Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells

It is well known that hydrogen peroxide (H2O2)-induced copper-catalyzed fragmentation of proteins follows a site-specific oxidative mechanism mediated by hydroxyl radical-like species (i.e. Cu(I)O, Cu(II)/*OH or Cu(III)) that ends in increased carbonyl formation and protein fragmentation. We have found that the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) prevented such processes by trapping human serum albumin (HSA)-centered radicals, in situ and in real time, before they reacted with oxygen. When (bi)carbonate (CO2, H2CO3, HCO3- and CO3(-2)) was added to the reaction mixture, it blocked fragmentation mediated by hydroxyl radical-like species but enhanced DMPO-trappable radical sites in HSA. In the past, this effect would have been explained by oxidation of (bi)carbonate to a carbonate radical anion (CO3*) by a bound hydroxyl radical-like species. We now propose that the CO3* radical is formed by the reduction of HOOCO2- (a complex of H2O2 with CO2) by the protein-Cu(I) complex. CO3* diffuses and produces more DMPO-trappable radical sites but does not fragment HSA. We were also able, for the first time, to detect discrete but highly specific H2O2-induced copper-catalyzed CO3*-mediated induction of DMPO-trappable protein radicals in functioning RAW 264.7 macrophages. We conclude that carbon dioxide modulates H2O2-induced copper-catalyzed oxidative damage to proteins by preventing site-specific fragmentation and enhancing DMPO-trappable protein radicals in functioning cells. The pathophysiological significance of our findings is discussed.     

Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II

Electron spin resonance spectroscopy and liquid chromatography have been used to detect radical formation and fragmentation of polypeptides during photoinhibition of purified major antenna proteins, free of protease contaminants. In the absence of oxygen and light, no radicals were observed and there was no damage to the proteins. Similarly illumination of the apoproteins did not induce any polypeptide fragmentation, suggesting that chlorophyll, light and atmospheric oxygen are all participating in antenna degradation. The use of TEMP and DMPO as spin traps showed that protein damage initiates with generation of (1)O(2), presumably from a triplet chlorophyll, acting as a Type II photosensitizer which attacks directly the amino acids causing a complete degradation of protein into small fragments, without the contribution of proteases. Through the use of scavengers, it was shown that superoxide and H(2)O(2) were not involved initially in the reaction mechanism. A higher production of radicals was observed in trimers than in monomeric antenna, while radical production is strongly reduced when antennae were organized in the photosystem II (PSII) complex. Thus, monomerization of antennae as well as their incorporation into the PSII complex seem to represent physiologically protected forms. A comparison is made of the photoinhibition mechanisms of different photosynthetic systems.     

Mechanism of hydrogen peroxide-induced Cu,Zn-superoxide dismutase-centered radical formation as explored by immuno-spin trapping: the role of copper- and carbonate radical anion-mediated oxidations

We have reinvestigated the biochemistry of H2O2-induced Cu,Zn-superoxide dismutase (SOD1)-centered radicals, detecting them by immuno-spin trapping. These radicals are involved in H2O2-induced structural and functional damage to SOD1, and their mechanism of generation depends on copper and/or (bi)carbonate (i.e., CO2, CO3(-2), or HCO3-). First, in the absence of DTPA and (bi)carbonate, Cu(II) was partially released and rebound at His, Cys, and Tyr residues in SOD1 with the generation of protein-copper-bound oxidants outside the SOD1 active site by reaction with excess H2O2. These species produced immuno-spin trapping-detectable SOD1-centered radicals associated with H2O2-induced active site ( approximately 5 and approximately 10 kDa fragments) and non-active site (smearing between 3 and 16 kDa) copper-dependent backbone oxidations and subsequent fragmentation of SOD1. Second, in the presence of DTPA, which inhibits H2O2-induced SOD1 non-active site fragmentation, (bi)carbonate scavenged the enzyme-bound oxidant at the SOD1 active site to produce the carbonate radical anion, CO3*-, thus protecting against active site SOD1 fragmentation. CO3*- diffuses and produces side chain oxidations forming DMPO-trappable radical sites outside the enzyme active site. Both mechanisms for generating immuno-spin trapping-detectable SOD1-centered radicals were susceptible to inhibition by cyanide and enhanced at high pH values. In addition, (bi)carbonate enhanced H2O2-induced SOD1 turnover as demonstrated by an enhancement in oxygen evolution and SOD1 inactivation. These results help clarify the free radical chemistry involved in the functional and structural oxidative damage to SOD1 by H2O2 with the intermediacy of copper- and CO3*--mediated oxidations.     

Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu,Zn-superoxide dismutase modification

Carnosine (beta-alanyl-L-histidine), homocarnosine (gamma-amino-butyryl-L-histidine) and anserine (beta-alanyl-1-methyl-L-histidine) have been proposed to act as anti-oxidants in vivo. The protective effects of carnosine and related compounds against the oxidative damage of human Cu,Zn-superoxide dismutase (SOD) by peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation, which is associated with the inactivation of enzyme. Carnosine, homocarnosine and anserine significantly inhibited the fragmentation and inactivation of Cu,Zn-SOD by AAPH. All three compounds also inhibited the release of copper ions from the enzyme and the formation of carbonyl compounds in AAPH-treated Cu,Zn-SOD. These compounds inhibited the fragmentation of other protein without copper ion. The results suggest that carnosine and related compounds act as the copper chelator and peroxyl radical scavenger to protect the protein fragmentation. Oxidation of amino acid residues in Cu,Zn-SOD induced by AAPH were significantly inhibited by carnosine and related compounds. It is proposed that carnosine and related dipeptides might be explored as potential therapeutic agents for pathologies that involve Cu,Zn-SOD modification mediated by peroxyl radicals.     

Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II

Electron spin resonance spectroscopy and liquid chromatography have been used to detect radical formation and fragmentation of polypeptides during photoinhibition of purified major antenna proteins, free of protease contaminants. In the absence of oxygen and light, no radicals were observed and there was no damage to the proteins. Similarly illumination of the apoproteins did not induce any polypeptide fragmentation, suggesting that chlorophyll, light and atmospheric oxygen are all participating in antenna degradation. The use of TEMP and DMPO as spin traps showed that protein damage initiates with generation of ¹O₂, presumably from a triplet chlorophyll, acting as a Type II photosensitizer which attacks directly the amino acids causing a complete degradation of protein into small fragments, without the contribution of proteases. Through the use of scavengers, it was shown that superoxide and H₂O₂ were not involved initially in the reaction mechanism. A higher production of radicals was observed in trimers than in monomeric antenna, while radical production is strongly reduced when antennae were organized in the photosystem II (PSII) complex. Thus, monomerization of antennae as well as their incorporation into the PSII complex seem to represent physiologically protected forms. A comparison is made of the photoinhibition mechanisms of different photosynthetic systems.     

Free radical-mediated degradation of proteins: the protective and deleterious effects of membranes

Lipid membranes have been shown to scavenge free radicals generated by various means. However, under oxidative conditions, unsaturated lipids within membranes can produce damaging free radicals. We have determined the relative importance of these two conflicting properties of lipid membranes with the use of liposomal membrane studies. (1) Liposome membranes can protect extra-liposomal albumin from free radicals derived from sources other than peroxidizing lipid. When albumin or copper (an essential component of the free radical generating systems used) were encapsulated, protein damage was further reduced. (2) Using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) we demonstrate that the exposure of albumin to peroxidizing liposome membranes results in both cross-linking and degradation. Our results indicate that protein damage is substantially less than in the case of other biologically relevant free radical generating systems. We discuss our findings with respect to membrane function and the in vivo exposure of cells to free radicals.     

