Cross-talk Between (Hydrogen)Sulfite and Metalloproteins: Impact on Human Health

Sulfite is a potent toxic substance causing harm to multi-organ in human. Despite toxicity, it is widely used as preservative, anti-browning and anti-oxidant in foods, beverages, and pharmaceuticals, which cause easy admission of sulfite in human. Sulfite is also produced endogenously during the catabolism of cysteine and methionine. In vivo, the serum sulfite level at physiological range is strictly maintained by a molybdenum dependent sulfite oxidase (SO), which catalyzes sulfite to sulfate oxidation via a two-electron oxidation pathway. The loss of SO activity causes high serum sulfite level that fosters several diseases, including asthma, neurological dysfunction, birth defects, and heart diseases. The cytotoxicity of (bi)sulfite is implicated as sulfite radicals, which are generated by mainly heme-peroxidases via a one-electron oxidation pathway. On the other hand, the toxic sulfite radicals are neutralized to sulfite by heme-globins. The enzymatic reduction of sulfite to sulfide is catalyzed by sulfite reductase, which contains an unusual metal cofactor, siroheme-[4Fe4S]-cluster. Overall, the interaction of sulfite with various metalloproteins in vivo is a close relation with human health. Therefore, this review describes the metabolic conversion of (bi)sulfite to sulfate, sulfite radical or sulfide via oxidation or reduction pathways by various metalloproteins (specially SOs, peroxidases, heme-globins, and sulfite reductases), and the potential applications of sulfite in biosensors/biofuel cells, anti-browning, and advance oxidation process.     

Determination of total sulfite in wine. Zone electrophoresis-isotachophoresis quantitation of sulfate on a chip after an in-sample oxidation of total sulfite

This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP-ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP-ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 microg/l concentration of sulfite in the loaded sample (0.9 microl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99-101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH).     

Structural analysis of missense mutations causing isolated sulfite oxidase deficiency

The physiologically essential oxidation of sulfite to sulfate is catalyzed by the molybdoheme enzyme, sulfite oxidase. Deficiencies of this enzyme in human patients lead to severe neurological symptoms, which usually result in death in early childhood. Up to date eleven missense mutations in the gene encoding sulfite oxidase have been identified from sulfite oxidase deficient patients. The structural characterization of these mutants is now possible after the chicken sulfite oxidase gene has been synthesized chemically and due to the high homology to the human enzyme it provides a good model of human sulfite oxidase. This review focuses on the possible effects of the sulfite oxidase deficiency causing mutations based on our new structures of recombinant chicken sulfite oxidase.     

Picomolar quantitation of free sulfite in foods by means of [57Co]hydroxocobalamin and radiometric chromatography of [57Co]sulfitocobalamin. Method, applications and significance of coexisting sulfides

The concentration dependent reaction of sulfite with 57Co-labeled hydroxocobalamin (OH57CoCbl) to produce a sulfitocobalamin (SO(3)57CoCbl) adduct served as a quantification strategy for foodborne sulfite residues freely extracted into pH 5.2, 0.05 M acetate buffer. SO(3)57CoCbl was then resolved using SP-Sephadex C-25 gel chromatography and its radiometric detection allowed calculation of a standard logit plot from which unknown sulfite concentrations could be determined. The sulfite detection range was 6.0 nM-0.3 pM with respective relative standard deviations of 4.4-29.4% for 50-microl samples. Individual incidences of foodborne sulfite intolerances provoked by L-cysteine or sulfite additive use in bakery products, which remained undetected using conventional sulfite analytical methods, underscored the quantitative value of the method. The analytical significance and occurrences of detectable sulfides coexisting with foodborne sulfite residues was also addressed.     

PHOTO- AND THERMAL-ACTIVATION OF BOVINE LIVER UROCANASE

Abstract— In the photoactivation of sulfite-modified urocanase from Pseudomonas putida, the sulfite adds to the nicotinamide adenine dinucleotide and photodissociation accompanies activation. Bovine urocanase contains the same coenzyme. The purpose was to find if sulfite can inactivate a mammalian urocanase and if UV light can reactivate the enzyme. It was inactivated by sulfite,10–100 i.M, and dialysis did not restore activity. Near-UV light (11 W m-²) reactivated the enzyme in 45 min. A competitive inhibitor protected urocanase from sulfite, showing that sulfite acts at the active site. The modification was dependent on temperature, time, and concentration of sulfite. The modification was reversed by incubation at 25°C for 24 h. These results resemble those found with the P. putida urocanase. It is likely that these thermal- and photo-reactions are the same for the bacterial and mammalian urocanases.     

