PHOTOLYSIS AND PHOTO-OXIDATION OF AMINO ACIDS AND PEPTIDES—VI. A STUDY OF THE INITIATION OF DISULPHIDE INTERCHANGE BY LIGHT IRRADIATION

Abstract— Investigations concerning the occurrence of disulphide interchange for disulphides related to cystine are made with either a single unsymmetrical disulphide or an equimolar mixture of two symmetrical disulphides. It is found that in the pH range 1–6 where spontaneous interchange can usually be disregarded, interchange can be initiated by the addition of a small amount of a thiol or by irradiation with ultraviolet light from either of two artificial sources, interchange being more rapid at pH 4–6 than at pH 1–3. Direct sunlight and, to a lesser extent, visible (filtered tungsten) light are also effective. Disulphide interchange in the solid state can also be promoted by ultraviolet light. A comparison is made between the roles' of S-S and C-S bond fission in the photochemistry of disulphides, with respect not only to the promotion of disulphide interchange, but to other phenomena such as the dismutation of disulphides to thioethers and trisulphides.     

Determination of glutathione, glutathione disulphide, ascorbic acid and dehydroascorbic acid in tissues by reversed-phase liquid chromatography with electrochemical detection

Summary High-performance liquid chromatography with electrochemical detection was applied to the estimation of glutathione, glutathione disulphide, ascorbic acid and dehydroascorbic acid in various tissues of man, animal, and plant. The simultaneous determination of glutathione and ascorbic acid in tissues was done by a coulometric method. Separation of glutathione and ascorbic acid and unequivocal substance identifications were performed on a 100×4.6 mm RP-18 Spheri 5 column. As mobile phase 0.015 mol/l o-phosphoric acid, pH 2.3 was used. Retention time of ascorbic acid was 5.0 min and of glutathione 10.0 min. Dehydroascorbic acid was determined after reduction to ascorbic acid with dithiothreitol. Glutathione disulphide was reduced at pH 7.5 by -nicotinamide-dinucleotide phosphate and glutathione reductase, EC 1.6.4.2., to regenerate glutathione. To exclude interfering substances, several other compounds present in tissues and foods were investigated. This coulometric method is highly sensitive, specific and simple. Very low concentrations of ascorbic acid, glutathione, dehydroascorbic acid, and glutathione disulphide (<500 pg/injection) could be analysed using this HPLC-ECD method.(on leave to Mexico)     

Controlled release matrix tablet formulation using synthesized N-acyl Thiolated Chitosan derivatives

Chitosan derivative was successively synthesized by initial preparation of acyl thiourea reagent using ammonium thiocyanate and making it to react with at primary amino groups of chitosan and then reduced to yield Thiolated Chitosan. Due to the formation of disulphide bonds with mFucus glycoproteins, mucoadhesiveness is augmented. The thiol groups were then quantified using Ellman’s reagent. The derivatives inherit good swelling property in neutral and alkaline media. The different derivatives containing thiol groups were formulated into tablets using reference drug for evaluation. The Thiolated Chitosan display in situ gelling features due to the pH-dependent (alkaline pH) formation of inter-molecular disulphide bonds which makes the application of Thiolated Chitosan on intestinal mucosa and can guarantee prolonged controlled release of embedded therapeutic ingredients.     

Electrochemistry of self-assembled monolayers of the blue copper protein Pseudomonas aeruginosa azurin on Au(111)

We report the self-assembly and electrochemical behaviour of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) electrodes in aqueous acetate buffer (pH=4.6). The formation of monolayers of this protein is substantiated by electrochemical measurements. Capacitance results indicate qualitatively that the protein is strongly adsorbed at sub-μM concentrations in a broad potential range (about 700 mV). This is further supported by the attenuation of a characteristic cyclic voltammetric peak of Au(111) in acetate solution with increasing azurin concentration. Reductive desorption is clearly disclosed in NaOH solution (pH=13), strongly suggesting that azurin is adsorbed via its disulphide group. An anodic peak and a cathodic peak associated with the copper centre of azurin are finally observed in the differential pulse voltammograms. These peaks are, interestingly, indicative of long-range electrochemical electron transfer such as paralleled by intramolecular electron transfer between the disulphide anion radical and the copper atom in homogeneous solution, and anticipated by theoretical frames. Together with reported in situ scanning tunnelling microscopy (STM) results they constitute the first case for electrochemistry of self-assembled monolayers of azurin, even redox proteins. This integrated investigation provides a new approach to both structure and function of adsorbed redox metalloproteins at the molecular level.     

