Inactivation of enteroviruses by ascorbic acid and sodium bisulfite.

Poliovirus type 1, coxsackievirus type A9, and echovirus type 7 were inactivated by sodium bisulfite and ascorbic acid. Inactivation rates depended upon concentration, temperature, and pH. RNA infectivity was lost during inactivation; the capsid was also altered by these inactivating agents, as determined by enzyme sensitivity assays and by tests of adsorption to cells. Structural modifications of the virus particles were not identical, suggesting that the mechanism of inactivation by ascorbic acid differs from that of sodium bisulfite.     

Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents

The C-8 position of deoxyguanosine (dGuo) was hydroxylated by ascorbic acid in the presence of oxygen (O2) in 0.1 M phosphate buffer (pH 6.8) at 37 degrees C. Addition of hydrogen peroxide (H2O2) remarkably enhanced this hydroxylation. The Udenfriend system [ascorbic acid, FeII, ethylenediaminetetraacetic acid (EDTA) and O2] was also effective for hydroxylation of dGuo in high yield. Guanine residues in DNA were also hydroxylated by ascorbic acid. Other reducing agents, such as hydroxylamine, hydrazine, dihydroxymaleic acid, sodium bisulfite and acetol, were also effective for the hydroxylation reaction, as were metal-EDTA complexes (FeII-, SnII-, TiIII-, CuI-EDTA). An OH radical seemed to be involved in this hydroxylation reaction in most of the above hydroxylating systems, but another reaction mechanism may also be involved, particularly when dGuo is hydroxylated by ascorbic acid alone or ascorbic acid plus H2O2. The possible biological significance of the hydroxylation of guanine residues in DNA in relation to mutagenesis and carcinogenesis is discussed.     

Enzymatic degradation of uracil-containing deoxyribonucleic acid. V. Survival of Escherichia coli and coliphages treated with sodium bisulfite.

A number of mutants of Escherichia coli defective in the ung gene (structural gene for uracil-deoxyribonucleic acid [ura-DNA] glycosylase) are shown to be abnormally sensitive to treatment with sodium bisulfite when compared with congenic ung+ strains. These results provide further evidence that sodium bisulfite causes the deamination of cytosine to uracil in DNA and that ura-DNA glycosylase is required for the repair of U-G mispairs. The effect of the chemical is apparently selective with respect to base damage; coliphages containing cytosine in their DNA are inactivated by treatment with sodium bisulfite, whereas those containing hydroxymethylcytosine are not. ura-DNA glycosylase and the major apurinic-apyrimidinic endonuclease of E. coli may function in the same repair pathway, since the extent of inactivation of a congenic set of strains which are ung xth (structural gene for the major apurinic-apyrimidinic endonuclease of E. coli) or ung xth+ is the same.     

Mechanism of Inactivation of Bacteriophage J1 by Ascorbic Acid

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacillus casei, by ascorbic acid was investigated.

Bubbling air, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by ascorbic acid. In contrast, bubbling nitrogen gas, other reducing agents and radical scavengers prevented the inactivation. The results indicated that the inactivating effect of ascorbic acid was oxygen dependent and caused by free radicals formed during the autoxidation of ascorbic acid.

The target of ascorbic acid in the phage particle was not the tail protein but DNA. Ascorbic acid caused single-strand scissions in phage DNA, as exhibited by alkaline sucrose density gradient centrifugation analysis, and caused a slight decrease in the viscosity of DNA.     

Calcium dependent reversible inactivation of erythrocyte transglutaminase by acrylamide

Purified transglutaminase from human erythrocytes was shown to undergo time dependent inactivation by acrylamide added at millimolar concentration: this effect was wholly dependent on calcium ions and some protection was produced by glutamine substrates and GTP. The enzyme activity was recovered by addition of thiol based reducing agents, while ascorbic acid and sodium borohydrate were ineffective. These data are suggestive of an active site directed action of acrylamide.     

Effect of sodium bisulfite modification on the arginine acceptance of E. coli tRNA Arg.