Conditions for sulfite stabilization and determination by ion chromatography

The efficiencies of three groups of potential sulfite-stabilizing compounds were found to be in the order: carbonyls > alcohols = saccharides. A mole ratio of 1:1 between formaldehyde and sulfite was sufficient for stabilizing a sulfite solution for at least 72 h. The lower stabilizing efficiencies of the alcohols and saccharides examined could be compensated by using large excesses of these compounds. For example, if a 100-fold excess of glycerol over sulfite was used, the recovery of sulfite was 96% after 72 h compared with only 40% without addition of stabilizer. During separations by ion chromatography, almost no oxidation of the sample occurs provided the sample solution is directly injected into a deaerated eluent. For formaldehyde, the peak heights were found to depend on the molar ratio of the stabilizer to sulfite as well as on the concentration of sulfite. This effect was not found for the other stabilizers tested.     

Determination of sulfite in dried garlic by reversed-phase ion-pairing liquid chromatography with post-column detection

A method is described for determining sulfite in dried garlic. Garlic is extracted with an HCl solution to inhibit the formation of allicin, which interferes with the determination of sulfite. After cleanup of the extract on a C18 solid-phase extraction column, sulfite is converted to hydroxymethylsulfonate (HMS) by adding formaldehyde and heating to 50 degrees C. HMS is determined by reversed-phase ion-pairing liquid chromatography with post-column detection. The post-column reaction system consists of the addition of KOH to convert HMS to sulfite ion, followed by the addition of 5,5'-dithiobis(2-nitrobenzoic acid) to produce 5-mercapto-2-nitrobenzoic acid which is detected spectrophotometrically at 450 nm. Background levels in unsulfited dried garlic equivalent to < 20 ppm SO2 were found. Recoveries of HMS from spiked garlic averaged 94.8% with a coefficient of variation of 3.8%. Sulfite was found in 13 of 21 samples of dried garlic produced in China, with sulfite ranging from 114 to 445 ppm. Sulfite was found in 60% of commercial dried garlic products purchased locally. The suitability of the Monier-Williams method for determining sulfite in garlic is discussed.     

A fiber optic biosensor for sulfite analysis in food

The sulfite fiber optic biosensor developed herein is based on the enzymatic oxidation reaction of sulfite catalyzed by sulfite oxidase (SOD). The consumption of O(2) is measured with an O(2) fiber optic sensor which monitors the fluorescence quenching of the indicator, perylene, by molecular oxygen. Perylene is immobilized into a polymer matrix and attached to the end of a fiber bundle forming an O(2) sensor. The enzyme, sulfite oxidase is immobilized on a pre-activated membrane and mounted onto the O(2) sensor. Several analytical characteristics of this sulfite biosensor were investigated including dynamic range, reversibility, reproducibility, stability and selectivity. The sulfite contents of various food samples, e.g. dried fruits, potato flakes, lemon juice were determined and the results obtained were in good agreement with the standard AOAC method.     

用电化学方法研究显影过程——pH对菲尼酮-对苯二酚体系中亚硫酸盐添加效应的影响

应用旋转环盘电极(RRDE)等电化学方法研究了在不同pH条件下,在银/溴化银电极上,亚硫酸盐(SO₃^2-)对菲尼酮-对苯二酚超加合体系(P-H₂Q体系)显影反应速率的影响.结果表明,显影液中的亚硫酸盐不仅起保护显影剂的作用,而且影响显影反应速率.在较低pH下亚硫酸盐起抑制显影的作用,而在较高的pH下,会促进显影反应.这种现象可以认为主要是由于亚硫酸盐存在时,不同pH条件下,对苯二酚的氧化还原性质不同而引起的.     

过氧乙酸催化氧化亚硫酸钙的反应动力学

利用间歇式反应装置, 研究了过氧乙酸催化氧化亚硫酸钠的本征反应动力学, 得到各反应物的分级数及表观活化能. 在此基础上, 利用鼓泡式反应装置, 通过改变pH、亚硫酸钙浓度、过氧乙酸浓度、空气流量及温度, 研究了过氧乙酸催化氧化亚硫酸钙的宏观反应动力学.     

