Biologically important thiols in various vegetables and fruits

Biological thiols are important antioxidants, and recent studies showed that their contents vary depending on the groups of foodstuffs. Therefore, we investigated the levels of some biological thiols in various vegetables and fruits by using a sensitive high-performance liquid chromatography (HPLC) technique. Biological thiols measured in some vegetables and fruits include glutathione (L-glutamyl-L-cysteinly glycine, GSH), N-acetylcysteine (NAC), captopril [CAP (C9H15NO3S)], homocysteine (HCYS), cysteine (CYS), and gamma-glutamyl cysteine (GGC). Our results show that biological thiol contents are between 3-349 nM/g wet weight in vegetables and 4-136 nM/g wet weight in fruits. CAP is only found in asparagus (28 nM/g wet weight). Furthermore, none of the biological thiols analyzed were found in cabbages, red grapes, blackberries, apples, and peaches. Therefore, various vegetables and fruits differ significantly in their thiol contents. Oxidation of these important thiols may occur and result in the production of toxic byproducts, if they are exposed to radiation and ozone treatment for sterilization purposes. Further studies should be performed to monitor the levels of these biological thiols.     

Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: kinetic study of adduct formation under chemical and enzymatic oxidation conditions

HPLC-MS and (1)H, (13)C, and 2D NMR analyses were used to identify new addition products between 3-sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (-)-epicatechin, and caftaric acid. The kinetics of formation of these adducts were monitored in a wine model solution and in a must-like medium by HPLC-UV-MS with the aim of understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(-)-epicatechin/3SH adducts were characterized. Caftaric acid was oxidized faster than (-)-epicatechin and (+)-catechin when these phenolic compounds were incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(-)-epicatechin in the absence of other nucleophilic species. Furthermore, o-quinone-(-)-epicatechin reacted faster than o-quinone-(+)-catechin with 3SH. Sulfur dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their vinification and aging process.     

Kinetic characterization of the oxidation of chlorogenic acid by polyphenol oxidase and peroxidase. Characteristics of the o-quinone

Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.     

Oxidation of porcine Myosin by hypervalent myoglobin: the role of thiol groups

Oxidation of the myofibrillar muscle protein myosin from pork by hypervalent myoglobin species (MbFe(III)/H 2O2 radical generating system) was investigated in aqueous solution in the pH range of 5.0-7.8 by electron spin resonance (ESR) spectroscopy using N- tert-butyl-alpha-phenylnitrone (PBN) as spin trap and indirectly by determination of the rate of reduction of hypervalent myoglobin species by UV spectroscopy. Cross-linking of myosin was examined by SDS-PAGE. The target for oxidative modification of myosin was studied by thiol blocking by N-acetylmaleimide (NEM) and by determining oxidative modification of myosin thiols. The reaction between myosin and hypervalent myoglobin was fast and showed little dependence on pH. The myosin radicals formed were observed to be short-lived. Myosin thiols are suggested to be the main target for oxidative modification, as NEM-treated myosin did not form radicals in the presence of hypervalent myoglobin. A significant decrease in thiol content was already demonstrated 25 s after initiation of oxidation of myosin. The majority of myosin heavy chain (MHC) was demonstrated to be cross-linked through intermolecular disulfide bonding 1 h after initiation of oxidation. This demonstrates that thiols are important for radical formation and cross-linking of myosin during oxidation with hypervalent myoglobin at the pH of meat products.     

Effect of ascorbic acid in dough: reaction of oxidized glutathione with reactive thiol groups of wheat glutelin

The reactions of oxidized glutathione generated from endogenous glutathione by the addition of ascorbic acid (AA) prior to dough mixing on free thiol groups of gluten proteins have been investigated. A small amount of (35)S-labeled glutathione was added as a tracer to identify the reaction products of GSSG and free protein thiols by radioactivity measurement. First, gluten was isolated from the dough, then the gliadins were extracted, and residual glutenin was partially hydrolyzed with thermolysin. After preseparation by gel permeation chromatography, the fractions with the highest radioactivity were separated by high-performance liquid chromatography. Radioactive peptides were identified, isolated, sequenced, and assigned to amino acid sequences of gluten protein components. The isolated peptides contained exclusively the cysteine residues C(b) and C(x) of low molecular weight subunits of glutenin, which are supposed to be highly reactive in forming intermolecular disulfide bonds. From these results it can be assumed that the cysteine residues C(b) and C(x) of the low molecular weight subunits of glutenin are at least partly present in the thiol form in flour. During dough mixing they are converted to protein-protein disulfides or glutathione-protein mixed disulfides by thiol/disulfide interchange reactions. Oxidized glutathione necessary for this reaction is generated from glutathione by the action of AA. These results are in accordance with the major hypothesis about the mechanism of action of AA.     

