Aromatic thiol pKa effects on the folding rate of a disulfide containing protein

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.     

Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers

Almost all therapeutic proteins and most extracellular proteins contain disulfide bonds. The production of these proteins in bacteria or in vitro is challenging due to the need to form the correctly matched disulfide bonds during folding. One important parameter for efficient in vitro folding is the composition of the redox buffer, a mixture of a small molecule thiol and small molecule disulfide. The effects of different redox buffers on protein folding, however, have received limited attention. The oxidative folding of denatured reduced lysozyme was followed in the presence of redox buffers containing varying concentrations of five different aromatic thiols or the traditional aliphatic thiol glutathione (GSH). Aromatic thiols eliminated the lag phase at low disulfide concentrations, increased the folding rate constant up to 11-fold, and improved the yield of active protein relative to GSH. The yield of active protein was similar for four of the five aromatic thiols and for glutathione at pH 7 as well as for glutathione at pH 8.2. At pH 6 the positively charged aromatic thiol provided a higher yield than the negatively charged thiols.     

Oxidative folding of lysozyme with aromatic dithiols, and aliphatic and aromatic monothiols

In vitro protein folding of disulfide containing proteins is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. In this study, we examined redox buffers containing asymmetric dithiols 1-5, which possess an aromatic and aliphatic thiol, and symmetric dithiols 6 and 7, which possess two aromatic thiols, for their ability to fold reduced lysozyme at pH 7.0 and 8.0. Most in vivo protein folding catalysts are dithiols. When compared to glutathione and glutathione disulfide, the standard redox buffer, dithiols 1-5 improved the protein folding rates but not the yields. However, dithiols 6 and 7, and the corresponding monothiol 8 increased the folding rates 8-17 times and improved the yields 15-42% at 1mg/mL lysozyme. Moreover, aromatic dithiol 6 increased the in vitro folding yield as compared to the corresponding aromatic monothiol 8. Therefore, aromatic dithiols should be useful for protein folding, especially at high protein concentrations.     

ortho- and meta-substituted aromatic thiols are efficient redox buffers that increase the folding rate of a disulfide-containing protein

Thiol based redox buffers are used to enhance the folding rates of disulfide-containing proteins in vitro. Traditionally, small molecule aliphatic thiols such as glutathione are employed. Recently, we have demonstrated that aromatic thiols can further enhance protein-folding rates. In the presence of para-substituted aromatic thiols the folding rate of a disulfide-containing protein was increased by 4-23 times over that measured for glutathione. However, several important practical issues remain to be addressed. Aromatic thiols have never been tested in the presence of denaturants such as guanidine hydrochloride. Only two of the para-substituted aromatic thiols previously examined are commercially available. To expand the number of aromatic thiols for protein folding, several commercially available meta- and ortho-substituted aromatic thiols were studied. Furthermore, an ortho-substituted aromatic thiol, easily obtained from inexpensive starting materials, was investigated. Folding rates of scrambled ribonuclease A at pH 6.0, 7.0 and 7.7, with ortho- and meta-substituted aromatic thiols, were up to 10 times greater than those with glutathione. In the presence of the common denaturant guanidine hydrochloride (0.5M) aromatic thiols provided 100% yield of active protein while maintaining equivalent folding rates.     

Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily

Thiols represent preferential targets of peroxynitrite in biological systems. In this work, we investigated the mechanisms and kinetics of the reaction of peroxynitrite with the dithiol dihydrolipoic acid (DHLA) and its oxidized form, lipoic acid (LA). Peroxynitrite reacted with DHLA being oxidation yields higher at alkaline pH. The stoichiometry for the reaction was two thiols oxidized per peroxynitrite. LA formation accounted for approximately 50% DHLA consumption at pH 7.4, probably reflecting secondary reactions between LA and peroxynitrite. Indeed, peroxynitrous acid reacted with LA with an apparent second-order rate constant (k(2app)) of 1400 M(-1) s(-1) at pH 7.4 and 37 degrees C. Nitrite and LA-thiosufinate were formed as reaction products. Surprisingly, the k(2app) for peroxynitrite-dependent DHLA oxidation was only 250 M(-1) s(-1) per thiol, at pH 7.4 and 37 degrees C. Testing various low-molecular-weight thiols, we found that an increase in the thiol pK (pK(SH)) value correlated with a decrease of k(2app) for the reaction with peroxynitrite at pH 7.4. The pK(SH) for DHLA is 10.7, in agreement with its modest reactivity with peroxynitrite.     

Catalytic properties, thiol pK value, and redox potential of Trypanosoma brucei tryparedoxin

The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides. Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness. As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins. The redox potential of T. brucei tryparedoxin of -249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin. The trypanothione/tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites. The pK value of Cys(40), the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation. Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to < or =4.0. The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions.     

Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide.