Escherichia coli tRNA Arg was treated with sodium bisulfite to convert exposed cytosine residues to uracil. This treatment resulted in the loss of amino acid acceptance of the tRNA Arg with pseudo first-order reaction kinetics. The active and inactive molecules were separated after about 60e active and inactive molecules were separated after about 60 percent inactivation and analyzed for U in various positions by finger-printing of the oligonucleotides produced by nucleases. The results show that C to U base transitions in the dihydrouridine loop and in the CCA terminus have no effect on the aminoacylation of this tRNA. Deamination of a cytosine residue at the second position of the anticodon resulted in the loss of amino acid acceptor activity of arginine transfer RNA.     

Co-operative mutagenic actions of bisulfite and nitrogen nucleophiles.

The combined effect of bisulfite and a nitrogen nucleophile, i.e. semicarbazide, methoxyamine or hydroxylamine, to chemically modify cytosine and to cause mutation and inactivation of bacteriophage lambda was investigated. A rapid transamination of cytidine with each of the amines took place in the presence of bisulfite, and the reaction product was solely the N(4)-transaminated 5,6-dihydrocytidine-6-sulfonate. Modifications of cytidine with bisulfite alone and with the nitrogen nucleophile alone were much slower reactions than those using a combination of bisulfite and the nucleophile. Whereas the product of the modification with the bisulfite/semicarbazide, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine ribofuranoside-6-sulfonate, is convertible to 4-semicarbazido-2-ketopyrimidine ribofuranoside by treatment with a phosphate buffer, the products of the modification with the bisulfite/methoxyamine and with the bisulfite/hydroxylamine, i.e. 4-methoxy-5,6-dihydrocytidine-6-sulfonate and 4-hydroxy-5,6-dihydrocytidine-6-sulfonate, were stable in phosphate buffer.Inactivation and the “clear” mutation of bacteriophage lambda were observed when the phage was treated with sodium bisulfite in the presence of semicarbazide, methoxyamine or hydroxylamine. Under the conditions used, only very small increases in the mutation frequency were obtained by treatment of the phage with bisulfite alone or with the base alone. It was concluded that the residues, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine-6-sulfonate, 4-methoxy-5, 6-dihydrocytosine-6-sulfonate and 4-hydroxy-5,6-dihydrocytosine-6-sulfonate in DNA are the causes of the mutation.When phage that had been inactivated by the semicarbazide/bisulfite reagent was subsequently treated with a phosphate buffer, a reactivation took place. The rate of the reactivation increased as the concentration of phosphate in the buffer increased. This reactivation was not accompanied by change in the mutation frequency. No reactivation was observed after a similar incubation when the prior inactivation had been induced by either methoxyamine/bisulfite or hydroxylamine/bisulfite. These results indicate that the 4-semicarbazido-2-ketopyrimidine residue is also mutagenic but is less lethal than the corresponding 5,6-dihydro-6-sulfonate structure.These results offer the first clear example of the co-operative mutagenic action of two different reagents.     

Antiviral effect of commercial juices and beverages.

Nineteen commercial juices or beverages were tested for inactivation of poliovirus type 1. Grape and apple juices and tea were particularly antiviral. Although antiviral in aqueous solution, ascorbic acid was ineffective after addition to juices.     

Antiviral effect of commercial juices and beverages.

Nineteen commercial juices or beverages were tested for inactivation of poliovirus type 1. Grape and apple juices and tea were particularly antiviral. Although antiviral in aqueous solution, ascorbic acid was ineffective after addition to juices.     

