Platinum(II) binding to metallothioneins

The reaction of equine renal metallothionein (MT) with excess K2PtCl4 at pH 2 results in a polymeric adduct containing 17 +/- 2 mol Pt/mol MT. A monomeric adduct containing 7 mol Pt/mol MT is obtained at neutral pH. Rates of reaction of Pt7MT with DTNB and iodoacetic acid are consistent with Pt2+ to cysteine thiolate coordination, and the extent of reaction in both cases is 11 +/- 2 mol cys/mol MT. Adducts from the reaction of K2PtCl4 with apoMT chemically modified at the N-terminal methionine residue, Cd7MT, and native MT are also reported. A structural model of Pt7MT is proposed in which the square planar tetrathiolate Pt(II) unit is incorporated into a three-metal beta cluster. Implications for the metabolism of platinum anticancer drugs are discussed.     

Synthesis of the fully protected phosphoramidite of the benzene-DNA adduct, N2-(4-Hydroxyphenyl)-2'-deoxyguanosine and incorporation of the later into DNA oligomers

N(2)- (4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N(2)-(4-hydroxyphenyl)-2'-dG (N(2)-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N(2)-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N(2)-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.     

Preparation and partial fractionation of nonhistone chromosomal proteins from human diploid fibroblasts

2,6-Dichlorophenolindophenol reacts with the sulfhydryl group at the active site of apo-glyceraldehyde-3-phosphate dehydrogenase isolated from chicken muscle to form a leuco dye-enzyme adduct which is yellow. The leuco dye-enzyme adduct is oxidized by 2,6-dichlorophenolindophenol to form an oxidized dye-enzyme adduct which is blue. NADH converts the oxidized enzyme-dye adduct to the leuco enzymedye adduct. The enzyme-dye adducts catalyze the oxidation of NADH by 2,6-dichlorophenolindophenol in a reaction which exhibits “ping-pong” kinetics. The pH rate behavior of the reaction catalyzed by the enzyme-dye adduct differs considerably from the non-enzymatic oxidation of NADH by 2,6-dichlorophenolindophenol. A scheme for the reaction catalyzed by the enzyme-dye adduct which is consistent with the experimental observations is presented.     

Synthesis of the Fully Protected Phosphoramidite of the Benzene-DNA Adduct,N 2-(4-Hydroxyphenyl)-2′-Deoxyguanosine and Incorporation of the Later into DNA Oligomers

N²- (4-Hydroxyphenyl)-2 ′-deoxyguanosine-5 ′-O-DMT-3 ′-phosphoramidite has been synthesized and used to incorporate the N²-(4-hydroxyphenyl)-2 ′-dG (N²-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N²-arylation (Buchwald-Hartwig reaction) of a fully protected 2 ′-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5 ′-O-DMT-3 ′-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N²-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.     

The effect of glyceraldehyde on red cells: Haemoglobin status,oxidative metabolism and glycolysis

Glyceraldehyde induces changes in the flux of glucose oxidised through the hexose monophosphate pathway, the concentrations of intermediates in the Embden-Meyerhoff pathway, the oxidative status of haemoglobin and levels of reduced and oxidised pyridine nucleotides and glutathione in red cells. Glyceraldehyde autoxidises in the cellular incubations, consuming oxygen and producing glyoxalase I- and II-reactive materials. Major fates of glyceraldehyde in red cells appear to be: (i) adduct formation with reduced glutathione and cellular protein; (ii) autoxidation and reaction with oxyhaemoglobin and pyridine nucleotides, and (iii) phosphorylation of d-glyceraldehyde and entry into the glycolytic pathway as glyceraldehyde 3-phosphate. The production of glycerol from glyceraldehyde by red cell l-hexonate dehydrogenase appears not to be a major reaction of glyceraldehyde in red cells. These results indicate that high concentrations of glyceraldehyde (1–50 mM) may induce oxidative stress in red cells by virtue of the spontaneous autoxidation of glyceraldehyde, forming hydrogen peroxide and α-ketoaldehydes (glyoxalase substrates). The implications of glyceraldehyde-induced oxidative stress for the in vitro anti-sickling effect of dl-glyceraldehyde and for the polyol pathway metabolism of glyceraldehyde are discussed.     

The development of SS''-polymethylenebis(methanethiosulphonates) as reversible cross-linking reagents for thiol groups and their use to form stable catalytically active cross-linked dimers within glyceraldehyde 3-phosphate dehydrogenase.