Spectrophotometric determination of copper as a dithiocarbamate by flow injection analysis

Copper(II) ions (1–20 mg l^?1) are determined at 385 nm by injection into an aqueous carrier solution containing EDTA (0.05 M), carbon disulphide (0.03%) and diethanolamine (0.1%) in aqueous ammonia solution (0.5%) adjusted to pH 8.0. Chromium(VI) and, to a minor extent, iron(III) interfere. In the absence of EDTA, cobalt, iron(II), nickel, and manganese ions interfere but the sensitivity to copper is higher.     

The three states criterion of nucleophilic reactivity in the conjugate acid of an anionic nucleophile and some problems of proton partitioning: implications for the reactions of 2,2′-dipyridyl disulphide with papain and with L-ergothioneine

The circumstances in which observation of three reactive protonic states of a reaction, involving an electrophile that increases its reactivity consequent on protonation, may be regarded as compelling evidence of nucleophilic character in the conjugate acid of an anionic nucleophile (”three states criterion“) are delineated. Aspects of the reaction of papain with 2,2′-dipyridyl disulphide not previously reported suggest that this reaction at pH 4 is best described as an intracomplex thiol-disulphide interchange involving the unionized thiol group of the cysteine-25-histidine-159-asparagine-175 hydrogen bonded system of papain with 2,2′-dipyridyl disulphide hydrogen bonded at one nitrogen atom to the carboxyl group of aspartic acid-158. The reaction appears to involve pre-transition state proton transfer in the thiol-imidazole hydrogen bond; the protonation of the side chains of aspartate-158 and histidine-159 may be positively cooperative. Rate equations for reactions involving up to three reactive protonic states are presented in an appendix.     

Spectrophotometric determination of cyanide with organic disulphides

The reaction of cyanide ion with the disulphides 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, 2,2'-dithiodipyrimidine, and 5,5'-dithiobis(2-nitrobenzoic acid) to displace an absorbing thiol anion has been evaluated for the spectrophotometric determination of cyanide. Reaction is somewhat slow and is applicable to cyanide determination in the range 0.2-5.0 mug ml . By variation of reactant ratios and pH the concentration range can be increased to 50 mug ml . The reaction is faster when cyanide is present at a higher concentration than the disulphide.     

Use of p-diethylaminophenylmercuric acetate for the determination of thiol groups in biological samples

A simple titrimetric method for the determination of thiol and disulphide groups in biological samples with p-diethylaminophenylmercuric acetate as titrant, is suggested. Milligram amounts of cysteine (1-25 mg) are determined with a relative standard error not higher than 1%. The lowest detenninable amount of cysteine is 0.1 mg (relative standard deviation 3%). The accuracy and the sensitivity are the same as in amperometric titration with mercuric chloride. The method has been successfully used for the determination of thiol groups in various biological samples.     

Thiomercurimetric determination of carbon disulphide, carbonyl sulphide, thiols and hydrogen sulphide by use of 1,3-diaminopropane and tributyltin chloride

A new approach to the determination of carbon disulphide and carbonyl sulphide in the presence of each other is based on the reaction with 1,3-diaminopropane (DAP), and titration with o-hydroxymercurybenzoie acid (HMB) before and after the selective decomposition of the COS-derivative at pH 4. Determination of hydrogen sulphide, thiols, carbon disulphide and carbonyl sulphide in the presence of each other in hydrocarbon solvents involves four titrations with HMB, viz. of all compounds after conversion of CS(2) and COS with DAP, of thiols plus H(2)S, of the thiols alone after removal of H(2)S by extraction, and of CS(2) alone after removal of other compounds by shaking with aqueous alkali. For selective trapping of H(2)S, HCN, RSH, CO(2) and CS(2) + COS, the sample gas is passed successively through a potassium antimonyl tartrate filter, a nickel carbonate filter, a tributyltin chloride filter, a bubbler containing 40% potassium hydroxide solution and a bubbler containing a benzene solution of DAP. The analysis is completed by titration with HMB with dithizone or dithiofluorescein as indicator.     

