Sulfite bound

It is important to note, however, that sulfonic acids formed by the addition of bisulfite to a double bond do not decompose on heating with acid, i.e., the Monier-Williams procedure will not assess sulfite bound in this form. 

Experiments with glucose solutions have shown that the amount of sulfite bound increases when the solutions are heated strongly beforehand. It seems as if the carbonyl compounds derived from glucose are responsible for this, having a greater affinity for sulfite than glucose, and competing successfully with it when only limited amounts of sulfite are available. 

At the anode, a chemical oxidation reaction is bound to take place. In normal fixers, sulfite (SOj ) is oxidized and acid (H ) is released as a consequence of this oxidation. Due to the decrease of the sulfite concentration and the decrease in the pH, the fixing solution becomes unstable and sulfur precipitation starts to occur when the pH of the fixer decreases below 4.0. In the case of hardening fixers, there is also an upper limit to the pH, since aluminum-hydroxides starts to precipitate when the pH exceeds 5.0. 

The handling of sulfate by protoaerobes depends upon the initial energised coupling to adenosine phosphate as APS, since sulfate is difficult to reduce. The reductase is a flavoprotein linked to Fe S electron transfer centres. Subsequently, released sulfite is reduced by a haem protein (SIR) in which haem is directly bound... 

Sulfite reductase catalyzes the six-electron reduction by NADPH of sol" to and NO2 to NH3. In E. coli this enzyme is a complex structure with subunit composition 0 8)84 (Siegel et al, 1982). The enzyme active site is on the /3 subunit, which contains both a 4Fe 4S cluster and a siroheme prophyrin. Substrates and ligands have been found to bind to the siroheme. The a subunit binds NADPH and serves to shuttle electrons to the active site through bound FAD and FMN groups. Isolated )8 subunits can catalyze sulfite reduction in the presence of a suitable electron donor. 

A considerable amount of information regarding flavin semiquinone reactivity as well as the environment of the bound flavin coenzyme has accumulated over the years from studies of flavoenzyme systems which produce semiquinones either on photochemical reduction or upon reduction by one electron equivalent of dithionite, but which do not form a detectable semiquinone intermediate during catalytic turnover. For example, the correlation of anionic semiquinone formation and the ability to bind sulfite at the N(5) position in a number of flavoenzyme... 

Spinach nitrite reductase,313 which is considered further in Chapter 24, utilizes reduced ferredoxin to carry out a six-electron reduction of N02 to NH3 or of SO-2 to S2. The 61-kDa monomeric enzyme contains one siroheme and one Fe4S4 cluster. A sulfite reductase from E. coli utilizes NADPH as the reductant. It is a large (38a4 oligomer.312 The 66-kDa a chains contain bound flavin... 

The enzymes from green plants and fungi are large multifunctional proteins,80 which may resemble assimilatory sulfite reductases (Fig. 16-19). These contain siroheme (Fig. 16-6), which accepts electrons from either reduced ferredoxin (in photosynthetic organisms) or from NADH or NADPH. FAD acts as an intermediate carrier. It seems likely that the nitrite N binds to Fe of the siroheme and remains there during the entire six-electron reduction to NH3. Nitroxyl (NOH) and hydroxylamine (NH2OH) may be bound intermediates as is suggested in steps a-c of Eq. 24-14. 

The reaction of sulfite with formaldehyde to form hydroxymethylsulfonate (HMS), which is very stable under the controlled conditions of this assay, was used as the first step in an analytical procedure to determine food-borne sulfite. The effect of mobile-phase pH on the stability of HMS during high-performance liquid chromatography was studied. It was found that on-column HMS dissociation to formaldehyde and bisulfite increased with the pH of the mobile phase therefore the relatively low pH 4.7, at which the dissociation of HMS was approximately 2%, was selected for the analysis. In addition, the release of sulfite from its reversibly bound forms in wine and other foods was examined as a function of the pH of the extraction medium by following the appearance of HMS formed from the reaction of the freed sulfite with formaldehyde. The rate of dissociation of the reversibly bound sulfite was relatively slow at pH 3 but very rapid at pH 7. 

This difference in kinetics was exploited to develop a procedure to determine free and reversibly bound sulfite in food. The mobile phase consisted of an aqueous solution of 0.05 M tetra-butylammonium hydroxide adjusted to the desired pH by the addition of glacial acetic acid (34). Fluorimetric detection is also possible, because a reaction of the formaldehyde-bisulfite complex with 5-aminofluorescein gives a nonfluorescent product. The sulfite is measured indirectly by its suppresion of the fluorescence of the reagent (31). This method is applicable to the determination of S02 at > 10 ppm and is not applicable to dark-colored foods or ingredients where SO, is strongly bound, e.g., caramel color. This method does not detect naturally occurring sulfite. Sulfur dioxide is released by direct alkali extraction. 

In one study, a modified Monier-Williams method has been utilized as a preparative procedure to obtain both the free and bound sulfite fractions. The two fractions were analyzed by HPLC with indirect photometric detection using a 250 X 4.6-mm LC-SAX column eluted with potassium hydrogen phtalate (0.15 g/L, pH 5.7) and detected at 280 nm. Levels of 5-10 ppm of S02 in foods, corresponding to 30 - 60 ng injected, were reliably detected by this method. The results confirmed that the chromatographic method, unlike the Monier-Williams method, is able to avoid the potential interference of volatile substances derived from matrices or utilized chemicals (36). The HPLC conditions are summarized in Table 3. 

The apocofactor is synthesized in the absence of molybdenum in E. coti and N. crassa. The cofactor from E. coli is soluble, but is isolated bound to a carrier protein from which it has to be separated.99 Tungstate competes with molybdate for the molybdenum site in the cofactor, leading either to the formation of a demolybdo species or to an inactive form containing tungsten. The tungsten protein has been characterized in the case of sulfite oxidase,1000 which, while inactive, is immunologically identical to the molybdenum cofactor. 

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