Oxidation Of Histidine Research Articles

Structure of a Transient Neutral Histidine Radical in Solution: EPR Continuous-Flow Studies in a Ti3+/EDTA−Fenton System and Density Functional Calculations

Oxidation of histidine by OH* radicals has been studied at room temperature over a large range of pH values in a Ti3+ /EDTA/H2O2−Fenton system using a special EPR continuous-flow setup. At pH 7 to 8, during fast flow, an EPR spectrum from a new transient histidine radical with a well-resolved hyperfine structure (hfs) has been observed at giso = 2.0026. The hfs changed significantly in the cases of methylene-deuterated histidine in H2O and of histidine in D2O. EPR spectral simulations resulted in an assignment of two slightly different splittings of the two methylene protons (1.27 and 1.59 mT); one large splitting from an out-of-plane proton of the imidazole ring (2.47 mT); and splittings of a ring proton (0.99 mT), a NH (or OH) proton (0.14 mT), and two ring nitrogens (0.27 and 0.11 mT). The large proton-splitting indicates an addition of an OH* radical to the neutral imidazole ring. Such an addition of an OH* radical was already observed for the histidine cation radical in a Ti3+/H2O2 Fenton system at pH 2.1 Density functional theory (DFT) calculations of hyperfine coupling constants were performed at the PCM/B3LYP/6-311G(2df,p) level for three isomeric forms of a histidine model (4-ethyl imidazole) in which an OH* radical is added to different positions (C2, C4, and C5) of the neutral imidazole ring. Theoretical hyperfine data are in excellent agreement with the experiment and clearly support an assignment to a neutral histidine radical formed by addition of an OH* radical at the C5 position of the imidazole ring. Extensive studies of histidine oxidation in a Ti3+/EDTA−Fenton system, at acidic pH as well as at neutral and basic pH values, have advanced our understanding of the Fenton chemistry of histidine and the electronic structure of involved paramagnetic species. The pH profile of the formation of histidine OH-addition radicals shows that the cation radical dominates at pH 2 to 4, and the neutral radical at pH 5 to 9. At pH ≥7, in the presence of histidine, a titanium complex with giso = 1.9632 and a significant hfs from naturally occurring 47Ti and 49Ti nuclei with aiso(Ti) = 1.54 mT has been observed. This complex, which dominates at pH 9 to 10, is also visible in the absence of H2O2 and without flow, and is thus assigned to a stable mixed-ligand Ti3+ complex containing EDTA and histidine. This is the first communication on a neutral histidine radical with a complete set of hyperfine coupling constants derived by EPR as well as by DFT calculations.

Mechanisms of metal-catalyzed oxidation of histidine to 2-oxo-histidine in peptides and proteins

The metal-catalyzed oxidation of histidine (His) to 2-oxo-histidine (2-oxo-His) represents an important pathway of protein oxidation in vivo and in vitro. In the pharmaceutical literature this pathway has received less attention. However, this fact may not necessarily represent reality as, in some cases, the analysis of His oxidation in proteins may be compromised by aggregation and precipitation of the target protein. For predicting the susceptibility of His residues in proteins it is important to understand in detail how protein sequence and conformation control the mechanisms of His oxidation to 2-oxo-His, reviewed in this paper.

Electronic Structure of a Transient Histidine Radical in Liquid Aqueous Solution: EPR Continuous-Flow Studies and Density Functional Calculations

Transient histidine radicals formed in aqueous solutions by oxidation of histidine with a Ti3+/H2O2 Fenton system at pH 2.0 have been studied by EPR using a fast continuous-flow setup and a dielectric ring resonator equipped with a mixing chamber. A histidine peroxy radical with a single EPR line at g = 2.0151 and a histidine cation radical with a complex hyperfine structure and g = 2.0023 have been detected. The hyperfine structure of the latter radical was analyzed by investigating two selectively deuterated histidines and using an EPR simulation and fit program for analysis of the spectra. Isotropic hyperfine coupling constants of two β-protons, three ring protons, and two nitrogen nuclei have been determined in this way and assigned to a histidine−OH adduct cation radical. Density functional theory (DFT) calculations at the B3LYP and PWP86 levels have been performed on protonated cation radicals of 4-ethylimidazole (as histidine models), yielding isotropic hyperfine coupling constants for three different positions of OH addition. The C5 position for OH addition (a 5-oxohistidine cation radical) is clearly supported by the calculated hyperfine coupling constants. The agreement between DFT and EPR is further improved when hydrogen-bonding interactions to the N1 and C2 protons are introduced in the calculations.