Electronic circular dichroism of disulphide bridge: ab initio quantum-chemical calculations

Electronic circular dichroism (ECD) parameters of the disulphide chromophore have been calculated for dihydrogen disulphide, dimethyl disulphide, and cystine using density-functional theory, coupled-cluster theory, and multiconfigurational self-consistent field theory. The objective is twofold: first, to examine the performance of the Coulomb-attenuated CAM-B3LYP functional for the calculation of ECD spectra; second, to investigate the dependence of the ECD parameters on the conformation around the disulphide bridge. The CAM-B3LYP functional improves considerably on the B3LYP functional, giving results comparable to CCSD theory and to MCSCF theory in an extended active space. The conformational dependence of the ECD parameters does not change much upon substitution, which is promising for the application of ECD in structural investigations of proteins containing disulphide bridges.     

纯化基因重组白细胞介素-2的初步研究

 采用羟基磷灰石 (HAP)作为填料 ,以pH 6 8的磷酸盐缓冲液为流动相 ,用制备型高效液相色谱来分离提纯基因重组白细胞介素 2 (IL 2 ) ,通过SDS 聚丙烯酰胺凝胶 (SDS PAGE)电泳测定IL 2的纯度 ,测定其活性为1× 1 0 6 U/mg。该法对IL 2的纯化、含量测定及活性测定等效果良好。     

Structural study of the stability of the captopril drug regarding the formation of its captopril disulphide dimer

Captopril disulphide is obtained under hydrothermal conditions. The IR and Raman spectra data are in agreement with the X-ray diffraction results. The disappearance of the band at 2566 cm^–1 (ν(SH)) in both spectra of captopril disulphide is consistent with the formation of the S–S bond. The degradation of the captopril drug is investigated by Raman spectroscopy and the results indicate that after 6 weeks of air exposure, a band at 512 cm^–1, assigned as ν(SS), is observed, suggesting the formation of captopril disulphide. DFT calculations in the solid state are performed for captopril and captopril disulphide. The results indicate that captopril disulphide is approximately 30 kcal?mol^–1 more stable than captopril. The analysis of the total density of states (DOS) reveals that the captopril valence band contains a significant contribution from the S atom, whereas for captopril disulphide, the O atom is the most important for the valence band.     

Titrimetric determination of thiols: Tetrathionate, iron(III), cystine and hexacyanoferrate(III) as thiol reagents

Four analytical reagents, tetrathionate, iron(III), cystine and hexacyanoferrate(III) have been tested with respect to their specificity for oxidation of thiols to disulphides. Of a number of thiols studied, most have a strong tendency to oxidize beyond the disulphide stage with several of the commonly employed reagents. Tetrathionate, cystine and hexacyanoferrate(III) function in phosphate buffer of pH 7, but iron(III) does not require rigid control of pH, although the solution should be only feebly acidic. The reagents were used in excess and the thiosulphate or cysteine formed in the reaction of thiols with tetrathionate or cystine respectively was determined. The residual iron(III) was measured by adding ascorbic acid or mercaptoacetic acid and titrating with 2,6-dichlorophenolindophenol or iodine monochloride respectively; surplus hexacyanoferrate(III) was back-titrated with ascorbic acid. All four reagents react selectively with thiols even in the presence of several possible interfering substances and afford results that are accurate and precise.     

Polarographic study of bis(2-pyridyl)disulphide di-N-oxide and bis(2-quinolyl)disulphide di-N-oxide in aqueous ethanol

The polarographic reduction of bis(2-pyridyl)- and of bis(2-quinolyl)disulphide di-N-oxide in aqueous alcohol yields aryl mercaptans. The electrode reaction is kinetically controlled by an initial one-electron transfer step, and values for the transfer coefficients and the specific heterogeneous rate constants for the forward reactions are presented. The polar N-oxide function in the alpha-position withdraws electrons from the disulphide bond, making the half-wave potential less negative than that of the diphenyl disulphide. The pk(a) values of the mercapto compounds formed have been evaluated and the low values obtained for the heterocyclic N-oxides are further evidence for tautomerism in these compounds.     

Method development and validation for the separation and determination of omeprazole enantiomers in pharmaceutical preparations by capillary electrophoresis

A new capillary zone electrophoresis (CZE) method for the separation of omeprazole enantiomers has been developed. Methyl-β-cyclodextrin (methyl-β-CD) was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated in order to optimize separation and run times. Analysis times, shorter than 8 min were found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM β-cyclodextrin and 5 mM sodium disulphide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/l for the omeprazole enantiomers. The proposed method was applied to five pharmaceutical preparations with recoveries between 84 and 104% of the labeled contents.     

