Redox-active cyclic bis(cysteinyl)peptides as catalysts for in vitro oxidative protein folding

The active-site hexapeptides of glutaredoxin (Grx), thioredoxin (Trx), protein disulfide isomerase (PDI), and thioredoxin-reductase (Trr) containing the common motif Cys-Xaa-Yaa-Cys were conformationally restricted by backbone cyclization, and their redox potentials were found to increase in the rank order of Trr < Grx < Trx < PDI peptide, with E'(0) values ranging between -204 mV and -130 mV. In each peptide the thiol pK(a) of one Cys residue was found to be lower than the other (e.g., 7.3 against 9.6 in the PDI peptide). Both the yield and rate of refolding of reduced RNase A in the presence of the bis(cysteinyl)peptides increased with the oxidizing character of the cyclic compounds. These results show that small peptides can function as adjuvants for the in vitro oxidative folding of proteins.     

Quantitative determination of SH groups using 19F NMR spectroscopy and disulfide of 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid

A new method of measurement of thiol concentration by 19F NMR spectroscopy is developed. The method is based on the detection of products of the exchange reaction of thiols with a newly synthesized fluorinated disulfide, 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (BSSB). A significant broadening of the 19F NMR signal of BSSB in the presence of thiols was observed and attributed to the exchange reaction between the parent disulfide and 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid. The rate constant for this reaction was found to be equal to (63 +/- 11) x 10(3) M(-1) s(-1) at pH 7.0. The method was applied for the measurement of concentration of glutathione and albumin in rat blood.     

Thermodynamic Stability of Ribonuclease B

The thermodynamic stability of pancreatic ribonuclease B (RNase B), which possesses identical protein structure of pancreatic ribonuclease A (RNase A), but differs by the presence of a carbohydrate chain attached to Asn 34, was studied by means of differential scanning calorimetry (DSC) at different pH conditions. The comparison between the two proteins has shown a little but significant stabilization of RNase B with respect to the unglycosylated one at pH values higher than 7.0. The thermodynamic analysis reveals the carbohydrate moiety to have a small stabilization effect of 3 kJ mol^–1 at pH 8.0 and 63°C on the protein. This revised version was published online in July 2006 with corrections to the Cover Date.     

还原变性核糖核酸酶在疏水性液-固界面上的复性

采用疏水相互作用色谱(HIC)对还原变性核糖核酸酶A (RNase A)在疏水性液-固界面上的复性进行了研究。详细讨论了流动相中脲的浓度、还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)的比例、流动相pH和变性蛋白质浓度对还原变性RNase A复性效率和质量回收率的影响。结果表明,在最优化的复性条件(流动相中含有2.0 mol/L脲,GSH/GSSG的浓度比为8:1,流动相pH为8.0)下,还原变性RNase A能完全复性。当变性蛋白质质量浓度为5.0 mg/mL时,还原脲变性RNase A的活性回收率和质量回收率分别为98.0%和61.9%,还原胍变性RNase A分别为100.1%和66.8%。研究表明HIC是还原变性蛋白质复性的有力工具之一,可为蛋白质复性研究提供新方法和新思路。     

Effects of aromatic thiols on thiol-disulfide interchange reactions that occur during protein folding

The folding of disulfide containing proteins from denatured protein to native protein involves numerous thiol-disulfide interchange reactions. Many of these reactions include a redox buffer, which is a mixture of a thiol (RSH) and the corresponding disulfide (RSSR). The relationship between the structure of RSH and its efficacy in folding proteins in vitro has been investigated only to a limited extent. Reported herein are the effects of aliphatic and especially aromatic thiols on reactions that occur during protein folding. Aromatic thiols may be particularly efficacious as their thiol pK(a) values and reactivities match those of the in vivo catalyst, protein disulfide isomerase (PDI). This investigation correlates the thiol pK(a) values of aromatic thiols with their reactivities toward small molecule disulfides and the protein insulin. The thiol pK(a) values of nine para-substituted aromatic thiols were measured; a Hammett plot constructed using sigma(p-) values yielded rho = -1.6 +/- 0.1. The reactivities of aromatic and aliphatic thiols with 2-pyridyldithioethanol (2-PDE), a small molecule disulfide, were determined. A plot of reactivity versus pK(a) of the aromatic thiols had a slope (beta) of 0.9. The ability of these thiols to reduce (unfold) the protein insulin correlates strongly with their ability to reduce 2-PDE. Since the reduction of protein disulfides occurs during protein folding to remove mismatched disulfides, aromatic thiols with high pK(a) values are expected to increase the rate not only of protein unfolding but protein folding as well.     