Photodegradation and phototoxicity studies of furosemide. Involvement of singlet oxygen in the photoinduced hemolysis and lipid peroxidation

The phototoxic diuretic drug furosemide ( 1), a 5-(aminosulfonyl)-4-chloro-2-[(2-furanylmethyl)-amino] benzoic acid is photolabile under aerobic and anaerobic conditions. Irradiation of a methanol solution of 1 under oxygen produces photoproducts 2, 3, 4 and singlet oxygen, while under argon the photoproducts 2 and 4 were isolated. A peroxidic unstable photoproduct was detected during the photolysis under oxygen atmosphere. The formation of singlet oxygen by photolysis of 1 was evidenced by trapping with 2,5-dimethylfuran (GC-mass), furfuryl alcohol and 1,3-cyclohexadiene-1,4-diethanoate (HPLC) as 1O 2 scavengers and by the histidine test. Furosemide was screened in vitro at different concentrations for UV-Vis-induced phototoxic effects in a photohemolysis test, in the presence and absence of different radical scavengers, singlet oxygen and hydroxyl radical quenchers. However, furosemide photosensitized the peroxidation of linoleic acid, as monitored by the UV-detection of dienic hydroperoxides and it also photosensitized the oxidation of histidine. The photodegradation was catalyzed in the presence of human serum albumin. Studies on peripheral blood mononuclear and polymorphonuclear cells (lymphocytes and neutrophils) demonstrated no phototoxicity on these cell lines.

Chemical pathways of peptide degradation: IX. Metal-catalyzed oxidation of histidine in model peptides.

To elucidate the nature of the reactive oxygen species (i.e., superoxide anion radical, hydroxyl radical, and hydrogen peroxide) involved in the metal-catalyzed oxidation of histidine (His) in two model peptides. The degradation of AcAla-His-ValNH2 (Ala-peptide) and AcCysNH2-S-S-AcCys-His-VaNH2 (Cys-peptide) was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen (ascorbate/ Cu(II)/O2) system, both in the absence and presence of selective scavengers (i.e., catalase, superoxide dismutase, mannitol, sodium formate, isopropanol, and thiourea) of the reactive oxygen species. All reactions were monitored by HPLC. The major degradation products were characterized by electrospray mass spectrometry. The Cys-peptide was more stable than the Ala-peptide at pH 5.3 and 7.4. Both peptides displayed greater stability at pH 5.3 than at 7.4. At pH 5.3, 35 +/- 0.7% of the Cys-peptide and 18 +/- 1% of the Ala-peptide remained after 7 hours, whereas at pH 7.4, 16 +/- 3% of the Cys-peptide and 4 +/- 1% of the Ala-peptide remained. Catalase, thiourea, bicinchoninic acid, and ethylenediaminetetraacetate were effective at stabilizing both peptides toward oxidation, while superoxide dismutase, mannitol, isopropanol, and sodium formate were ineffective. The main degradation products of the Ala- and Cys-peptides at pH 7.4 appeared to be AcAla-2-oxo-His-ValNH2 and AcCysNH2-S-S-AcCys-2-oxo-His-ValNH2, respectively. Hydrogen peroxide, Cu(I), and superoxide anion radical were deduced to be intermediates involved in the oxidation of the Ala- and Cys-peptides. Hydrogen peroxide degradation to secondary reactive oxygen species may have led to the oxidation of the peptides. The degradation of hydrogen peroxide by a Fenton-type reaction was speculated to form a complexed form of hydroxyl radical that reacts with the peptide before diffusion into the bulk solution.

Quantitation of the singlet oxygen produced by UVA irradiation of human lens proteins.