Inactivation of Brain Tryptophan Hydroxylase by Nitric Oxide

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by nitric oxide (NO) and by the NO generators sodium nitroprusside, diethylamine/NO, S -nitroso- N -acetylpenicillamine, and S -nitrosocysteine. The inactivation occurs in an oxygen-free environment and is enhanced by dithiothreitol and ascorbic acid. Protection against the effect of NO on tryptophan hydroxylase is afforded by oxyhemoglobin, reduced glutathione, and exogenous Fe(II). Catalase partially protects the enzyme from NO-induced inactivation, whereas both superoxide dismutase and uric acid are without effect. These findings indicate that tryptophan hydroxylase is a target for NO and suggest that critical iron-sulfur groups in this enzyme serve as the substrate for NO-induced nitrosylation of the protein, resulting in enzyme inactivation.     

Anisotropic staining of apurinic acid with toluidine blue.

Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

Anisotropic staining of apurinic acid with toluidine blue

Summary Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

Interaction between 2,4-Dichlorophenoxyacetic Acid and Vitamin K in Datura stramonium

Plants of the cross Datura stramonium L. var. stramonium×Datura stramonium L. var. inermis (Juss. ex Jacq.) Timm. were sprayed either with a water solution of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D) or with a combination of 2,4-D (sodium salt) and the vitamin K compound menadione sodium bisulfite in water solution. The plants were found to differ in one of their responses to the treatments, namely the development of necrotic spots on the leaf blades. Many necroses appeared on the plants treated with 2,4-D plus menadione sodium bisulfite while only a few necroses or none at all developed on the plants treated with 2,4-D alone. Furthermore, 2,4-D in combination with menadione sodium bisulfite induced much larger necrotic spots than did 2,4-D by itself. Spraying with menadione sodium bisulfite in water solution did not produce any observable effects upon Datura plants. The great increase in necrotic lesion formation was probably due to an interaction between 2,4-D and menadione sodium bisulfite (or a metabolite of this compound) within leaf cells.     

ON THE INACTIVATION OF ASCORBIC ACID OXIDASE

1. In the absence of protective agents, highly purified ascorbic acid oxidase is rapidly inactivated during the enzymatic oxidation of ascorbic acid under optimum experimental conditions. This inactivation, called reaction inactivation to distinguish it from the loss in enzyme activity that frequently occurs in diluted solutions of the oxidase prior to the reaction, is indicated by incomplete oxidation of the ascorbic acid as measured by oxygen uptake; i.e., "inactivation totals." 2. A minor portion of the reaction inactivation appears to be due to environmental factors such as rate of shaking of the manometers, pH of the system, substrate concentration, and oxidase concentration. The presence of inert protein (gelatin) in the system ameliorates the environmental inactivation to a considerable extent, and variation of the above factors in the presence of gelatin has much less effect on the inactivation totals than in the absence of gelatin. 3. A major portion of the reaction inactivation of the oxidase appears to be due to some factor inherent in the ascorbic acid-ascorbic acid oxidase-oxygen system, possibly a highly reactive "redox" form of oxygen other than H₂O₂ or H₂O. The inactivation cannot be attributed to dehydroascorbic acid, the oxidation product of ascorbic acid. 4. Small amounts of native catalase, native peroxidase, native or denatured methemoglobin, and hemin when added to the system, markedly protect the oxidase against inactivation. Cytochrome c has no such protective action. Likewise proteins such as egg albumin, gelatin, denatured catalase, or denatured peroxidase show no such protective action. 5. None of the protective agents mentioned above affect the initial rate of oxygen uptake or change the total oxygen absorbed for complete oxidation of the ascorbic acid, and hence do not act by removal of hydrogen peroxide, per se. 6. Sodium azide and hydroxylamine hydrochloride which inhibit catalase and peroxidase activity also inhibit the protective action of these iron-porphyrin enzymes.     

Alkaline phosphatase expression in human chorionic villi

The physicochemical properties and electrophoretic mobility of different isoforms of alkaline phosphatase were studied in chorionic villi. Based on selective inactivation and inhibition studies (thermal stability, inactivation by urea, EDTA and L(+)ascorbic acid and L-amino acid inhibition), evidence was obtained for the existence of two distinct types of alkaline phosphatase in trophoblast cells. One type is peculiar to chorionic villi while the other is also found in term placenta. Both show two isoforms. These two isoforms were observed with polyacrylamide gel electrophoresis, carried out at pH 6.0 and 9.5. It is suggested that the qualitative and quantitative methods of alkaline phosphatase analysis could be used for first trimester fetal diagnosis of severe infantile hypophosphatasia and for understanding genetic control during early fetal development.     