The effect of trimethylamine N-oxide on RNase a stability

The thermal stability of bovine pancreatic ribonuclease (RNase A) has been investigated in the presence of trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements at neutral and acid pH conditions. It is well known that compatible osmolytes such as TMAO are effective in stabilizing protein structure and counteracting the denaturing the effect of urea and guanidinium hydrochloride (GuHCl). Calorimetric results show that TMAO stabilizes RNase A at pH 7.0 and does not stabilize the protein at pH 4.0. RNase A thermal denaturation in the presence of TMAO is a reversible two-state N ⇆ D process. We also show that TMAO counteracts the urea and GuHCl denaturing effect at neutral pH, whereas the counteracting ability is lost at acid pH.     

Noncovalent associations of glutathione S-transferase and ligands: A study using electrospray quadrupole/time-of-flight mass spectrometry

Human glutathione S-transferase A1-1 was observed predominantly as dimeric ions (51 kDa) during electrospray mass spectrometric analysis from aqueous solution at pH 7.4, in keeping with the known dimeric structure in solution. When analyses were performed on solutions of the enzyme containing glutathione (GSH), noncovalent adducts of protein dimer and one or two ligand molecules were observed; each mass increment, which exceeded the mass of GSH alone, was provisionally interpreted to indicate concomitant association of two water molecules per bound GSH. Noncovalent adducts of ligand and protein dimer were similarly observed for oxidized glutathione and for two glutathione inhibitors, both incorporating substituted thiol structures. In these instances, the mass increments exactly matched the ligand masses, suggesting that the apparent concomitant binding of water was associated with the presence in the ligand of a free thiol group. Collisionally activated decomposition during tandem mass spectrometry analyses of noncovalent adducts incorporating protein dimer and ligands yielded initially the denuded dimer; at higher collision energies the monomer and a protein fragment were formed.     

Substituent effects upon the catalytic activity of aromatic cyclic seleninate esters and spirodioxyselenuranes that act as glutathione peroxidase mimetics

Substituent effects were studied in a series of aromatic cyclic seleninate esters and spirodioxyselenuranes that function as mimetics of the antioxidant selenoenzyme glutathione peroxidase. The methoxy-substituted selenurane proved the most efficacious catalyst for the reduction of hydrogen peroxide with benzyl thiol, and the reaction rates were enhanced for both classes by electron-donating substituents. Hammett plots indicated rho = -0.45 and -3.1 for the seleninates and selenuranes, respectively, suggesting that oxidation at Se is the rate-determining step in their catalytic cycles.     

Kinetics and mechanism for reduction of the anticancer prodrug trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by thiols

The reduction of the platinum(IV) prodrug trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by L-cysteine, DL-penicillamine, DL-homocysteine, N-acetyl-L-cysteine, 2-mercaptopropanoic acid, 2-mercaptosuccinic acid, and glutathione has been investigated at 25 degrees C in a 1.0 M aqueous perchlorate medium with 6.8 < or = pH < or = 11.2 using stopped-flow spectrophotometry. The stoichiometry of Pt(IV):thiol is 1:2, and the redox reactions follow the second-order rate law -d[Pt(IV)]/dt = k[Pt(IV)][RSH]tot, where k denotes the pH-dependent second-order rate constant and [RSH]tot the total concentration of thiol. The pH dependence of k is ascribed to parallel reductions of JM335 by the various protolytic species of the thiols, the relative contributions of which change with pH. Electron transfer from thiol (RSH) or thiolate (RS-) to JM335 is suggested to take place as a reductive elimination process through an attack by sulfur at one of the mutually trans chloride ligands, yielding trans-[Pt(OH)2(c-C6H11NH2)(NH3)] and RSSR as the reaction products, as confirmed by 1H NMR. Second-order rate constants for the reduction of JM335 by the various protolytic species of the thiols span more than 3 orders of magnitude. Reduction with RS- is approximately 30-2000 times faster than with RSH. The linear correlation log(kRS) = (0.52 +/- 0.06)-pKRSH--(2.8 +/- 0.5) is observed, where kRS denotes the second-order rate constant for reduction of JM335 by a particular thiolate RS- and KRSH is the acid dissociation constant for the corresponding thiol RSH. The slope of the linear correlation indicates that the reactivity of the various thiolate species is governed by their proton basicity, and no significant steric effects are observed. The half-life for reduction of JM335 by 6 mM glutathione (40-fold excess) at physiologically relevant conditions of 37 degrees C and pH 7.30 is 23 s. This implies that JM335, in clinical use, is likely to undergo in vivo reduction by intracellular reducing agents such as glutathione prior to binding to DNA. Reduction results in the immediate formation of a highly reactive platinum(II) species, i.e., the bishydroxo complex in rapid protolytic equilibrium with its aqua form.     