Ultraviolet irradiation of aged human lens proteins in vitro causes extensive photolytic damage of His and Trp residues. Protection by sodium azide argues for a process mediated by singlet oxygen (1O2). In the work described here, the synthesis of 1O2 was measured by the bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), the oxidation of added histidine and the oxidation of furfuryl alcohol. To obtain a more accurate value for 1O2 generation, a known quantity of 1O2 was generated by the thermal dissociation of 3-(4-methyl-naphthyl)propionic acid endoperoxide, and the efficiency of each assay method to report on the 1O2 generated was determined. The values obtained were 0.003 mol of RNO bleached/mol of 1O2 generated, 0.55 mol of furfuryl alcohol oxidized/mol 1O2 and 0.5 mol of His oxidized/mol 1O2 generated. Irradiation of the human lens proteins with UVA light produced from 2.1 to 2.4 mM of 1O2 by RNO bleaching, 2.6-2.8 mM 1O2 by furfuryl alcohol oxidation and up to 1.9 mM of 1O2 by histidine oxidation during a 1 h irradiation period. The average value (2.2 mM of 1O2) corresponds to the theoretical production of 30 nmol of singlet oxygen at UVA light intensities equivalent to a 1 h exposure to sunlight at noon in the northern hemisphere.

Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid‐Glycated Lens Proteins

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol. 62, 454-463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/cm2 of absorbed UVA light (gamma > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 microM as determined by the superoxide dismutase (SOD)-dependent increase in hydrogen peroxide formation and of 52 microM by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 microM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system. Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N-dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H2O2 were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.

Inactivation of vanadium bromoperoxidase: formation of 2-oxohistidine.

The basis of the irreversible inactivation of the vanadium bromoperoxidase (V-BrPO) isolated from the marine alga Ascophyllum nodosum under turnover conditions at low pH (i.e., 15 to 100 mM H2O2, 0.1 KBr, ca. 15 nM V-BrPO in 0.1 M citrate, pH 4) has been investigated. Inactivation under these conditions was found to produce 2-oxohistidine as identified by HPLC using electrochemical detection. Formation of 2-oxohistidine requires all the components of turnover (i.e., bromide, hydrogen peroxide, and V-BrPO) as well as low pH; inactivation does not occur nor is significant 2-oxohistidine formed in the presence of hydrogen peroxide alone. The oxidation of histidine did not occur by singlet oxygen generated by V-BrPO, because neither 2-oxohistidine nor inactivation occur under the conditions in which singlet oxygen is produced quantitatively by V-BrPO. The addition of aqueous bromine to N alpha-benzoylhistidine at low pH formed N alpha-benzoyl-2-oxohistidine. cis-Dioxovanadium(V) (VO2+) in strong acid and MoO(O2)2(ox)2- (ox2- is oxalate) at pH 5, both of which are functional mimics of V-BrPO by oxidizing bromide by hydrogen peroxide, catalyzed the oxidation of N alpha-benzoylhistidine to N alpha-benzoyl-2-oxohistidine. Furthermore, when hypobromite was added to N alpha-benzoylhistidine in the presence of hydrogen peroxide at neutral pH, conditions under which HOBr would react first with H2O2 to produce singlet oxygen, no N alpha-benzoyl-2-oxohistidine was formed. Thus the oxidation of histidine in V-BrPO is proposed to occur via oxidized bromine species. Irreversible inactivation V-BrPO was also found to be accompanied by release of vanadium.

Folate-mediated incorporation of ring-2-carbon of histidine into nucleic acids: Influence of thyroid hormone

Folate-mediated incorporation of [ring-2- 14C]histidine into DNA and its modulation under thyroid stress have been studied. Both hyperthyroidism and hypothyroidism decrease the folate-mediated incorporation of the one-carbon unit derived from histidine into DNA significantly, resulting in growth retardation. In concurrence with previous reports, in vivo oxidation of histidine is decreased in hyperthyroidism and increased in hypothyroidism. 5,10-Methylenetetrahydrofolate reductase levels are elevated in hyperthyroidism and decreased in hypothyroidism, whereas methionine synthase levels are decreased in hyperthyroidism and increased in hypothyroidism. It is discussed how in both hyperthyroidism and hypothyroidism folate cofactor-mediated reactions are adversely affected.

Amyloidogenicity of rodent and human βA4 sequences

Previously we have shown that aggregation of the C-terminal 100 residues (A4CT) of the βA4 amyloid protein precursor (APP) and also of βA4 itself depends on the presence of metal-catalyzed oxidation systems [T. Dyrks et al. (1988) EMBO J. 7, 949-957]. We showed that aggregation of the amyloidogenic peptides induced by radical generation systems requires amino acid oxidation and protein cross-linking. Here we report that aggregation of A4CT and βA4 induced by radical generation systems involves oxidation of histidine, tyrosine and methionine residues. The roden βA4 sequence lacking the single tyrosine and one of the three histidine residues of human βA4 and a βA4 variant in which the tyrosine and the three histidine residues were replaced showed a reduced tendency for aggregation. Thus our results may explain why βA4 amyloid deposits could so far not been detected in the rodent brain.