Proteolytic activation of latent Paraguaya peach PPO. Characterization of monophenolase activity

The kinetics of the activation process of latent peach PPO by trypsin was studied. By coupling this activation process to the oxidation of 4-tert-butylcatechol (TBC) to its corresponding quinone, it was possible to evaluate the specific rate constant of active PPO formation, k(3), which showed a value of 0.04 s(-1). This proteolytic activation of latent peach PPO permitted us to characterize the monophenolase activity of peach PPO for the first time using p-cresol as substrate, and it showed the characteristic lag period of the kinetic mechanism of monophenols hydroxylation, which depended on the enzyme and substrate concentration, the pH and the presence of catalytic amounts of o-diphenol (4-methylcatechol). The enzyme activation constant, k(act), was 2 microM.     

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Thiol-quinone adduct formation in myofibrillar proteins detected by LC-MS

Protein oxidation in meat is considered to decrease meat tenderness due to protein disulfide cross-link formation of thiol-containing amino acid residues. An LC-MS method for detection of thiol-quinone adducts (RS-QH(2)) in myofibrillar proteins was developed to investigate the interaction between phenols, as protective antioxidants, and proteins from meat under oxidative conditions using aqueous solutions of (i) cysteine (Cys), (ii) glutathione (GSH), (iii) bovine serum albumin (BSA), or (iv) a myofibrillar protein isolate (MPI). The aqueous solutions were incubated at room temperature (30 min) with 4-methyl-1,2-benzoquinone (4MBQ) prepared from oxidation of 4-methylcatechol (4MC) by periodate resin or incubated at room temperature (5 h) with 4MC and Fe(II)/H(2)O(2). GSH, BSA, and MPI were hydrolyzed (6 N HCl, 110 °C, 22 h) after incubation, and the cysteine-quinone adduct, Cys-QH(2) (m/z 244.2) was identified according to UV and mass spectra after separation on an RP-C18 column. The thiol-quinone adduct was present in all thiol systems after incubation with 4MBQ or 4MC oxidized by Fe(II)/H(2)O(2). Direct reaction with 4MBQ resulted in each case in increased Cys-QH(2) formation compared to simultaneous oxidation of thiol source and 4MC with Fe(II)/H(2)O(2). The covalent bonds between quinones and thiol groups may act as a potential antioxidant by inhibiting disulfide protein cross-link formation.     

Determination by liquid chromatography with electrochemical detection of cysteamine and cysteine, possible precursors of N-nitrosothiazolidine

A method is described that is selective, sensitive, rapid, and accurate for the quantitative measurement in meat products of both cysteamine and cysteine, potential precursors for N-nitrosothiazolidine (NTHZ) and N-nitrosothiazolidine-4-carboxylic acid (NTHZC), respectively. In general, a ground meat sample is homogenized with acetonitrile-formate buffer in the presence of dithiothreitol, and then is centrifuged, filtered, and recentrifuged in a disposable microfilter. The thiols are quantitated by liquid chromatography using an amperometric detector equipped with a gold/mercury electrode, operated in the oxidative mode. Cysteamine was found in 6 of 20 samples of raw pork belly in concentrations ranging from 150 to 450 ppb, and cysteine was found in all samples in concentrations ranging from 2.4 to 36.5 ppm. Analysis for the thiols and their corresponding nitrosamines--NTHZ and NTHZC--of bacon before and after processing showed no correlation between cysteamine and cysteine levels before processing nor with nitrosamine levels after processing. Liquid chromatography with electrochemical detection was found to be an extremely selective technique to measure the 2 free sulfhydryl compounds in a complex food substrate.     

