Gerald L. Newton

Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography

Abstract A method is described for determination of biological thiols at the picomole level based upon conversion of thiols to fluorescent derivatives by reaction with monobromobimane and separation of the derivatives by reverse-phase high-performance liquid chromatography. Thiols separated by the procedure include N -acetylcysteine, coenzyme A, coenzyme M, cysteamine, cysteine, cysteinylglycine, ergothioneine, ethanethiol, glutathione, γ-glutamylcysteine, homocysteine, hydrogen sulfide, 2-mercaptoethanol, mercaptopyrimidine, methanethiol, pantetheine, 4′-phosphopantetheine, thiosulfate, and 2-thiouracil. Since monobromobimane has little fluorescence and reacts very selectively with thiols to produce fluorescent derivatives, crude extracts can be derivatized and analyzed without prepurification of the thiols, the entire process requiring only 1 to 2 h. The technique is illustrated by determination of the thiol levels in red blood cells.

Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography

Publisher Summary This chapter describes methods for the preparation and high-performance liquid chromatography (HPLC) analysis of monobromobimane derivatives of low molecular weight thiols in extracts of biological samples and discusses typical problems encountered in the development and application of these methods. Several different types of samples are typically needed in the course of a given application, and these include the thiol standard sample, the reagent blank, the unknown sample, and the unknown control. The thiol standard solutions were prepared from authentic stock solutions of the individual thiols of interest. Three HPLC protocols have proved useful for different types of sample analysis. Separation of the greatest number of thiol derivatives is achieved with Method 1, which utilizes a 4.6 × 250 mm Altex Ultrasphere ODS 5 μm analytical column equipped with a Brownley MPLC guard column containing an OD-GU 5 μm C 18 cartridge. Method 2 is developed to provide quantitative analytical data for coenzyme A and dephospho-CoA, which elute as broad peaks with method 1, while retaining the capacity to simultaneously analyze for glutathione and cysteine. Method 3 utilizes sodium perchlorate ion pairing to enhance the analysis of cysteamine and WR-1065 derivatives in samples also containing glutathione (GSH) and cysteine.

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

SUMMARY Mycothiol (MSH; AcCys-GlcN-Ins) is the major thiol found in Actinobacteria and has many of the functions of glutathione, which is the dominant thiol in other bacteria and eukaryotes but is absent in Actinobacteria. MSH functions as a protected reserve of cysteine and in the detoxification of alkylating agents, reactive oxygen and nitrogen species, and antibiotics. MSH also acts as a thiol buffer which is important in maintaining the highly reducing environment within the cell and protecting against disulfide stress. The pathway of MSH biosynthesis involves production of GlcNAc-Ins-P by MSH glycosyltransferase (MshA), dephosphorylation by the MSH phosphatase MshA2 (not yet identified), deacetylation by MshB to produce GlcN-Ins, linkage to Cys by the MSH ligase MshC, and acetylation by MSH synthase (MshD), yielding MSH. Studies of MSH mutants have shown that the MSH glycosyltransferase MshA and the MSH ligase MshC are required for MSH production, whereas mutants in the MSH deacetylase MshB and the acetyltransferase (MSH synthase) MshD produce some MSH and/or a closely related thiol. Current evidence indicates that MSH biosynthesis is controlled by transcriptional regulation mediated by σB and σR in Streptomyces coelicolor. Identified enzymes of MSH metabolism include mycothione reductase (disulfide reductase; Mtr), the S-nitrosomycothiol reductase MscR, the MSH S-conjugate amidase Mca, and an MSH-dependent maleylpyruvate isomerase. Mca cleaves MSH S-conjugates to generate mercapturic acids (AcCySR), excreted from the cell, and GlcN-Ins, used for resynthesis of MSH. The phenotypes of MSH-deficient mutants indicate the occurrence of one or more MSH-dependent S-transferases, peroxidases, and mycoredoxins, which are important targets for future studies. Current evidence suggests that several MSH biosynthetic and metabolic enzymes are potential targets for drugs against tuberculosis. The functions of MSH in antibiotic-producing streptomycetes and in bioremediation are areas for future study.

Analysis of biological thiols: Quantitative determination of thiols at the picomole level based upon derivatization with monobromobimanes and separation by cation-exchange chromatography

A method for quantitative determination of biological thiols is presented. Thiols are converted to fluorescent derivatives by reaction with monobromobimane or monobromotrimethylammoniobimane. The derivatives are separated by ion-exchange chromatography and detected by fluorometry. Thiols that can be separated and quantitated by combined use of mBBr and qBBr derivatives include N-acetylcysteine, coenzyme A, coenzyme M, cysteine, cysteamine, cysteinylglycine, ergothioneine, ethanethiol, glutathione, γ-glutamylcysteine, homocysteine, 2-mercaptoethanol, methanethiol, pantetheine, 4′-phosphopantetheine, thiosulfate, thiouracil, and the mono- and diderivatives of dithiothreitol. Most thiols can be detected at the 1-pmol level and quantitated when present at the 10-pmol or higher level. Some, such as ergothioneine, exhibited low fluorescent yields and can be measured only at levels an order of magnitude higher. The method was applied to human red blood cell where the main thiols were found to be glutathione (2.4 mm) and ergothioneine (120 μm), in accord with earlier reports.