Determination of sulfite in beer samples using an amperometric fill and flow channel biosensor employing sulfite oxidase

A simple method is described to determine sulfite in beer samples using a fill and flow channel biosensor. A droplet of sample is placed into the inlet of a rectangular flow cell and begins to flow through the channel by capillarity. The flow is maintained and controlled by a porous outlet plug of defined porosity. In a rectangular flow cell, the sample solution flows through three consecutive zones: over a predictor electrode, an enzyme layer and a detector electrode. Together these three zones enable the differentiation between current due to sulfite and current due to other electroactive species in the sample. The predictor electrode is located upstream, and on the opposite channel wall to the enzyme layer and detector electrode, and is poised at the same potential (+0.65 V versus Ag/AgCl) as the detector electrode. On this electrode, the current contribution from all species in the sample solution that are oxidized at that potential is determined. The enzyme layer contains sulfite oxidase, which, in the process of oxidizing sulfite, produces hydrogen peroxide, which itself is reduced by excess sulfite. The current at the downstream detector electrode is therefore different from that at the predictor electrode as a result of the enzyme reaction and the difference of the currents, corrected for the dimensions of the electrodes, is proportional to the concentration of sulfite. The method enables a straightforward correction of the interfering current at the detector electrode and a determination of the analyte concentration. The effect of interferences from ascorbic acid, ethanol, sorbic acid and tartaric acid in the detection of sulfite is efficiently removed. The concentration of sulfite in a sample of beer measured by the biosensor is equivalent to that measured using a reference method based on the AOAC-recommended Monier-Williams method.     

Galvanostatic Entrapment of Sulfite Oxidase into Ultrathin Polypyrrole Films for Improved Amperometric Biosensing of Sulfite

The entrapment of sulfite oxidase (SOx) into ultrathin polypyrrole (PPy) films of 27–135 nm thickness has been successfully used for amperometric biosensing of sulfite with considerably improved performance. Optimum galvanostatic entrapment was accomplished in an electrolyte‐free solution which contained 0.1 M pyrrole and 5 U/mL of SOx with a polymerization period of 120 seconds and an applied current density of 0.2 mA cm^?2. Evidence of the incorporation and retention of SOx in the ultrathin PPy film was obtained by scanning electron microscopy, cyclic voltammetry and amperometric measurements. Entrapment of the enzyme in a 54 nm thick PPy‐SOx film gave optimum amperometric response for sulfite and enabled the detection of as little as 0.9 μM of sulfite with a linear concentration range of 0.9 to 400 μM. The successful application of the biosensor to the determination of sulfite in beer and wine samples is reported. Comparison with a spectrophotometric method indicates that the biosensor was more superior for the determination of sulfite in red wine.     

High performance liquid chromatographic determination of bound sulfide and sulfite and thiosulfate at their low levels in human serum by pre-column fluorescence derivatization with monobromobimane.

A sensitive and specific high performance liquid chromatographic method for the determination of sulfide, sulfite, and thiosulfate was established. Inorganic sulfur anions were converted into fluorescent derivatives with monobromobimane. The derivatives were separated on a coupled column chromatography with a reversed-phase octadecyl silica column connected with a weakly basic anion exchanger column by isocratic elution with acetic acid solution (pH 3)-acetonitrile (13:3, v/v) containing 25 mM NaClO4. The method was applied to the determination of bound sulfide and sulfite and thiosulfate in normal human serum. Thiosulfate could be determined directly by use of an ultrafiltered sample. For the determination of bound sulfide and sulfite, the pretreatment step with continuous flow gas dialysis was effective for the sample after releasing sulfide and sulfite by reduction with dithiothreitol. The limits of quantification by the present method were 0.05 microM for thiosulfate, 0.5 microM for bound sulfide, and 0.2 microM for bound sulfite.     

Rapid fermentation of beer using an immobilized yeast multistage bioreactor

The characteristics of yeast sulfite metabolism in a multistage bioreactor system for beer fermentation were investigated. No sulfite was produced in the continuous stirred-tank reactor (CSTR). However, large amounts were produced in the packed-bed reactor (PBR). Production of sulfite in the PBR seems to be inevitable when it is operated continuously. In order to control the sulfite level in the young beer, the yeast needs to be reactivated into the growth phase. One possible strategy to achieve this is to aerate and periodically remove yeast clogged in the reactor once every 6–7 months before the sulfite level exceeds a given concentration (e.g., 20 mg/L). It was confirmed that sulfite production is closely related to the growth condition of the yeast and is therefore important to consider in the control strategy for sulfite when using the immobilized yeast reactor for beer production.     

Esr spin trapping evidence for SO.- 3 and .OH radicals in sulfite oxidation

Electron spin resonanee (ESR) spin trapping experimenss have been carried out to investigate the mechanism of sulfite oxidation employing 5,5-dimethyl-1-pyrroine-1-oxide (DMPO) as a spin trap. The results show that sulfite autoxidation, catalyzed by Mn(II), involves not only the SO^.- ₃ radicals but also the .OH radicals. An addition of H₂O₂ to the sulfite aqueous medium significantly increases the .OH radical formation. This result provides new clues to the chemical mechanism of the sulfite oxidation and the sulfite toxicity.     