Aminoparathion: a highly reactive metabolite of parathion. 1. Reactions with polyphenols and polyphenol oxidase

Spiking of tomato and apple fruits with parathion at different levels of about 1-4 mg/kg irradiation and under simulated sunlight conditions resulted in nearly complete photodegradation within 13 h, but extractable parathion degradation products could not be found in any case. However, after irradiation of an unrealistically spiked apple (134 mg/kg) different photoproducts including aminoparathion (AP) were detectable by HPLC, proving that the hitherto postulated photochemistry of parathion indeed takes place in the fruit cuticle environment. Besides the photoreduction pathway it was shown for the first time that AP is also easily formed by reduction of the primary photoproduct nitrosoparathion with thiols (cysteine, glutathione), while ascorbic acid only leaves hydroxylaminoparathion. In the presence of polyphenols, AP was effectively bound to quinone intermediates formed by both silver oxide and polyphenol oxidases. For pyrocatechol, a disubstituted o-quinone derivative could be isolated as a dark red addition product and structurally be elucidated. However, in the presence of caffeic acid, catechol, naringin, and quercetin, respectively, insoluble dark colored polymers precipitated within 48 h, while in the supernatants AP was not detectable any more. Polymer-bound and nonextractable AP was proven by transesterification with sodium ethoxide releasing O,O,O-triethyl thiophosphate which was determined by GC. Additionally, AP itself was a substrate for polyphenol oxidases, resulting in a quinone imine intermediate which in turn reacted with excessive AP yielding deep red colored di- and trimerization products.     

Degradation of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase

The degradation mechanism of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase (PPO) was investigated using several model systems. The enzymically generated (-)-epicatechin o-quinone could induce cyanidin 3-rutinoside degradation. The results obtained in this study allowed us to propose a pathway for cyanidin 3-rutinoside degradation in the presence of (-)-epicatechin and litchi pericarp PPO. First, enzymatic oxidation of (-)-epicatechin produced the corresponding o-quinone, and then cyanidin 3-rutinoside and (-)-epicatechin competed for (-)-epicatechin o-quinone, resulting in degradation of cyanidin 3-rutinoside and regeneration of (-)-epicatechin. Moreover, the results of kinetic studies indicated this competition was influenced by both (-)-epicatechin concentration and cyanidin 3-rutinoside concentration in the model system.     

Identification and quantitation of 3-S-cysteinylglycinehexan-1-ol (Cysgly-3-MH) in Sauvignon blanc grape juice by HPLC-MS/MS

Precursors to varietal wine thiols are a key area of grape and wine research. Several such precursors, in the form of odorless conjugates, have been closely studied in recent years. A new conjugate has now been identified as 3-S-cysteinylglycinehexan-1-ol (Cysgly-3-MH), being the dipeptide intermediate between cysteine and glutathione precursors of tropical thiol 3-mercaptohexan-1-ol (3-MH). Authentic Cysgly-3-MH was produced via enzymatic transformation of the glutathione conjugate and used to verify the presence of both diastereomers of Cysgly-3-MH in Sauvignon blanc juice extracts. Cysgly-3-MH was added into our HPLC-MS/MS precursor method, and the validated method was used to quantify this new analyte in a selection of Sauvignon blanc juice extracts. Cysgly-3-MH was found in the highest concentrations (10-28.5 μg/L combined diastereomer total) in extracts from berries that had been machine-harvested and transported for 800 km in 12 h. This dipeptide conjugate was much less abundant than the glutathione and cysteine conjugates in the samples studied. On the basis of the results, the new cysteinylglycine conjugate of 3-MH seemingly has a short existence as an intermediate precursor, which may explain why it has not been identified as a natural juice component until now.     

Kinetic model for the antiradical activity of the isolated p-catechol group in flavanone type structures using the free stable radical 2,2-diphenyl-1-picrylhydrazyl as the antiradical probe

The time evolution of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH*) concentration in four solvents (methanol, ethanol, propanol, and acetonitrile) during its reduction by three flavanones containing an isolated p-catechol group (taxifolin, eriodyctiol, and fustin) as well as the time evolution of the mass spectra of the reaction mixture has been determined by spectrophotometry and liquid mass spectrometry, respectively. In alcoholic solvents the reduction curves consisted of an initial short but fast kinetics step followed by a longer slow kinetics step; in contrast, in acetonitrile the reduction curves completely lacked the slow kinetics step. From the results, a kinetic model for the reaction of reduction of the DPPH* by the isolated p-catechol group in flavanone type structures is proposed. According to this model, the p-catechol group rapidly transfers two hydrogen atoms to DPPH*, through a fast rate constant k1, yielding the corresponding o-quinone. Then, the intermediate o-quinone forms an adduct with the alcoholic solvent, through a slow rate constant k2, and regenerates the p-catechol group. The regenerated p-catechol group reduces additional DPPH* through a fast rate constant k3, yielding the corresponding o-quinone, which can form a new adduct with the solvent to regenerate the p-catechol group, and so on. From the kinetics model, two explicit kinetics equations have been derived that fit very well the experimental data points acquired from all assayed compounds in all of the experiments carried out, thus allowing an accurate determination of the corresponding rate and stoichiometric constants.     