The synthesis of a series of SS'-polymethylenebis(methanethiosulphonates) including the pentane, hexane, octane, decane and dodecane derivatives is described. These derivatives were synthesized by condensation between dibromoalkanes and potassium methanethiosulphonate in refluxing methanol and this seems an especially versatile reaction for the synthesis of asymmetric thiosulphonate derivatives. The synthesis of SS'-[1,8-3H4]-octamethylenebis(methanethiosulphonate) was also perfomed. Cross-linking was demonstrated in the four enzymes lactate dehydrogenase, phosphofructokinase, pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase. For all four enzymes cross-linking was efficiently reversed by reducing conditions in denaturing solvents. The reaction with glyceraldehyde 3-phosphate dehydrogenase was unique in that only the cross-linked dimer was produced in significant amounts (greater than 90% of total products as dimer). This reaction was followed in detail with radioactive cross-linking reagent. Inhibition of enzyme activity was extremely fast and showed an asymmetric distribution of enzyme activity on subunits. Thus complete modification of only one subunit resulted in up to 75% inhibition of enzyme activity. Reaction of glyceraldehyde 3-phosphate dehydrogenase with 1.25 mol of SS'-octamethylenebis(methanethiosulphonate) per mol of enzyme subunit produced two species of protein. The first species was obtained in 20% yield and was only partially re-activated on mild reduction with 2-mercaptoethanol. The second species was isolated in 66% yield and was completely re-activated on mild reduction. Before reduction there was 4 mol of inhibitor per tetramer for the latter species, and more than 95% of the enzyme was present as a dimer on non-reducing electrophoresis. After mild reduction 2 mol of inhibitor was still bound per tetramer, the enzyme was now catalytically active and the dimer was still the major structure on non-reducing electrophoresis. Thus mild reduction of SS'-octamethylenebis(methanethiosulphonate-treated glyceraldehyde 3-phosphate dehydrogenase enabled the production of active enzyme in which there is a stable cross-link across one of the molecular axes of the tetrameric enzyme. This cross-link was only reversed if reduction was performed when the enzyme was denatured. The molecular weight of cross-linked and re-activated cross-linked glyceraldehyde 3-phosphate dehydrogenase was established as 144000 (tetramer) by sucrose-density-gradient centrifugation. These observations are interpreted in terms of the molecular structure of glyceraldehyde 3-phosphate dehydrogenase.     

Multiple pathways of the reaction of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) with (+)-catechin: evidence for the formation of a covalent adduct between DPPH· and the oxidized form of the polyphenol

The pathways of the reaction of 2,2-diphenyl picrylhydrazyl radicals (DPPH·) with (+)-catechin were studied in alcoholic solvents. The reaction mixtures were analysed by using reversed-phase liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The intermediate o-quinone of catechin, yellow dimers, trimers and, interestingly, an adduct of the oxidized form of catechin with DPPH radicals were identified. The mass of this adduct was 681 Da, suggesting that one molecule of the DPPH radical complexes with the oxidized form of catechin. It is concluded that once the intermediate o-quinone is formed, the reaction proceeds in two pathways, either the o-quinone reacts with catechin to form a hydrophilic dimer (type B), which is further oxidized to hydrophobic dimers (type A) and consequently to oligomers of higher molecular weights; or the A-ring of the o-quinone is further oxidized by a DPPH radical and that this oxidized intermediate then reacts with another DPPH radical to form the observed adduct. The identification of the latter mechanism could explain the contradictory results reported in the literature for the reaction of polyphenols with DPPH radicals.     

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation.

We previously demonstrated by X-ray crystallography and electrospray mass spectrometry that D52E mutant hen lysozyme formed a covalent enzyme-substrate adduct on reaction with N-acetylglucosamine oligomer. This observation indicates that D52E lysozyme may acquire a catalytic pathway via a covalent adduct. To explain this pathway, the formation and hydrolysis reactions of the covalent adduct were investigated. Kinetic analysis indicated that the hydrolysis step was the rate-limiting step, 60-fold slower than the formation reaction. In the formation reaction, the pH dependence was bell-shaped, which was plausibly explained by the functions of the two catalytic pKas of Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis was sigmoidal with a transition at pH 4. 5, which was identical with the experimentally determined pKa of Glu35 in the covalent adduct, indicating that Glu35 functions as a general base to hydrolyze the adduct. To improve the turnover rate of D52E lysozyme, the mutation of N46D was designed and introduced to D52E lysozyme. This mutation reduced the activation energy in the hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40 degrees C but did not affect the formation reaction. Our data may provide a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.     