Whey protein soluble aggregates from heating with NaCl: physicochemical, interfacial, and foaming properties

Whey protein isolate was heat-treated at 85 degrees C for 15 min at pH ranging from 6.0 to 7.0 in the presence of NaCl in order to generate the highest possible amount of soluble aggregates before insolubility occurred. These whey protein soluble aggregates were characterized for composition, hydrodynamic diameter, apparent molecular weight, zeta-potential, surface hydrophobicity index, activated thiol group content, and microstructure. The adsorption kinetics and rheological properties (E', etad) of these soluble aggregates were probed at the air/water interface. In addition, the gas permeability of a single bubble stabilized by the whey protein soluble aggregates was determined. Finally, the foaming and foam-stabilizing properties of these aggregates were measured. The amount of whey protein soluble aggregates after heat treatment was increased from 75% to 95% from pH 6.0 to pH 7.0 by addition of 5 mM to 120 mM NaCl, respectively. These soluble aggregates involved major whey protein fractions and exhibited a maximum of activated thiol group content at pH > 6.6. The hydrodynamic radius and the surface hydrophobicity index of the soluble aggregates increased from pH 6.0 to 7.0, but the molecular weight and zeta-potential decreased. This loss of apparent density was clearly confirmed by microscopy as the soluble aggregates shifted from a spherical/compact structure at pH 6.0 to a more fibrillar/elongated structure at pH 7.0. Surface adsorption was faster for soluble aggregates formed at pH 6.8 and 7.0 in the presence of 100 and 120 mM NaCl, respectively. However, interfacial elasticity and viscosity measured at 0.01 Hz were similar from pH 6.0 to 7.0. Single bubble gas permeability significantly decreased for aggregates generated at pH > 6.6. Furthermore, these aggregates exhibited the highest foamability and foam liquid stability. Air bubble size within the foam was the lowest at pH 7.0. The coarsening exponent, alpha, fell within predicted values of 1/3 and 1/2, except for very dry foams where it was 1/5.     

Modeling the adsorption of Cd(II) onto goethite in the presence of citric acid

The adsorption of cadmium onto goethite in the presence of citric acid was measured as a function of pH and cadmium concentration at 25 degrees C. Potentiometric titrations were also performed on the system. Cadmium adsorption onto goethite was enhanced above pH 4 in the presence of 50 microM, 100 microM and 1 mM citric acid. While there was little difference between the enhancements caused by 50 and 100 microM citric acid below pH 6, above pH 6 further enhancement is seen in the presence of 100 microM citric acid. When 1 mM citric acid was present, the enhancement of cadmium adsorption was greater below pH 6, with increased Cd(II) adsorption down to pH 3.5. However, above pH 6, 1 mM of citric acid caused slightly less enhancement than the lower citric acid concentrations. ATR-FTIR spectra of soluble and adsorbed citrate-cadmium species were measured as a function of pH. At pH 4.6 there was very little difference between the ternary Cd(II)-citric acid-goethite spectrum and the binary citric acid-goethite spectrum. However, spectra of the ternary system at pH 7.0 and 8.7 indicated the presence of additional surface species. Further analysis of the spectra suggested that these were metal-ligand outer-sphere complexes. Data from the adsorption experiments and potentiometric titrations of the ternary Cd(II)-citric acid-goethite system were fitted by an extended constant-capacitance surface complexation model. The spectroscopic data were used to inform the choice of surface species. Three reactions in addition to those for the binary Cd(II)-goethite and citric acid-goethite systems were required to describe all of the data. They were [formula in text], [formula in text], and [formula in text]. Neither the spectroscopy nor the modeling suggested the formation of a ternary inner-sphere complex or a surface precipitate under the conditions used in this study.     