Bacillithiol is an antioxidant thiol produced in Bacilli

Glutathione is a nearly ubiquitous low-molecular-weight thiol and antioxidant, although it is conspicuously absent from most Gram-positive bacteria. We identify here the structure of bacillithiol, a novel and abundant thiol produced by Bacillus species, Staphylococcus aureus, and Deinococcus radiodurans. Bacillithiol is the α-anomeric glycoside of l-cysteinyl-d-glucosamine with l-malic acid and likely functions as an antioxidant. Bacillithiol, like structurally similar mycothiol, may serve as a substitute for glutathione.

Mycothiol-Deficient Mycobacterium smegmatis Mutants Are Hypersensitive to Alkylating Agents, Free Radicals, and Antibiotics

ABSTRACT Mycothiol (MSH; 1d-myo-inosityl 2-[N-acetyl-l-cysteinyl]amido-2-deoxy-α-d-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1d-myo-inosityl 2-deoxy-α-d-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside ligase (MshC) activities of the three mutant strains were ≤2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics.

Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli

Bacillithiol (BSH), the α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid, is a major low-molecular-weight thiol in Bacillus subtilis and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electrophiles, the BshA glycosyltransferase and BshB1 deacetylase are encoded in an operon with methylglyoxal synthase. BshB1 is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identified a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that co-occurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshB1 and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxide-containing antibiotic detoxified by FosB, a prototype for bacillithiol-S-transferase enzymes.

Radioprotection of DNA by Thiols: Relationship between the Net Charge on a Thiol and Its Ability to Protect DNA

Release of free bases from calf thymus DNA upon irradiation in aerated 0.1 mol dm-3NaClO4 at pH 7 has been measured by HPLC and shown to be markedly influenced by the presence of thiols during irradiation. The ability of thiols to protect DNA was shown to depend upon the net charge (Z) at pH 7 in the order WR 1065 (Z = +2) greater than cysteamine (Z = +1) greater than 2-mercaptoethanol (Z = 0) approximately equal to dithiothreitol (Z = 0) greater than GSH (Z = -1) approximately equal to 2-mercaptoethanesulfonic acid (Z = -1) approximately equal to 2-mercaptosuccinate (Z = -2). A similar dependence of protection upon net charge was found for disulfides: cystamine (Z = +2) greater than 2-mercaptoethyl disulfide (Z = 0) greater than GSSG (Z = -2). Protection by WR 1065, but not by 2-mercaptoethanol or GSH, was found to decrease significantly with increasing ionic strength. Protection by WR 1065 and GSH was not markedly dependent upon pH between pH 6 and 8. The results are explained in terms of electrostatic interaction of the thiols with DNA, leading to high concentrations of cations near DNA, which allow them to scavenge hydroxyl radicals and repair DNA radicals effectively and to low concentrations of anionic thiols near DNA, which limit their effectiveness as protectors. Poly(dG,dC) and calf thymus DNA exhibited comparable release of G and C upon changing from 0.1 to 0.7 mol dm-3 MgSO4. Since this change causes poly(dG,dC), but not calf thymus DNA, to undergo a change from the B-form to the Z-form of DNA, both forms must have a comparable susceptibility to radiation-induced base release.

Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics

Mycothiol, MSH or 1d‐myo‐inosityl 2‐(N‐acetyl‐l‐cysteinyl)amido‐2‐deoxy‐α‐d‐glucopyranoside, is an unusual conjugate of N‐acetylcysteine (AcCys) with 1d‐myo‐inosityl 2‐acetamido‐2‐deoxy‐α‐d‐glucopyranoside (GlcN‐Ins), and is the major low‐molecular‐mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced ≈ 20% of wild‐type levels of MSH. Levels of the Rv1170 substrate, GlcNAc‐Ins, were elevated, whereas those of the product, GlcN‐Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc‐Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first‐line antituberculosis drugs.

Mycothiol Is Essential for Growth of Mycobacterium tuberculosis Erdman

Mycothiol (MSH) is the major low-molecular-mass thiol in mycobacteria and is associated with the protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. The biosynthesis of MSH is a multistep process, with the enzymatic reaction designated MshC being the ligase step in MSH production. A targeted disruption of the native mshC gene in M. tuberculosis Erdman produced no viable clones possessing either a disrupted mshC gene or reduced levels of MSH. However, when a second copy of the mshC gene was incorporated into the chromosome prior to the targeted disruption, multiple clones having the native gene disrupted and the second copy of mshC intact were obtained. These clones produced normal levels of MSH. These results demonstrate that the mshC gene and, more generally, the production of MSH are essential for the growth of M. tuberculosis Erdman under laboratory conditions.