Esr Detection of Free Radical Intermediates During Autoxidation Of 5-Hydroxyprimaquine

Autoxidation of 5–hydroxyprimaquine, a putative metabolite of the antimalarial primaquine, was studied by oxygen consumption and ESR spectroscopy. 5–Hydroxyprirnaquine undenvent fast autoxidation under mild conditions (pH 7.4-8. 5, 25°C. and presence of I mM diethylenetriamine pentaacetic acid); each mol of the drug consumed 0.75 mol of oxygen and formed 0.5 mol of hydrogen peroxide. Direct-ESR experiments demonstrated that 5–hydroxyprimaquine autoxidation was accompanied by generation of a drug-derived free radical that is oxygen sensitive. Generation of hydroxyl radical was also established by spin-trapping experiments in the presence of 5,5–dimethyl-l-pyrroline N-oxide. The effect of antioxidant enzymes on hydroxyl radical adduct yield and analysis of autoxidation stoichiometry suggest that the main route for hydroxyl radical generation is the iron-catalyzed reaction between the drug-derived free radical and hydrogen peroxide.     

A new spectrofluorimetric method for determination of trace amounts histamine in human urine and serum

A new spectrofluorimetric method was developed for the determination of trace amounts of histamine in human urine and serum samples. In NaAc–HAc buffer solution of pH 4.0, histamine can react with the acetylacetone–formaldehyde system to produce a fluorescent derivative which emits yellow‐green fluorescence at 476 nm, according to the Hantzsch reaction, and the enhanced fluorescence intensity is in proportion to the concentration of histamine. Optimum conditions for the determination of histamine were also investigated. The dynamic range and detection limit for the determination of histamine is 5.96 × 10^–8–1.50 × 10^–5 mol/L and 4.35 × 10^–8mol/L, respectively. This method is practical and can be successfully applied to determination of histamine in human urine and serum samples. A proposal of the reaction pathway is suggested. Copyright © 2009 John Wiley & Sons, Ltd.     

Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus

We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.     

The effect of glyceraldehyde on red cells. Haemoglobin status, oxidative metabolism and glycolysis

Glyceraldehyde induces changes in the flux of glucose oxidised through the hexose monophosphate pathway, the concentrations of intermediates in the Embden-Meyerhoff pathway, the oxidative status of haemoglobin and levels of reduced and oxidised pyridine nucleotides and glutathione in red cells. Glyceraldehyde autoxidises in the cellular incubations, consuming oxygen and producing glyoxalase I- and II-reactive materials. Major fates of glyceraldehyde in red cells appear to be: (i) adduct formation with reduced glutathione and cellular protein; (ii) autoxidation and reaction with oxyhaemoglobin and pyridine nucleotides, and (iii) phosphorylation of D-glyceraldehyde and entry into the glycolytic pathway as glyceraldehyde 3-phosphate. The production of glycerol from glyceraldehyde by red cell L-hexonate dehydrogenase appears not to be a major reaction of glyceraldehyde in red cells. These results indicate that high concentrations of glyceraldehyde (1-50 mM) may induce oxidative stress in red cells by virtue of the spontaneous autoxidation of glyceraldehyde, forming hydrogen peroxide and alpha-ketoaldehydes (glyoxalase substrates). The implications of glyceraldehyde-induced oxidative stress for the in vitro anti-sickling effect of DL-glyceraldehyde and for the polyol pathway metabolism of glyceraldehyde are discussed.     

Selective modification of cytosines in oligodeoxyribonucleotides.

A single deoxycytidine residing in an oligodeoxyribonucleotide which also contains 5-methyldeoxycytidines can be selectively derivatized with various alkylamines by sodium bisulfite-catalyzed transamination. Selective transamination results because 5-methylcytosine, unlike cytosine, does not form a bisulfite adduct. When the reaction is carried out at pH 7.1, transamination in the oligomer appears to occur to greater than 95% with little or no deamination. This procedure has been used to introduce aminoalkyl or carboxyalkyl side chains at the N4-position of a deoxycytidine in oligonucleotides. These side chains contain potentially reactive amine or carboxy groups which could serve as a sites for further conjugation of the oligomer with a variety functional groups. Oligonucleotides which carry these side chain form duplexes and triplexes with appropriate complementary single-stranded or double-stranded oligodeoxyribonucleotide target molecules. The stabilities of the duplexes are similar to those formed by unmodified oligomers, whereas the stability of the triplexes is approximately 18 degrees C lower than that formed by unmodified oligomers.     