Influence of pH for the determination of serum albumin by a dye-binding method in the presence of a detergent

In the dye-binding method, the absorbance increase caused by a protein error of a pH indicator is observed only in a restricted pH range. However, this pH range in the presence of a detergent has not yet been examined. Thus, the author investigated the pH (pH(UL)) where the absorbance increase becomes zero by a calculation based on the chemical equilibrium of a protein error of a pH indicator, and by experiments using four sulfonephthalein dyes. The pH(UL) value changed only with the detergent concentration, but did not change at all due to the dye, buffer solution or protein concentrations. Although the pH(UL) value was different according to the kind of dye used, it correlated well with the pK(D) values (dissociation constant) of BPB, BCG, BCP and BTB. The characteristics of pH(UL) in the reactions of the four dyes indicated good agreement with that obtained by a calculation.     

Influence of tartrate ions on the coprecipitation of some trace metal inner complex compounds with bis(8-quinolyl)disulphide

The coprecipitation of Fe(III) quinoline-8-thiolate (QT) with bis (8-quinolyl) disulphide has been investigated with 1 mol × dm^–3 potassium tartrate and 0.001 mol × dm^–3 aqueous tartaric acid solutions in dependence on their pH. In return QTs of Cu(II), Cd(II) and Hg(II) have been coprecipitated from 0.2, 0.1, 0.01 and 0.001 mol × dm^–3 aqueous solutions of tartaric acid after adjusting the pH to 6–7. These tartaric acid concentrations relate to their initial concentrations before the coprecipitation. The presence of tartrate ions influences the recovery of the coprecipitated Fe(III), Cd(II) and Hg(II) QTs. A complete coprecipitation of the Cu(II) inner complex compound can be achieved from the aqueous solutions indicated.     

Photocatalytical reduction of disulphide bonds in peptides on Ag-loaded nano-TiO2 for subsequent derivatization and determination

We reported an alternative strategy to reduce disulphide bonds in peptides with Ag-nanoparticle loaded nano-TiO(2) (Ag/TiO(2)) under UV irradiation. The feasibility of this strategy was adequately demonstrated using the model peptides oxidized glutathione, vasopressin and insulin, which contain various disulphide bonds, as well as by its application to the determination of Cd-induced phytochelatins in Phaeodactylum tricornutum.     

Two-dimensional electrophoresis of cereal prolamins: applications to biochemical and genetic analyses

Three complementary two-dimensional systems for the analysis of cereal prolamins are described. These are electrophoresis at pH 3.1 followed by electrophoresis at pH 9.2, isoelectric focusing (IEF) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE under non-reducing conditions followed by SDS-PAGE with reduction. They together give information on the pIs, Mrs and charge properties of the individual prolamin components, and on their interactions to form oligomers stabilized by inter-chain disulphide bonds. The three systems are then compared for their effectiveness in analysing unreduced prolamin I fractions from wheat and rye, and the IEF/SDS-PAGE system for analysing reduced and pyridylethylated prolamin fractions from all the major cereals. Finally, applications of the systems in biochemical and genetic studies are discussed and illustrated with three examples: analysis of the structural relationships of the S-rich prolamins (B hordeins and gamma-type hordeins) of barley, determination of the interactions of wheat and rye prolamin subunits in a 2RS/2BL translocation line, and the mapping of genes for alpha-type prolamins in the wild grass Haynaldia villosa.     

Determination of sulphide, polysulphide, thiocarbonates and sulphur by extraction with tributyltin hydroxide, and of thiosulphate by hydrogenation

The methods presented involve the separation of sulphur compounds by means of a hexane solution of tributyltin hydroxide (TBT), followed by titration with o-hydroxymercuribenzoic acid in the presence of dithizone as indicator. Free sulphide is selectively extracted from strongly alkaline solution, whereas the polysulphides and thiocarbonates are extracted at pH 9-10. The polysulphide and thiocarbonate extracts decompose to form TBT-sulphide, sulphur and carbon disulphide. Treatment with sulphite, stannate(II), copper, vinyl cyanide and ethylenediamine, and hydrogenation with zinc and hydrobromic acid have been applied in the course of the analysis. The sulphur is determined by its attack on copper to form copper(I) sulphide which is subsequently dissolved in aqueous potassium cyanide solution and the sulphide separated by extraction.