On the structure and dynamics of the hydrated sulfite ion in aqueous solution--an ab initio QMCF MD simulation and large angle X-ray scattering study

Theoretical ab initio quantum mechanical charge field molecular dynamics (QMCF MD) formalism has been applied in conjunction to experimental large angle X-ray scattering to study the structure and dynamics of the hydrated sulfite ion in aqueous solution. The results show that there is a considerable effect of the lone electron-pair on sulfur concerning structure and dynamics in comparison with the sulfate ion with higher oxidation number and symmetry of the hydration shell. The S-O bond distance in the hydrated sulfite ion has been determined to 1.53(1) ? by both methods. The hydrogen bonds between the three water molecules bound to each sulfite oxygen are only slightly stronger than those in bulk water. The sulfite ion can therefore be regarded as a weak structure maker. The water exchange rate is somewhat slower for the sulfite ion than for the sulfate ion, τ(0.5) = 3.2 and 2.6 ps, respectively. An even more striking observation in the angular radial distribution (ARD) functions is that the for sulfite ion the water exchange takes place in close vicinity of the lone electron-pair directed at its sides, while in principle no water exchange did take place of the water molecules hydrogen bound to sulfite oxygens during the simulation time. This is also confirmed when detailed pathway analysis is conducted. The simulation showed that the water molecules hydrogen bound to the sulfite oxygens can move inside the hydration shell to the area outside the lone electron-pair and there be exchanged. On the other hand, for the hydrated sulfate ion in aqueous solution one can clearly see from the ARD that the distribution of exchange events is symmetrical around the entire hydration sphere.     

多相条件下亚硫酸镁非催化氧化反应动力学及机理

副产物的氧化回收利用是影响镁法脱硫工艺发展的关键.本文通过真空旋转蒸发的方式制备了高纯度的亚硫酸镁样品,并利用鼓泡式反应装置,研究了亚硫酸镁非催化氧化的反应动力学,考察了pH、氧分压、亚硫酸镁浓度、气体流量、温度等因素对亚硫酸镁氧化反应速率的影响,得到了各反应物的分级数及表观活化能.结合三相反应过程的数学模型推断出亚硫酸镁氧化的本征反应在慢反应区进行,且氧的传质扩散是总反应的速率控制步骤.研究结果为氧化回收镁法脱硫副产物提供了理论依据.     

Siroheme‐containing sulfite reductase: A density functional investigation of the mechanism

Siroheme‐containing sulfite reductases (SiR) catalyze the six‐electron reduction of sulfite to sulfide via a mechanism involving sulfite binding at the heme iron. The exact sequence in which the required electrons and protons are delivered to the heme‐bound sulfite has received little attention to date. Here, a detailed account is given of these steps, based on density functional theory, thus providing data for the first attempt to draw a detailed picture of sulfite reduction in SiR by theoretical methods. Parallels are shown with reduction of other small molecules at heme centers: dioxygen (including generation of sulfide high‐valent iron centers akin to hemoproteins Compounds I and II), nitrite (including linkage isomerism akin to the nitro/nitrito known for nitrite reducing proteins), or nitric oxide. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Thermal behaviour and S-O bonding state of alkaline-earth metal sulfites

The thermal behaviours of magnesium sulfite, strontium sulfite and barium sulfite were investigated in the atmospheres of argon and air. the thermal behaviours of magnesium sulfite were different from those of the other two sulfites. The oxidation of magnesium sulfite in air does not occur.The bonding state of the SO ₃ ^2– in each sulfite was compared. The SO ₃ ^2– in magnesium sulfite was coordinated through the sulfur, while those in the other sulfites were coordinated through the oxygen.It appears that the difference in thermal behaviour between magnesium sulfite and the other sulfites depend upon the difference in bonding state of the SO ₃ ^2– We wish to thank Mr. K. Takahashi of MAC Science Inc. for TG and DTA measurements, mass spectra in Ar and his valuable discussions.     

亚硫酸根离子在硅和二氧化硅的湿法腐蚀中的作用

运用光刻技术和原子力显微技术(AFM)研究了亚硫酸根离子对硅和二氧化硅在40％(w)氟化铵水溶液中腐蚀速率的影响.结果表明硅和二氧化硅的腐蚀速率和亚硫酸根离子浓度有关.高分辨X射线光电子能谱(XPS)分析在有/没有亚硫酸根的溶液中腐蚀后的硅和二氧化硅表面氟元素的结果表明在这两种溶液中腐蚀得到的表面化学成分是有差别的.实验结果证明亚硫酸根离子在硅和二氧化硅的湿腐蚀中不只是表现为一种除氧剂，还干预了表面腐蚀反应过程.