Selective detection of the reduced form of glutathione (GSH) over the oxidized (GSSG) form using a combination of glutathione reductase and a Tb(III)-cyclen maleimide based lanthanide luminescent 'switch on' assay

The synthesis of a novel Tb(III) luminescent probe for the detection of thiols is presented. The probe 1.Tb, possessing a maleimide moiety, as its sulfhydryl acceptor, was poorly emitting in aqueous pH 7 solution in the absence of a thiol. However, upon addition of thiols such as glutathione (GSH), large enhancements were observed, particularly within the physiological pH range. In contrast no enhancements were observed in the presence of the oxidized form of glutathione (GSSG), except in the presence of the enzyme glutathione reductase and NADPH which enabled 1.Tb to be used to observe the enzymatic reduction of GSSG to GSH in real time.     

Assemblage of signaling DNA enzymes with intriguing metal-ion specificities and pH dependences

We report a group of new DNA enzymes that possess a synchronized RNA-cleavage/fluorescence-signaling ability and exhibit wide-ranging metal-ion and pH dependences. These DNA catalysts were derived from a random-sequence DNA pool in a two-stage process: (1) establishment of a catalytic DNA population through repetitive rounds of in vitro selection at pH 4.0, and (2) sequence-diversification and catalytic-activity optimization through five parallel paths of in vitro evolution conducted at pH 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. The deoxyribozymes were evolved to cleave the phosphodiester bond of a single ribonucleotide embedded in DNA and flanked immediately by two deoxyribonucleotides modified with a fluorophore and a quencher, respectively--a setting that synchronizes catalysis with fluorescence signaling. The most dominant catalyst from each pool was examined for metal-ion specificity, catalytic efficiency, pH dependence, and fluorescence-signaling capability. Individual catalysts have different metal-ion requirements and can generate as much as a 12-fold fluorescence enhancement upon RNA cleavage. Most of the DNA enzymes have a pH optimum coinciding with the selection pH and exhibit a rate constant approximating 1 min(-)(1) under optimal reaction conditions. The demonstration of DNA enzymes that are functional under extremely high acidity (such as pH 3 and 4) indicates that DNA has the ability to perform efficient catalysis even under harsh reaction conditions. The isolation of many new signaling DNA enzymes with broad pH optima and metal-ion specificities should facilitate the development of diverse deoxyribozyme-based biosensors.     

A fluorescence turn-on probe for the detection of thiol-containing amino acids in aqueous solution and bioimaging in cells

A turn-on fluorescent probe, based on a water-soluble terphenyl derivative, for the detection of cysteine and homocysteine is reported. The aldehyde groups in the probe play crucial roles in providing reaction with thiol groups in the amino acids, leading to a formation of thiazolidine (from cysteine) or thiazinane ring (from homocysteine). As a result, the new formation of such rings alters the electronic property of the conjugated system in the probe and results in emission enhancement. The probe in aqueous solution exhibits a remarkable increase in its quantum yield upon exposure to cysteine (up to 20-fold) and to homocysteine (up to 700-fold), while slight quenching is observed in the presence of glutathione. Moreover, an investigation on time-resolved fluorescence spectra of the probe in the presence of cysteine and homocysteine reveals potential discriminatory detection of cysteine and homocysteine. Bioimaging of the thiols in live HeLa cells was successfully applied.     

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.     

