No abstract

Methylene blue interacts with carrageenans and other anionic hydrocolloids to form water-soluble metachromatic complexes at low concentrations of the reactants. The complexation results in a color change of the dye from blue (absorption maxima at 610 and 664 nm) to purple (absorption maximum at 559 nm). The anionic sites of the hydrocolloids appear to be primarily responsible for the dye-binding. The interaction between methylene blue and polyanions is reversible, electrostatic, and stoichiometric (1:1 ratio between the anionic sites and the bound dye molecules). The proportionality of the absorbance of the complex at 559 nm to polyanion concentrations allows a simple quantitative determination of the polymers. The high sensitivity of the assay (i.e., (0.2-2.0) x 10~1 23% polyanion) may require a high dilution of samples. Consequently, potentially interfering compounds (e.g., acids, sugars, salts, milk, proteins, dyes, emulsifiers, and neutral hydrocolloids) may be diluted out and become compatible with the assay. In addition, a selective dye-binding inhibition of carboxylated polymers by phosphate allows the analysis of mixed carboxylated and sulfated hydrocolloids. Because the colorimetric assay is simple, highly sensitive, fast, and reproducible, it can be useful for routine quantitative analysis.Carrageenans, sulfated polysaccharides that occur as intercellular matrix materials in numerous species of red seaweeds, are extensively used as gelling, thickening, and stabilizing agents in the food, pharmaceutical, and cosmetics industries. Among the existing methods for determination of carrageenans and other polysaccharides are gas and liquid chromatography,12 which require hydrolysis of the samples prior to analysis. Other methods involve precipitation of the anionic polysaccharides using barium chloride,3 barium chloranilate,4 cetyl pyridinium chloride,5 alkyldimethylbenzylammonium chloride,6•7 and cationic dyes.8-10 In these methods, the polyanions are estimated from either the precipitate (e.g., analyzed gravimetrically or spectrophotometrically after solubilization in appropriate reagents) or the excess (1)

Hydrogen peroxide, which is a substrate of vanadium bromoperoxidase (V-BrPO), has been shown to be a noncompetitive inhibitor of V-BrPO. Hydrogen peroxide inhibition increases with increasing pH. The inhibition is reversible under the conditions of the initial steady-state kinetic experiments. Analysis of the inhibition constants (KiiH2O2, KisH2O2) versus H+ concentration indicates that an ionizable group with a pKa between 6.5 and 7 is involved in the inhibition. The origin of the oxygen atoms in the dioxygen produced by the V-BrPO-catalyzed bromide-assisted disproportionation of hydrogen peroxide has been shown through H2(18)O2 labeling experiments to originate from the same molecule of hydrogen peroxide. V-BrPO-catalyzed bromination is shown to be an electrophilic (Br+) as opposed to a radical (Br.) process. The stoichiometry of H2O2 consumed to MCD reacted or to O2 produced is reported. The concentration of hydrogen peroxide also affects the competition of dioxygen formation during MCD bromination; competitive dioxygen formation is strongly enhanced at high pH. Turnover of V-BrPO under conditions of very high hydrogen peroxide concentration leads to irreversible inactivation at pH 4 and pH 5. Much less inactivation occurs during turnover at long reaction times at higher pH (> pH 6), and the inactivation can be fully reversed by subsequent addition of vanadate.

No abstract

Vanadium bromoperoxidase (V-BrPO) has been isolated and purified from the marine brown algae Fucus distichus and Macrocystis pyrifera. V-BrPO catalyzes the oxidation of bromide by hydrogen peroxide, resulting in the bromination of certain organic acceptors or the formation of dioxygen. V-BrPO from F. distichus and M. pyrifera have subunit molecular weights of 65,000 and 74,000, respectively, and specific activities of 1580 units/mg (pH 6.5) and 1730 units/mg (pH 6) for the bromination of monochlorodimedone, respectively. As isolated, the enzymes contain a substoichiometric vanadium/subunit ratio; the vanadium content and specific activity are increased by addition of vanadate. V-BrPO (F. distichus, M. pyrifera, and Ascophyllum nodosum) also catalyzes the oxidation of bromide using peracetic acid. In the absence of an organic acceptor, a mixture of oxidized bromine species (e.g., hypobromous acid, bromine, and tribromide) is formed. Bromamine derivatives are formed from the corresponding amines, while 5-bromocytosine is formed from cytosine. In all cases, the rate of the V-BrPO-catalyzed reaction is much faster than that of the uncatalyzed oxidation of bromide by peracetic acid, at pH 8.5, 1 mM bromide, and 2 mM peracetic acid. In contrast to hydrogen peroxide, V-BrPO does not catalyze formation of dioxygen from peracetic acid in either the presence or absence of bromide. V-BrPO also uses phenylperacetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid to catalyze the oxidation of bromide; dioxygen is not formed with these peracids. V-BrPO does not catalyze bromide oxidation or dioxygen formation with the alkyl peroxides ethyl hydroperoxide, tert-butyl hydroperoxide, and cuminyl hydroperoxide.