Cyclodextrins as activator and inhibitor of latent banana pulp polyphenol oxidase

The effect of cyclodextrins (CDs) on o-diphenol oxidation catalyzed by banana polyphenol oxidase (PPO) was studied. The oxidation of dopamine, the natural substrate of banana, in the presence of cyclodextrins was unaffected, because this hydrophilic phenol does not form inclusion complexes with CDs. However, when a hydrophobic phenol such as tert-butylcatechol (TBC) was used, a marked inhibition was observed with beta-, hydroxypropyl-beta-, and maltosyl-beta- CDs. This inhibition was due to the complexation of TBC in the CD core, demonstrating that banana pulp PPO worked only toward free substrate and not toward the complex TBC-CDs. In addition, the effect of some inhibitors in the presence of CDs and dopamine as substrate was studied. Increasing concentrations of CDs, in the presence of two inhibitors (4-iodophenol and cinnamic acid) were able to activate the inhibited enzyme to reach the noninhibited level by complexing the inhibitors in the hydrophobic core of the CDs. This dual effect of CDs as activator and inhibitor was tested in crude banana pulp extracts, with surprising activation effects never before described being observed.     

A novel method for quantification of 2-methyl-3-furanthiol and 2-furanmethanethiol in wines made from Vitis vinifera grape varieties

A rapid, easy method has been developed for isolating and quantifying 2-methyl-3-furanthiol (2M3F) in wines. Until now, it was not possible to quantify this highly odoriferous compound, with a smell reminiscent of cooked meat, in wine. The original aspect of this method is the specific release of volatile thiols using a cysteamine solution applied in reverse flow to sample percolation on the basis of a p-hydroxymercuribenzoate (pHMB)-volatile thiol conjugate formed by the direct addition of pHMB to 50 mL of wine. Purification of volatile thiols in wines is much faster and easier than our previous method. This method may also be used to assay 2-furanmethanethiol in wine. This thiol's strong aroma of roasted coffee has been shown to contribute to the "roast coffee" aroma of certain wines. Assaying 2M3F by this method showed that it was present in the wines analyzed (red and white Bordeaux, Loire Valley Sauvignon blanc, white Burgundy, and Champagne) at concentrations up to 100 ng/L, i.e., significantly above the olfactory perception threshold for this compound in model dilute alcohol solution.     

Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins.

It has been reported earlier that when macerated tea leaf is fermented at lower pH, the resultant black tea contains increased levels of theaflavin, an important quality marker in black tea. In an attempt to investigate the biochemistry and chemistry underlying this observation, in vitro oxidation experiments using polyphenol oxidase (PPO) from fresh tea leaves, horseradish peroxidase (POD), and tea catechins, precursors for theaflavins, were carried out. In vitro oxidation experiments using crude tea PPO resulted in higher content of theaflavins at pH 4.5 in comparison with pH 5.5, the normal pH of the macerated tea leaf. When purified PPO was used in the in vitro system, surprisingly a reversal of this trend was observed, with more theaflavins being formed at the higher pH. A combination of pure tea PPO and POD led to an observation similar to that with the crude enzyme preparation, suggesting a possible role for POD in the formation or degradation of theaflavin. POD was observed to oxidize theaflavins in the presence of H(2)O(2), leading to the formation of thearubigin, another black tea pigment. This paper demonstrates that tea PPO, while oxidizing catechins, generates H(2)O(2). The amount of H(2)O(2) produced is greater at pH 5.5, the optimum pH for PPO activity, than at pH 4.5. Hence, an observed increase of theaflavins in black teas fermented at pH 4.5 appears to be due to lower turnover of formed theaflavins into thearubigins.     

Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation conditions

Chemical interaction between polyphenols and thiols was investigated under radical oxidation conditions using a model cysteinyl thiol derivative, N-benzoylcysteine methyl ester. The radical oxidation was carried out with a stoichiometric amount of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the decreases in the amounts of polyphenols and the thiol were measured by HPLC analysis. Cross-coupling products between various polyphenols and the thiol were examined by LC-MS in reactions that showed decreases in both the polyphenols and the thiol. The LC-MS results indicated that three phenolic acid esters (methyl caffeate, methyl dihydrocaffeate, and methyl protocatechuate) and six flavonoids (kaempferol, myricetin, luteolin, morin, taxifolin, and catechin) gave corresponding thiol adducts, whereas three polyphenols (methyl ferulate, methyl sinapate, and quercetin) gave only dimers or simple oxidation products without thiol substituents. Thiol adducts of the structurally related compounds methyl caffeate and methyl dihydrocaffeate were isolated, and their chemical structures were determined by NMR analysis. The mechanism for the thiol addition was discussed on the basis of the structures of the products.     

Approaches to wine aroma: release of aroma compounds from reactions between cysteine and carbonyl compounds in wine

Under conditions close to those of wine, that is, low pH, aqueous medium, and low temperatures, this work examines the role of carbonyl (acetoin and acetol) and dicarbonyl (glyoxal, methylglyoxal, diacetyl, and pentane-2,3-dione) compounds associated with cysteine in the formation of odorous products. In particular, thiazole, 4-methylthiazole, 2-acetylthiazole, and trimethyloxazole and two sulfur and oxygenated heterocyclic compounds, 2-furanmethanethiol and thiophene-2-thiol, are examined. For thiophene-2-thiol, the reactional mechanism is proposed. Attempts were made to detect these compounds in wines from various origins. Certain molecules were identified for the first time in wine.     

Investigation of the reaction between 4-hydroxy-5-methyl-3(2H)-furanone and cysteine or hydrogen sulfide at pH 4.5

Reaction of 4-hydroxy-5-methyl-3(2H)-furanone (HMF) with cysteine or hydrogen sulfide at pH 4.5 for 60 min at 140 degrees C produced complex mixtures of volatile compounds, the majority of which contained sulfur. Sixty-nine compounds were identified, some tentatively, by GC/MS. These included disulfides (26), thiols (7), dithiolanones (6), thiophenones (4), dithianones (3), and thienothiophenes (6). The main non-sulfur compounds were 2, 3-pentanedione, 2,4-pentanedione, and 3,4-hexanedione. Both systems produced approximately the same total quantity of volatile compounds, but the reaction containing cysteine gave the larger number of individual compounds, with thiols quantitatively the dominant components. By comparison, the major products formed in the reaction with hydrogen sulfide were the dithiolanones. Reaction pathways are presented for the major products and, where applicable, possible reasons for the differences in composition of the two systems are discussed. The contribution of these reactions, and their products, to the flavor of roasted foods is considered.     

Structural Modifications of Gluten Proteins in Strong and Weak Wheat Dough During Mixing

The network‐forming attributes of gluten have been investigated for decades, but no study has comprehensively addressed the differences in gluten network evolution between strong and weak wheat types (hard and soft wheat). This study monitored changes in SDS protein extractability, SDS‐accessible thiols, protein surface hydrophobicity, molecular weight distribution, and secondary structural features of proteins during mixing to bring out the molecular determinants of protein network formation in hard and soft wheat dough. Soft wheat flour and dough exhibited greater protein extractability and more accessible thiols than hard wheat flour and dough. The addition of the thiol‐blocking agent N‐ethylmaleimide (NEM) resulted in similar results for protein extractability and accessible thiols in hard and soft wheat samples. Soft wheat dough had greater protein surface hydrophobicity than hard wheat and exhibited a larger decrease in surface hydrophobicity in the presence of NEM. Formation of high‐molecular‐weight (HMW) protein in soft wheat dough was primarily because of formation of disulfides among low‐molecular‐weight (LMW) proteins, as indicated by the absence of changes in protein distribution when NEM was present, whereas in hard wheat dough the LMW fraction formed disulfide interaction with the HMW fraction. Fourier transform infrared spectroscopy indicated formation of β‐sheets in dough from either wheat type at peak mixing torque. Formation of β‐sheets in soft wheat dough appears to be driven by hydrophobic interactions, whereas disulfide linkages stabilize secondary structure elements in hard wheat dough.