Ormosil Encapsulated Pyrroloquinoline Quinone‐Modified Electrochemical Sensor for Thiols

An organically modified sol‐gel glass (ORMOSIL) encapsulating pyrroloquinoline quinone (PQQ)‐modified electrode for the rapid, sensitive and simple determination of thiol‐containing compounds such as cysteine and glutathione is reported. The effect of applied potential, nature of thiol compound and pH on the response of the sensor was examined and optimum conditions were determined. The electrochemical responses and detection limits were found to be sensitive to the nature of thiols and pH. The electrochemical responses for cysteine and glutathione at an applied potential of ?0.2 V (vs. Ag/AgCl) were found to be linear with detection limits of 18 nM for cysteine and 36 nM for glutathione at pH 3.5, whereas the detection limits at pH 8.5 were 0.5 μM for cysteine and 1 μM for glutathione. The electrode retained 95% of the original response for 7 days when stored at 4 °C. The ORMOSIL‐encapsulated PQQ was also characterized by spectrophotometry. The absorbance measurement using 5,5′‐dithiobis(2‐nitrobenzoic acid) at 412 nm justify the PQQ‐mediated oxidation of glutathione whereas fluorescence measurements (excitation wavelength=380 nm; emission wavelength=480 nm) justify the successful encapsulation of PQQ in ORMOSIL matrix.     

Immobilization of Candida antarctica lipase B by covalent attachment to green coconut fiber

The objective of this study was to covalently immobilize Candida antarctica type B lipase (CALB) onto silanized green coconut fibers. Variables known to control the number of bonds between enzyme and support were evaluated including contact time, pH, and final reduction with sodium borohydride. Optimal conditions for lipase immobilization were found to be 2 h incubation at both pH 7.0 and 10.0. Thermal stability studies at 60 degrees C showed that the immobilized lipase prepared at pH 10.0 (CALB-10) was 363-fold more stable than the soluble enzyme and 5.4-fold more stable than the biocatalyst prepared at pH 7.0 (CALB-7). CALB-7 was found to have higher specific activity and better stability when stored at 5 degrees C. When sodium borohydride was used as reducing agent on CALB-10 there were no improvement in storage stability and at 60 degrees C stability was reduced for both CALB-7 and CALB-10.     

Kinetics and mechanism of the addition of nucleophiles to alpha,beta-unsaturated thiol esters

Compounds containing the UV-absorbing chromophores p-methoxycinnamate, p-methoxycinnamide, or anthranilate and an alpha,beta- or alpha,beta,gamma,delta-unsaturated thiol ester (crotonyl or sorboyl) have been prepared. These compounds are subject to nucleophilic attack at the C=C conjugated to the thiol ester carbonyl group. The kinetics of the reactions of these thiol esters with N-acetyl-l-cysteine (NAC), N-acetylcysteamine, and N(2)-acetyl-L-lysine (NAL) have been studied, and the thiol addition products have been identified. The reaction rates increased at higher pH, and the reaction of NAC thiolate with a crotonyl thiol ester in 1:1 (v/v) acetonitrile/aqueous HEPES exhibited buffer catalysis as a result of protonation of the enolate intermediate. At the same concentration, NAC underwent approximately 300-fold more reaction than NAL with a crotonyl thiol ester at pH 9.8. Additionally, a crotonyl thiol ester was found to be 7.9 times more reactive than a sorboyl thiol ester toward NAC addition. These unsaturated thiol esters may serve as a means of covalently binding UVA and UVB sunscreens to the outer layer of skin to provide long-lasting protection.     

Synthesis and Properties of Diuridine Phosphate Analogues Containing Thio and Amino Modifications

Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).     

Determination of glutathione in pharmaceuticals and cosmetics by HPLC with UV and fluorescence detection

Summary A selective HPLC determination of reduced glutathione (GSH) in pharmaceutical and cosmetic formulations is described based on prechromatographic derivatization with 4-(6-methylnaphthalen-2-yl)-4-oxo-2-buteneoic acid. The derivatization reaction is rapid under mild reaction conditions (10 min at ambient temperature and pH 7.5) and the excess reagent can be removed by liquid-liquid extraction. The thiol adducts were chromatographed on a C-8 column using 0.05 M triethylammonium phosphate (pH 4.0) — acetonitrile 68∶32 (v/v), as the mobile phase; UV and fluorescence detection (l_em 450 nm, l_exc 300 nm) were both used. The structure of the thiol adducts was confirmed by¹H and¹³C NMR spectra using sodium methanethiolate as thiol nucleophile. This work constitutes part of the thesis for the “Dottorato di ricerche” of Dr. Roberto Gotti.