Catalysis of dehydrogenation of 4-trans-(N,N-dimethylamino)cinnamaldehyde by aldehyde dehydrogenase

4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) is a chromophoric and fluorogenic substrate of aldehyde dehydrogenase. Fluorescence of DACA is enhanced by binding to aldehyde dehydrogenase in the absence of catalysis both in the presence and absence of the coenzyme analogue 5′AMP. DACA binds to aldehyde dehydrogenase with a dissociation constant of 1–3 μM and stoichiometry of 2 mol mol⁻¹ enzyme. Incorporation of DACA during catalysis was also investigated and found to be 2 mol DACA mol⁻¹ enzyme. Effect of pH on the stoichiometry of DACA incorporation during catalysis has shown that DACA incorporation remained constant at 2 mol DACA mol⁻¹ enzyme, despite a 74-fold velocity enhancement between pH 5.0 and 9.0. Increase of pH increased decomposition of enzyme–acyl intermediate without affecting the rate-limiting step of the reaction. At pH 7.0 the pH stimulated velocity enhancement was 10-fold over that at pH 5.0; further velocity enhancement (11.5-fold that of pH 7.0) was achieved by 150 μM Mg²⁺ ions. The velocity at pH 7.0 with Mg²⁺ exceeded that of pH 9.0, and that at maximal pH stimulation at pH 9.5. It was observed that level of intermediate decreased to about 1 mol mol⁻¹ enzyme, indicating that Mg²⁺ ions increased the rate of decomposition of the enzyme–acyl intermediate and shifted the rate-limiting step of the reaction to another step in the reaction sequence.     

Effect of bipolar membrane electrobasification on chitosanase activity during chitosan hydrolysis

Chitosanase is an enzyme that hydrolyzes chitosan, a beta-(1-4) glucosamine polymer, into size-specific oligomers that have pharmaceutical and biological properties. The aim of the present work was to use the bipolar membrane technology, in particular the OH(-) stream produced by water splitting, for inactivation of chitosanase at alkaline pH in order to terminate the enzymatic reaction producing chitosan oligomers. The objectives consisted of studying the effect of pH: (a) on the stability of chitosanase, and (b) on the catalytic activity of chitosanase during chitosan hydrolysis. The enzyme was found to be stable in the pH range of 3-8 during at least 7h, and partially lost its activity after 1h at pH 8. The catalytic activity of chitosanase during chitosan hydrolysis decreased after pH adjustment by electrobasification. The reaction rate decreased by 50% from pH 5.5 to 6, whereas the reaction was completely inhibited at pH>7. The decrease of reaction rate was due to chitosan substrate insolubilization and chitosanase denaturation at alkaline pH values.     

Active site rearrangement of the 2-hydrazinopyridine adduct in Escherichia coli amine oxidase to an azo copper(II) chelate form: a key role for tyrosine 369 in controlling the mobility of the TPQ-2HP adduct

Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

Reactions of d-glyceraldehyde 3-phosphate dehydrogenase facilitated by oxidized nicotinamide–adenine dinucleotide

Transient kinetic methods have been used to study the influence of NAD(+) on the rate of elementary processes of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. In the pH range 5-8 NAD(+) is bound to the enzyme during the following elementary processes of the mechanism: phosphorolysis of the acyl-enzyme, its formation from 1,3-diphosphoglycerate and the enzyme and the formation and breakdown of the glyceraldehyde 3-phosphate-enzyme complex. The rates of these four elementary processes only equal or exceed the turnover rate of the enzyme when NAD(+) is bound and are as much as 10(4) times the rates in the absence of NAD(+). Autocatalysis of the reductive dephosphorylation of 1,3-diphosphoglycerate occurs when glyceraldehyde 3-phosphate release is rate determining because NAD(+) is a reaction product. An important feature of the enzyme mechanism is that the negative-free-energy change of a chemical reaction, acyl-enzyme formation, is linked in a simple way to the positive-free-energy change of a dissociation reaction, NAD(+) release.     

A ratiometric fluorescent probe for bisulphite anion,employing intramolecular charge transfer

4‐(1H‐benzimidazol‐2‐yl)benzaldehyde (1) has been developed as a new ratiometric fluorescent probe for bisulphite, based on the modulation of intramolecular charge transfer (ICT). Upon mixing with bisulphite in aqueous ethanol, an aldehyde–bisulphite adduct was formed and the ICT of the probe was switched off, which resulted in a ratiometric fluorescence response with an enhancement of the ratios of emission intensities at 368 and 498 nm. The detection range of the probe for bisulphite is in the 2.0–200 µmol/L concentration range and the detection limit is 0.4 µmol/L. Probe 1 produces a ratiometric fluorescent response to bisulphite with a marked emission wavelength shift (130 nm) and displays high selectivity for bisulphite over other anions. Copyright © 2012 John Wiley & Sons, Ltd.