The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

The production of hydroxyl radical from hydrogen peroxide

The formation of hydroxyl radical (OH·) from the oxidation of glutathione, ascorbic acid, NADPH, hydroquinone, catechol, and riboflavin by hydrogen peroxide was studied using a range of enzymes and copper and iron complexes as possible catalysts. Copper-1,10-phenanthroline appears to catalyze the production of OH· from hydrogen peroxide without superoxide radical being formed as an intermediate, and without the involvement of a catalyzed Haber-Weiss (Fenton) reaction. Superoxide radical is involved, however, in the Cu²⁺ -catalyzed decomposition of hydrogen peroxide, and in the oxidation of glutathione by atmospheric oxygen. For this latter oxidation, copper-4,7-dimethyl-1,10-phenanthroline was found to be a much more effective catalyst than the copper complex of 1,10-phenanthroline, which is normally used. Mechanisms for these reactions are proposed, and the toxicological significance of the ability of a variety of biological reductants to provide a prolific source of OH· when oxidized by hydrogen peroxide is discussed.     

Lactoferrin-catalysed hydroxyl radical production. Additional requirement for a chelating agent.

The ability of lactoferrin to catalyse hydroxyl radical production was determined by measuring ethylene production from methional (2-amino-4-methylthiobutyraldehyde) or 4-methylthio-2-oxobutyrate. Lactoferrin, isolated from human milk and saturated by adding the exact equivalents of Fe3+-nitrilotriacetic acid and dialysing, give little if any catalysis of the reaction between H2O2 and either O2-. or ascorbic acid at either pH 7.4 or pH 5.0. However, in the presence of chelating agents such as EDTA or nitrilotriacetic acid that can complex with lactoferrin, hydroxyl radical production by both mechanisms was observed.     

The reaction of methionine with hydroxyl radical: reactive intermediates and methanethiol production

The mechanisms of reaction of methionine with hydroxyl radical are not fully understood. Here, we unequivocally show using electron paramagnetic resonance spin-trapping spectroscopy and GC-FID and GC-MS, the presence of specific carbon-, nitrogen- and sulfur-centered radicals as intermediates of this reaction, as well as the liberation of methanethiol as a gaseous end product. Taking into account the many roles that methionine has in eco- and biosystems, our results may elucidate redox chemistry of this amino acid and processes that methionine is involved in.     

Detection of hydroxyl radicals by D-phenylalanine hydroxylation: a specific assay for hydroxyl radical generation in biological systems

Hydroxylation of l-phenylalanine (Phe) by hydroxyl radical (*OH) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively). Phe derivative measurements have been employed to detect *OH formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. d-Phe, the d-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether d-Phe reacts with *OH as well as l-Phe, providing a more reliable probe for *OH generation in biological systems. With *OH generated by a Fenton reaction or xanthine oxidase, d- and l-Phe equally gave rise to p, m, o-tyr and this could be prevented by *OH scavengers. Resting human neutrophils (PMNs) markedly converted l-Phe to p-tyr, through non-oxidant-mediated reactions, whereas d-Phe was unaffected. In contrast, when PMNs were stimulated in the presence of redox cycling iron the *OH formed resulted in more significant rise of p-tyr from d-Phe (9.4-fold) than l-Phe (3.6-fold) due to the significant background formation of p-tyr from l-Phe. Together, these data indicated that d- and l-Phe were equally hydroxylated by *OH. Using d-Phe instead of l-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of *OH in biological systems.     

Enhanced hydroxyl radical production by dihydroxybenzene-driven Fenton reactions: implications for wood biodegradation

Brown rot fungi degrade wood, in initial stages, mainly through hydroxyl radicals (•OH) produced by Fenton reactions. These Fenton reactions can be promoted by dihydroxybenzenes (DHBs), which can chelate and reduce Fe(III), increasing the reactivity for different substrates. This mechanism allows the extensive degradation of carbohydrates and the oxidation of lignin during wood biodegradation by brown rot fungi. To understand the enhanced reactivity in these systems, kinetics experiments were carried out, measuring •OH formation by the spin-trapping technique of electron paramagnetic resonance spectroscopy. As models of the fungal DHBs, 1,2-dihydroxybenzene (catechol), 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid were utilized as well as 1,2-dihydroxy-3,5-benzenedisulfonate as a non-Fe(III)-reducing substance for comparison. Higher amounts and maintained concentrations of •OH were observed in the driven Fenton reactions versus the unmodified Fenton process. A linear correlation between the logarithms of complex stability constants and the •OH production was observed, suggesting participation of such complexes in the radical production.     

Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation.

Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10^?5m) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe²⁺-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction.     

Factors that influence the deoxyribose oxidation assay for Fenton reaction products.