Investigation of the origin of the "S3" EPR signal from the oxygen-evolving complex of photosystem 2: the role of tyrosine Z.

The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161).

Kinetics and mechanism of the oxidation of histidine by dodecatungstocobaltate(III) and trans-cyclohexane-1,2-diamine-N,N,N′,N′-tetraacetatomanganate (III) in aqueous medium

The oxidation of histidine with dodecatungstocobaltate(III), CoW12O405– and trans-cyclohexane-1,2-diamine-N,N,N′,N′-tetraacetatomanganate(III), [MnIII(cdta)]– have been investigated in the range pH 3.0–9.5 at variable reductant concentrations, at a constant ionic strength and temperature. Both reactions are found to be dependent on the first powers of the concentration of the substrate and oxidants and follow the general rate law, -d[Ox]/dt= 2k[Ox][Hist], where 2 is the stoichiometric factor. In the reduction of [MnIII(cdta)]–, a bell-shaped curve is obtained for the variation of second-order rate constant (K) as a function of pH, and this has been explained by considering the hydroxo form of the complex as being unreactive towards the reductant. An attempt has been made to verify the effect of alkali cation catalysis on the reaction rate. The observed alkali cation catalysis for the reduction of CoW12O405– is consistent with a rate law, k=k0+kc[M+] where k0 and kc are the rate constants for the spontaneous and catalysed paths respectively. For the reduction of [MnIII(cdta)]–, a negligible dependence of rate on [M+] was noted. All the observations are successfully explained by considering outer-sphere mechanistic pathways for both reactions.

Metabolism and Urinary Excretion of Folates in Hypothyroid Rats.

Hypothyroidism results in decreased urinary excretion of folates leading to enhanced tissue retention of folates. The proportion of polyglutamylfolates, which are better retained by tissues, is elevated in liver, blood, and bone marrow of hypothyroid rats. The hypothyroid condition is discussed in the light of the enhanced proportion of polyglutamylfolate cofactors and increased in vivo oxidation of histidine possibly resulting in under-utilization of one-carbon units of the folate pool leading to a decrease in weight gain.

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.

Biochemical evidence for histidine oxidation in photosystem II depleted of the Mn-cluster for O 2-evolution

Flash excitation of Tris-treated photosystem II (PSII) membranes generated a thermoluminescene band peaking -25°C. Indicating that a positively charged oxidizing equavalent is stably accumulated on donor side of PSII even in the Mn-cluster. This oxidizing equivalent was sensitive to low concentrations of exogenous Mn ???, and its stable accumulation was reversibly inhibited by diethylpyrocarbonate treatment that modifies histidine residues with high specificity. It was inferred that there is a photooxidizable histidine residue(s) on donor side of PSII and it provides a redox-active ligand for Mn

Effect of Hormones and Protein Level on Induction of Enzymes Involved in Histidine Degradation and Folate Metabolism

Summary The effects of the hormones thyroxine, glucagon and hydrocortisone, and of protein level on hepatic histidase, urocanase, and certain folate enzymes were studied in rats. Hypothyroidism, induced either by feeding thiouracil or by thyroidectomy, increased histidase, urocanase, and the folate dependent enzyme formiminotransferase, and increased histidine metabolism as measured by oxidation of the 2-ring carbon of histidine to carbon dioxide. Hyperthyroidism, produced by feeding thyroid powder, decreased histidine oxidation and liver levels of this enzyme. Glucagon and hydrocortisone increased histidine oxidation and increased levels of histidase, urocanase and formiminotransferase. Elevated levels of casein and soy protein produced four to eight fold increases in histidase and urocanase, and 50% increases in formiminotransferase. Thus, increases in histidase and urocanase involved in the formation of formiminoglutamic acid (FIGlu) were accompanied by increases in the FIGlu metabolizing enzyme formiminotransferase.