The reactivities of glutathione, cysteine, cysteamine, penicillamine, N-acetylcysteine, dithiothreitol and captopril with superoxide generated from xanthine oxidase and hypoxanthine, and with reagent hydrogen peroxide, have been investigated. Rates of thiol loss on adding hydrogen peroxide, and superoxide-dependent thiol loss and oxygen uptake were measured. The relative reactivities of the different thiols with both oxidants were inversely related to the pK of the thiol group, such that at pH 7.4, penicillamine was the most reactive. N-acetylcysteine weakly reactive and no reaction was seen with captopril. For hydrogen peroxide, the calculated rate constants for the reaction with the thiolate anion all fell within the range 18-26 M(-1) s(-1). With superoxide, our results are consistent with each thiol reacting via a short chain that consumes oxygen and regenerates superoxide. Only with some of the thiols, was the consumed oxygen recovered as hydrogen peroxide. Reported values for the rate constant for the reaction of thiols with superoxide vary over four orders of magnitude, with the highest being > 10(5) M(-1) s(-1). Due to the complexity of the chain reaction, no study so far has been able to obtain accurate values and we consider the best estimates to be in the 30 to 1000 M(-1) s(-1) range.     

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ～threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.     

Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.     

Ionization and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds of Bence Jones proteins and an Fab(t) fragment

The pK values and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds in Bence Jones proteins and the Fab(t) fragment of a myeloma protein (Jo) (IgGl, kappa) were determined by means of the reactions with chloroacetamide and DTNB, and of spectrophotometric titration. The two thiol groups of partially reduced type kappa Bence Jones protein dimers had the same pK values (pK = 9.76 at 0.2 ionic strength and 25 degrees C) and the same true second-order rate constants (k) toward chloroacetamide (k = 18.8 x 10(-2) M-1 . S-1). The two thiol groups of partially reduced type lambda Bence Jones protein dimers had different pK values but the variation of the pK values among the specimens was small (pK1 = 8.5-8.6 and pK2 = 9.5-9.7 at 0.2 ionic strength and 25 degrees C). The spectrophotometric titration of partially reduced Nag protein (type lambda) also showed that the two thiol groups have different pK values. The pK values of two thiol groups of the partially reduced Fab(t) fragment were determined as 8.51 and 9.76 at 0.2 ionic strength and 25 degrees C. The effect of ionic strength on the pK values of the thiol groups of partially reduced Nag protein and the pK values of the thiol groups in partially reduced Ta protein (type kappa) and in a hybrid molecule formed between partially reduced Ta protein and partially reduced and alkylated H chains indicated that the difference in pK values did not arise from electrostatic interaction between the two thiol groups, but that the pK values are intrinsically different. The true rate constants, k1 and k2, of the two thiol groups of type lambda Bence Jones proteins varied with the specimen (k1 = 1.9-5.7 x 10(-2) M-1 . S-1 and k2 = 18.5-25.0 x 10(-2) M-1 . S-1). The k1 and k2 values for Jo-Fab(t) were 7.21 x 10(-2) and 23.1 x 10(-2) M-1 . S-1, respectively. On the basis of these pK values and reactivities, we discuss the reformation of the interchain disulfide bonds from partially reduced Bence Jones proteins and immunoglobulins in the presence of oxidized glutathione.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

Comprehensive plasma thiol redox status determination for metabolomics

Thiol homeostasis plays an important role in human health and aging by regulation of cellular responses to oxidative stress. Due to major constraints that hamper reliable plasma thiol/disulfide redox status assessment in clinical research, we introduce an improved strategy for comprehensive thiol speciation using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that overcomes sensitivity, selectivity and dynamic range constraints of conventional techniques. This method integrates both specific and nonspecific approaches toward sensitivity enhancement for artifact-free quantification of labile plasma thiols without complicated sample handling. A multivariate model was developed to predict increases in ionization efficiency for reduced thiols when conjugated to various maleimide analogs based on their intrinsic physicochemical properties. Optimization of maleimide labeling in conjunction with online sample preconcentration allowed for simultaneous analysis of nanomolar levels of reduced thiols and free oxidized thiols as their intact symmetric or mixed disulfides. Identification of low-abundance thiols and various other polar metabolites detected in plasma was supported by prediction of their relative migration times using CE as a qualitative tool complementary to ESI-MS. Plasma thiol redox status determination together with untargeted metabolite profiling offers a systemic approach for elucidation of the causal role of dysregulated thiol metabolism in the etiology of human diseases.     

Increased catalytic activity of protein disulfide isomerase using aromatic thiol based redox buffers

PDI is an enzyme that acts as a chaperone, shufflase, and oxidase during the folding of disulfide-containing proteins. The ability of aromatic thiols to increase the activity of PDI-catalyzed protein folding over that of the standard thiol glutathione (GSH) was measured. 4-Mercaptobenzoic acid (ArSH) increased the activity of PDI by a factor of three.     

Kinetics of the reactions of hypochlorous acid and amino acid chloramines with thiols, methionine, and ascorbate

Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.     

Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase

The reactivity of simple alkyl thiolates with N-ethylmaleimide (NEM) follows the Br?nsted equation, log kS- = log G + beta pK, with G = 790 M-1 min-1 and beta = 0.43. The rate constant for the reaction of the thiolate of 2-mercaptoethanol with NEM is 10(7) M-1 min-1, whereas the rate constant for the reaction of the protonated thiol is less than 0.0002 M-1 min-1. The intrinsic reactivity of the protonated thiol (SH) is over (5 X 10(10]-fold less than the thiolate (S-) and makes a negligible contribution to the reactivity of thiols toward NEM. The rate of NEM modification of chalcone isomerase was conveniently measured by following the concomitant loss in enzymatic activity. The pseudo-first-order rate constants for inactivation show a linear dependence on the concentration of NEM up to 200 mM and yield no evidence for noncovalent binding of NEM to the enzyme. Evidence is presented demonstrating that the modification of chalcone isomerase by NEM is limited to a single cysteine residue over a wide range of pH. Kinetic protection against inactivation and modification by NEM is provided by competitive inhibitors and supports the assignment of this cysteine residue to be at or near the active site of chalcone isomerase. The pH dependence of inactivation of the enzyme by NEM indicates a pK of 9.2 for the cysteine residue in chalcone isomerase. At high pH, the enzymatic thiolate is only (3 X 10(-5))-fold as reactive as a low molecular weight alkyl thiolate of the same pK, suggesting a large steric inhibition of reaction on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and mechanistic insights into unusual thiol disulfide oxidoreductase

Cytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism.     

The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.     

Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes

Quantitative structure toxicity relationship (QSTR) equations were obtained to predict and describe the cytotoxicity of 31 phenols using logLD(50) as a concentration to induce 50% cytotoxicity of isolated rat hepatocytes in 2 h and logP as octanol/water partitioning: logLD(50) (microM)=-0.588(+/-0.059)logP+4.652(+/-0.153) (n=27, r(2)=0.801, s=0.261, P<1 x 10(-9)). Hydroquinone, catechol, 4-nitrophenol, and 2,4-dinitrophenol were outliers for this equation. When the ionization constant pK(a) was considered as a contributing factor a two-parameter QSTR equation was derived: logLD(50) (microM)=-0.595(+/-0.051)logP+0.197(+/-0.029)pK(a)+2.665(+/-0.281) (n=28, r(2)=0.859, s=0.218, P<1 x 10(-6)). Using sigma+, the Brown variation of the Hammet electronic constant, as a contributing parameter, the cytotoxicity of phenols towards hepatocytes were defined by logLD(50) (microM)=-0.594(+/-0.052)logP-0.552(+/-0.085)sigma+ +4.540(+/-0.132) (n=28, r(2)=0.853, s=0.223, P<1 x 10(-6)). Replacing sigma+ with the homolytic bond dissociation energy (BDE) for (X-PhOH+PhO.-->X-PhO.+PhOH) led to logLD(50) (microM)=-0.601(+/-0.066)logP-0.040(+/-0.018)BDE+4.611(+/-0.166) (n=23, r(2)=0.827, s=0.223, P<0.05). Hydroquinone, catechol and 2-nitrophenol were outliers for the above equations. Using redox potential and logP led to a new correlation: logLD(50) (microM)=-0.529(+/-0.135)logP+2.077(+/-0.892)E(p/2)+2.806(+/-0.592) (n=15, r(2)=0.561, s=0.383, P<0.05) with 4-nitrophenol as an outlier. Our findings indicate that phenols with higher lipophilicity, BDE, or sigma+ values or with lower pK(a) and redox potential were more toxic towards hepatocytes. We also showed that a collapse of hepatocyte mitochondrial membrane potential preceded the cytotoxicity of most phenols. Our study indicates that one or a combination of mechanisms; i.e. mitochondrial uncoupling, phenoxy radicals, or phenol metabolism to quinone methides and quinones, contribute to phenol cytotoxicity towards hepatocytes depending on the phenol chemical structure.     

Delocalized electronic structure of the thiol sulfur substantially prevents nucleic acid damage induced by neocarzinostatin.

Neocarzinostatin is a potent antitumor antibiotic and is a prodrug, which induces genome damage after activation by a thiol. The prodrug is stored as a protein-bound chromophore that contains an enediyne nucleus. A thiolate attack on the chromophore cyclizes the nucleus and produces radicals that abstract hydrogen from DNA. Because thiol is the only cofactor in the vital activation process, the structure of the thiol plays an important role in the activity of the drug. Here we systematically examine the effect of the electronic structure of some thiols on the efficiency of the drug, and compare particularly aromatic with aliphatic thiols. The values of drug-induced base release from DNA are remarkably different between thiophenol (3.6%) and benzyl mercaptan (12.5%), the activity of which is comparable with those of aliphatic thiols. Cleavage results determined by DNA electrophoresis are consistent with the results of base release; they show that the total number of DNA lesions is more than 3-fold lower for thiophenol than for aliphatic thiols or benzyl mercaptan. We conclude that among aromatic thiols, only those that have delocalized thiol sulfur electrons can substantially reduce the DNA cleavage activity. This result suggests that the effect of an aromatic ring arises from an inductive effect imposed on the thiol sulfur electron through pi-resonance rather than through effects such as aromatic stacking, steric hindrance, or hydrophobic interaction. Replacing thiophenol with substituted derivatives with electron-releasing or -withdrawing groups changes the drug activity and supports the important role of the electronic structure of the thiol sulfur in determining the drug activity.