehydrogenases is discussed.     

A selective postcolumn o-phthalaldehyde-derivatization system for the determination of histamine in biological material by high-performance liquid chromatography

Based on studies of the reaction between histamine and o-phthalaldehyde in alkaline solution, a method optimized for the determination of histamine in biological samples by means of HPLC and postcolumn o-phthalaldehyde derivatization has been developed. The method permits determination of histamine even at low-picomolar levels. By means of a valve, placed immediately after the column outlet, the eluent stream can be switched between the fluorimetric and an electrochemical detector system whereby electroactive biogenic amines may also be studied under the same chromatographic conditions.     

Screening of Thiol Compounds: Depolarization-Induced Release of Glutathione and Cysteine from Rat Brain Slices

Superfusates from rat brain slices were screened for thiol compounds after derivatization with monobromobimane by reversed-phase HPLC. Only glutathione and cysteine were detected. The Ca(2+)-dependent release of these compounds from slices of different regions of rat brain was investigated, applying a highly sensitive and reproducible quantification method, based on reduction of superfusates with dithiothreitol, reaction of thiols with iodoacetic acid, precolumn derivatization with o-phthalaldehyde reagent solution, and analysis with reversed-phase HPLC. This methodology allowed determination of reduced and total thiols in aliquots of the same superfusates. Mostly reduced glutathione and cysteine were released upon K+ depolarization and the Ca2+ dependency suggests that they originate from a neuronal compartment. The GSH release was most prominent in the mesodiencephalon, cortex, hippocampus, and striatum and lowest in the pons-medulla and cerebellum. This underscores a physiologically significant role for glutathione in CNS neurotransmission.     

Cleavage of DNA by model complexes of cytochrome P-450

Thiolate-hemin complexes as chemical models for cytochrome P-450 have been shown to cause cleavage of DNA. The cleavage of DNA to open-circular and linear forms depended on the structure of thiol ligand and the thiol ligand:hemin ratio at pH 7.8. Complete cleavage of DNA was observed by complexes containing thioglycolate ethylester and mercaptoethanol at 400-600 moles excess of thiol ligand to hemin, those containing cysteine, cysteine methylester and cysteine ethylester at 50-200 moles excess, and those containing mercaptopropionylglycine, glutathione, glutathione dimethylester, penta- and nonapeptides at 5-100 moles excess. Inhibition experiments suggested the involvement of active oxygen species in the cleavage of DNA.     

Generating favorable nano-environments for thermal and solvent stabilization of immobilized beta-galactosidase.

beta-Galactosidase (Escherichia coli) was immobilized through its thiol groups on thiolsulfinate-agarose gel. After enzyme immobilization, different nano-environments were generated by reacting the excess of gel-bound thiolsulfinate moieties with 2-mercaptoethanesulfonic acid (S-gel), glutathione (G-gel), cysteamine (C-gel), and mercaptoethanol (M-gel). Concerning thermal stability at 50 degrees C, the G-gel and the M-gel derivatives were the most stable with residual activity values of 67% and 45%, respectively. The stability in several solvent systems was studied: ethyl acetate (1.6% vol/vol), ethylene glycol (50% vol/vol), and 2-propanol (50% vol/vol). In ethyl acetate, both the M-gel and S-gel were highly stabilized; the time required for activity to decay to 80% of the initial activity was increased 29-fold for the M-gel and 20-fold for the S-gel with respect to the soluble enzyme. The G-gel was the least stable of all the derivatives. The different behaviors of the derivatives in thermal and solvent stability studies suggest that each nano-environment contributes differently to the enzyme stability, depending on the denaturing conditions. Therefore, it may be possible to tailor the matrix surface to maximize enzyme stability in particular applications.     

Fluorescence stopped-flow study of the o-phthaldialdehyde reaction

The fluorogenic reaction involving three species, namely, a primary amine, o-phthaldialdehyde (OPA), and a thiol compound was studied with the fluorescence stopped-flow technique. The results are consistent with the reaction of the amine with a 1:1 adduct of OPA and the thiol compound. The equilibrium constant for the formation of the adduct, OPAME, from OPA and mercaptoethanol (ME) was determined to be 164 m^?1. A survey of the rates of reaction of OPAME with various amino acids demonstrated that with OPA: ME:amine equal to 1:2.4:1 (total OPA concentration 0.5 to 3.0 × 10^?3m), the reaction followed second-order kinetics, with k = 150 to 450 m^?1s^?1 at pH 9.O. The differences in rates are discussed in relation to structural differences between the amines. The reaction, when conducted under conditions of excess OPAME yielded pseudo-first-order kinetics, with rates consistent with the second-order rate constants. The rate of reaction of OPAME with alanine was maximal at pH 10.5–11, and a great excess of ME resulted in a slower rate. Slower rates were also observed if ME was replaced by dithiothreitol or 1-propanethiol.     

Sequential inactivation of zeta-crystallin by o-phthalaldehyde

o-Phthalaldehyde, a bifunctional cross-linking reagent, is commonly used as a probe for the active site of enzymes. In this study, the interaction of o-phthalaldehyde with camel lens zeta-crystallin was examined by activity and fluorescence measurements. Predictably, the oxidoreductase activity of zeta-crystallin was inhibited irreversibly by o-phthalaldehyde in a time- and concentration-dependent manner, and the presence of NADPH with the enzyme appeared to provide a high degree of protection against o-phthalaldehyde inactivation. Interaction of o-phthalaldehyde with zeta-crystallin resulted in formation of isoindole adduct, which exhibited characteristic fluorescence at 415 nm. However, neither inactivation nor modification of the enzyme showed the expected pseudo-first-order kinetics; both events were highly sequential reaching different levels of saturation at different concentrations of o-phthalaldehyde. The modified enzyme had a maximum stoichiometry of 1 mol isoindole/subunit, and bound NADPH to nearly the same extent as unmodified enzyme. Gel filtration experiments suggested that o-phthalaldehyde-modified zeta-crystallin had higher apparent molecular weight than unmodified enzyme, even though the enzyme remained largely monomeric as revealed by electrophoresis on denaturing gel. These results suggested that modification by o-phthalaldehyde might have been so intrusive as to sequentially modify the tetrameric structure of zeta-crystallin.     

Reaction of thiols with 7-methylbenzopentathiepin

Polysulfides typically react readily with thiols, thus, reactions of endogenous cellular thiols with the polysulfide linkage in naturally-occuring pentathiepin cytotoxins are likely to be an important aspect of their biological chemistry. Here, it is reported that the reaction of thiols with the pentathiepin ring system initially produces a complex mixture of polysulfides that further decomposes in the presence of excess thiol to yield the corresponding 1,2-benzenedithiol with concomitant production of H(2)S and dimerized thiol. In this reaction, a single molecule of the pentathiepin consumes approximately six equivalents of thiol. The reaction of thiols with the pentathiepin ring system is faster than the analogous reaction involving typical di- and trisulfides.