The mechanism of oxidation of deoxyribose to thiobarbituric acid-reactive products by Fenton systems consisting of H2O2 and either Fe2+ or Fe2+ (EDTA) has been studied. With Fe2+ (EDTA), dependences of product yield on reactant concentrations are consistent with a reaction involving OH.. With Fe2+ in 5-50 mM phosphate buffer, yields of oxidation products were much higher and increased with increasing deoxyribose concentration up to 30 mM. The product yield varied with H2O2 and Fe2+ concentrations in a way to suggest competition between deoxyribose and both reactants. Deoxyribose oxidation by Fe2+ and H2O2 was enhanced 1.5-fold by adding superoxide dismutase, even though superoxide generated by xanthine oxidase increased deoxyribose oxidation. These results are not as expected for a reaction involving free OH. or site localized OH. product on the deoxyribose. They can be accommodated by a mechanism of deoxyribose oxidation involving an iron(IV) species formed from H2O2 and Fe2+, but the overall conclusion is that the system is too complex for definitive identification of the Fenton oxidant.     

The oxidation of some polysaccharides by the hydroxyl radical: an e.s.r. investigation

E.s.r. Experiments employing a flow system in conjunction with the TiIII-H2O2 couple show that dextrans react with the hydroxyl radical (HO.) via indiscriminate attack (except that abstraction of hydrogen atoms from carbons which are both linked by glycosidic bonds and included in the pyranose ring may be inhibited, possibly for steric reasons). Acid- and base-catalysed transformations of first-formed radicals have been demonstrated; the suggestion that such reactions can lead to glycosidic cleavage is supported by viscosity studies which confirm the pH-dependence of radical-initiated degradation. For galacturonan and related compounds, e.s.r. results indicate that reaction with HO. proceeds preferentially via abstraction of the hydrogen on the carbon adjacent to the carboxyl group. The crucial step in the subsequent degradation pathway probably involves a pH-independent rearrangement.     

Synthesis, hydroxyl radical production and cytotoxicity of analogues of bleomycin

Two pyridine analogues of the metal complexing region of the anticancer drug bleomycin and two related but deactivated prodrugs have been linked to a 2,6-diphenylpyridine derivative as a DNA binding unit. The 2,6-diphenylpyridine system is structurally related to known amplifiers of the cytotoxicity of bleomycin. The conjugates were found to bind to DNA more strongly than bleomycin-A2 and were more cytotoxic than the corresponding compounds lacking the DNA binding unit. On exposure of a mixture of cells and prodrugs to hypoxia and then air, the prodrug containing the nitrohistidine unit was not bioreductively activated but the prodrug having an N-oxide group was bioreductively activated. This result represents a novel approach to the improvement of the therapeutic ratio of bleomycin analogues.     

Conversion of superoxide generated by polymorphonuclear leukocytes to hydroxyl radical: a direct spectrophotometric detection system based on degradation of deoxyribose

Hydroxyl radical is produced secondarily after phagocytic cells have been stimulated to generate superoxide anion. The systems used most commonly for detection of cell-generated hydroxyl radical are often inconvenient for routine biomedical research. We have modified an assay used heretofore in cell-free systems, that is, the degradation of deoxyribose, and adapted it for use with neutrophils. The time and dose responses of the system, requirement for chelated iron, inhibition profiles with various scavengers, and correlation with superoxide production have been ascertained. The method correlated strongly with a standard but more cumbersome technique. Values for a normal population are provided. The method can readily be used to study the parameters of superoxide-hydroxyl radical conversion by cells in various disease or treatment states.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

DNA polymerase insertion fidelity. Gel assay for site-specific kinetics

A quantitative assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase alpha holoenzyme complex and M13 primer-template DNA. Km and Vmax values are reported for correct insertion of A and misinsertion of G, C, and T opposite a single template T site. The misinsertion frequencies are 2 X 10(-4) for G-T and 5 X 10(-5) for both C-T and T-T relative to normal A-T base pairs. The dissociation constant of the polymerase-DNA-dNTP complex, as measured by Km, plays a dominant role in determining the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 +/- 0.7 microM), the Km value is 1100-fold greater for misinsertion of G opposite T (4.2 +/- 0.4 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 +/- 4.2 mM). These Km differences indicate that in the enzyme binding site the stability of A-T base pairs is 4.3 kcal/mol greater than G-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax are relatively small. There is only a 4-fold reduction in Vmax for insertion of G opposite T and an 8-fold reduction for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase alpha seems to be governed primarily by a Km discrimination mechanism.     

Iron-dependent free radical damage to DNA and deoxyribose. Separation of TBA-reactive intermediates

1. Iron-dependent free radical damage to DNA and deoxyribose results in the formation of thiobarbituric acid (TBA) reactive intermediates. 2. These intermediates have been compared chromatographically and spectrophotometrically after incubation with the enzymes xanthine oxidase and peroxidase. 3. Loss of TBA-reactivity occurred in the bleomycin-iron(II) derived products incubated with xanthine oxidase and in a standard solution of sodium malondialdehyde incubated with peroxidase.