Study of the interaction of sulfur dioxide derivative with cardiac sodium channel

The effects of sulfur dioxide (SO(2)) derivatives (bisulfite and sulfite, 1:3 M/M) on voltage-dependent sodium channel in isolated rat ventricular myocyte were studied using the whole cell patch-clamp technique. SO(2) derivatives increased sodium current (I(Na)) in a concentration-dependent manner. SO(2) derivatives at 10 microM significantly shifted steady-state inactivation curve of I(Na) to more positive potentials, but did not affect the activation curve. SO(2) derivatives markedly shifted the curve of time-dependent recovery of I(Na) from inactivation to the left, and accelerated the recovery of I(Na). SO(2) derivatives also significantly shortened the activation and inactivation time constants of I(Na). These results indicated that SO(2) derivatives produced concentration-dependent stimulation of cardiac sodium channels, which due mainly to the interaction of the drug with sodium channels in the inactivated state.     

Modification of polyuridylic acid by bisulfite. II: Studies on ribosomal binding and enzymatic hydrolysis

The reaction of polyuridylic acid with sodium bisulfite produces modified polymers in which up to 95% of the uracil residues have been converted to uracil-6-sulfonate residues. A 91.6% bisulfite-saturated polymer was found to resist hydrolysis by spleen phosphodiesterase and phosphorolysis by polynucleotide phosphorylase. Digestion by pancreatic ribonuclease was successful and gave the bisulfite adduct of uridine-3-phosphate. Treatment of this nucleotide adduct with acid phosphatase afforded the bisulfite adduct of uridine. The ability of polyuridylic acid to bind to ribosomes, and to stimulate the binding of phenylalanine tRNA to ribosomes was abolished by progressive bisulfite saturation of the polymer. The rate of decline of these functionsf with increasing bisulfite content, was less sharp than the loss of phenyl-alanine coding ability o, the modified polymer.     

Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase

We have previously studied purified human myeloperoxidase-hydrogen peroxide-halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl- was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189-191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase-hydrogen peroxide-halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase-dependent mechanism.     

Stereospecific binding of 3H-dopamine in neostriatal membrane preparations: inhibitory effects of sodium ascorbate

It has been pointed out by several different groups of investigators in the past several years that ascorbic acid was a potent inhibitor of the binding of dopamine (DA) agonists including 3H-DA itself and 3H-ADTN, 3H-apomorphine and 3H-norpropylapomorphine to neostriatal membrane preparations. However, the significance of this effect of ascorbic acid has been controversial. For example, it has recently been claimed that the stereospecific binding of DA agonists is facilitated by ascorbic acid and can be measured only in its presence. In the present study in neostriatal membrane preparations in the absence of ascorbic acid, the binding of 3H-DA was very potently inhibited by potent DA agonists (DA, ADTN, apomorphine). Considerably weaker effects were obtained with norepinephrine, isoproterenol, serotonin, catechol and pyrogallol. Stereospecific effects were clearly observed in that the binding of 3H-DA was inhibited to a much greater extent by several biologically active enantiomers than by their less active counterparts. For example, (-)-2-hydroxyapomorphine and (-)-norpropylapomorphine were much more potent inhibitors than their corresponding (+) isomers. This binding of 3H-DA was also very strongly inhibited by sodium ascorbate and several other reducing agents. In control experiments in the neostriatal membrane preparation in the absence of ascorbic acid, there was no detectable decomposition of 3H-DA. The data suggest that 3H-DA can, in the absence of sodium ascorbate, bind stereospecifically to a site that has the properties of a DA receptor. Furthermore, sodium ascorbate is a potent inhibitor of this stereospecific binding.