Histidine oxidation in the oxygen-evolving photosystem-II enzyme

THE evolution of oxygen as a result of light-driven water oxidation occurs in plants and is catalysed by photosystem-II (PS-II). A manganese-cluster probably acts both as the active site and as a charge-accumulating device (for a review, see ref. 1). The enzyme cycle involves five redox states which are denoted as S0–S4, depend-ing on the number of positive equivalents stored2. Oxygen is released after formation of the transient S4 state. Ca2+ is an obligatory cofactor in this process and its depletion inhibits the enzyme cycle at the step before water oxidation, that is, after formation of the S3 state3. In chelator-treated, Ca2+-depleted PS-II, a new electron paramagnetic resonance (EPR) signal arising from a formal S3 state has been reported4. It was suggested that the S3 EPR signal could originate from the oxidation of an amino acid, interacting magnetically with the manganese cluster4. There are only a few examples of amino-acid oxidation in enzyme chemistry and these are limited to tyrosine5 and tryptophan6. Here we report evidence that the S2 to S3 transition occurring in Ca2+-depleted PS-II corresponds to the oxidation of histidine.

INFLUENCE OF FLUOROCARBON EMULSIONS ON PORPHYRIN‐SENSITISED OXIDATION OF HISTIDINE

The influence of fluorocarbon emulsions on the efficiency of photosensitized oxidation of histidine in solution has been studied, using haematoporphyrin and dihaematoporphyrin derivatives as sensitisers. It is shown that the fluorocarbon emulsions at low concentrations efficiently disaggregate porphyrins, and thereby enhance photosensitised oxidation. The high solubility of oxygen in fluorocarbon emulsions maintains solution oxygen tension, optimising photooxidative damage. It is suggested that fluorocarbon emulsions might find a role in photodynamic therapy, both as carriers for sensitising dyes, and also to maintain tissue oxygenation in hypoxic regions of solid tumours.

Reaction of a histidyl residue analog with hydrogen peroxide in the presence of copper(II) ion

Site-specific damage to a specific amino acid residue of a protein by Cu{sup 2+}/H{sub 2}O{sub 2} was investigated. Observation of specific damage to the histidine residue of the protein was fully supported by a model experiment using N-benzoylhistidine. The reaction of H{sub 2}O{sub 2} toward the substrate was characteristic of a Cu{sup 2+}-catalyzed system and was significantly influenced by pH. Other peroxides and peracids were slightly reactive to the substrate even in the presence of Cu{sup 2+}. Under simulated physiological conditions (pH 7.2, room temperature), incubation with Cu{sup 2+}/H{sub 2}O{sub 2} (0.5 mM CuSO{sub 4}, 50 mM H{sub 2}O{sub 2}) resulted in complete destruction of the substrate within 1 h. Four products including N-benzoyl derivatives of asparagine, aspartic acid, aspartylurea, and formylasparagine were detected as the ring-ruptured products, and three products including 5-(2-benzamidovinyl)imidazole, benzamide, and benzoate were detected as the oxidation products at the {alpha}- and {beta}-positions of the substrate. Plausible mechanisms for oxidation of histidine are proposed.

SINGLET OXYGEN INDUCES FRANK STRAND BREAKS AS WELL AS ALKALI‐ AND PIPERIDINE‐LABILE SITES IN SUPERCOILED PLASMID DNA

A covalently closed, circular, supercoiled plasmid was exposed to singlet oxygen by a separated-surface sensitizer. For each exposure, the quantity of single oxygen entering the DNA target solution was estimated by its oxidation of histidine. After singlet oxygen exposure, some DNA samples were treated to disclose occult lesions. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed circular and linear species, and all bands were quantitated fluorometrically. Exposure of supercoiled plasmid DNA to singlet oxygen induced frank DNA strand breaks, alkali-labile sites (pH 12.5, 90 degrees C, 30 min), and piperidine-labile sites (0.4 M, 60 degrees C, 30 min), all in a dose-dependent manner. Yields of alkali-labile and piperidine-labile sites ranged from one to four times the frank strand break yield. Replacement of buffered H2O by buffered D2O as the DNA solvent for singlet oxygen exposures increased DNA lesion yields by a factor of 2.6 (averaged over lesion classes). Our data for the detection of frank strand breaks is at variance with published results from studies in which singlet oxygen was derived from a thermolabile endoperoxide dissolved in the DNA solution.