Free radical scavenging and copper chelation: a potentially beneficial action of captopril

Captopril (CpSH), an angiotensin converting enzyme (ACE) inhibitor, is reported to provide protection against free-radical mediated damage. The purpose of this study was to investigate, by means of pulse radiolysis technique, the behaviour of CpSH towards radiation-induced radicals in the absence and in the presence of copper(II) ions, which can play a relevant role in the metal catalysed generation of reactive oxygen species. The results indicate that the -SH group is crucial in determining the radical scavenging action of CpSH and the nature of the resulting CpSH transient products in the absence or in the presence of oxygen.

In the presence of Cu(II), the -SH group is still involved in the biological action of the molecule participating both in the one-electron reduction of Cu(II) with formation of CpSSCp, and in Cu(I) chelation. This conclusion is supported by the Raman spectroscopic data which allow to identify the CpSH sites involved in the copper complex at different pH.

These results suggest that CpSH may potentially inhibit oxidative damage both through free radical scavenging and metal chelation. Considering the low CpSH concentration in vivo, the metal chelation mechanism, more than the direct radical scavenging, could play the major role in moderating the toxicological effects of free radicals.     

Salsolinol, a tetrahydroisoquinoline catechol neurotoxin, induces human Cu,Zn-superoxidie dismutase modificaiton

The endogenous neurotoxin, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), has been considered a potential causative factor for the pathogenesis of Parkinsonos disease (PD). In the present study, we examined the pattern of human Cu,Zn-superoxide dismutase (SOD) modification elicited by salsolinol. When Cu,Zn-SOD was incubated with salsolinol, some protein fragmentation and some higher molecular weight aggregates were occurred. Salsolinol led to inactivation of Cu,Zn-SOD in a concentration-dependent manner. Free radical scavengers and catalase inhibited the salsolinolmediated Cu,Zn-SOD modificaiton. Exposure of Cu,Zn-SOD to salsolinol led also to the generation of protein carbonyl compounds. The deoxyribose assay showed that hydroxyl radicals were generated during the oxidation of salsolinol in the presence of Cu,Zn-SOD. Therefore, the results indicate that free radical may play a role in the modification and inactivation of Cu,Zn-SOD by salsolinol.     

The role of reactive N-bromo species and radical intermediates in hypobromous acid-induced protein oxidation

Activated eosinophils, and hypobromous acid (HOBr) generated by these cells, have been implicated in the tissue injury in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but limited information is available on the mechanisms of damage and intermediates formed. Reaction of HOBr with proteins is shown to result in the formation of bromamines and bromamides, from side-chain and backbone amines and amides, and 3-bromo- and 3,5-dibromo-Tyr, from Tyr residues; these materials account for ca. 70% of the oxidant consumed. Protein carbonyls, dityrosine, and 3,4-dihydroxyphenylalanine are also formed, though these are minor products (<5% of HOBr added). With BSA, extensive (selective and nonspecific) protein fragmentation and limited aggregation are also observed. The bromamines/bromamides are unstable and induce further oxidation and free radical formation as detected by EPR spin trapping. Evidence was obtained for the generation of nitrogen-centered radicals on side-chain and backbone amide groups of amino acids, peptides, and proteins. These radicals readily undergo rearrangement reactions to give carbon-centered radicals. With proteins, alpha-carbon (backbone) radicals are detected, which may play a role in protein fragmentation. A novel damage transfer pathway from Gln side-chain amide groups to backbone sites was also observed.     

Free radical scavenging and copper chelation: A potentially beneficial action of captopril

Captopril (CpSH), an angiotensin converting enzyme (ACE) inhibitor, is reported to provide protection against free-radical mediated damage. The purpose of this study was to investigate, by means of pulse radiolysis technique, the behaviour of CpSH towards radiation-induced radicals in the absence and in the presence of copper(II) ions, which can play a relevant role in the metal catalysed generation of reactive oxygen species. The results indicate that the -SH group is crucial in determining the radical scavenging action of CpSH and the nature of the resulting CpSH transient products in the absence or in the presence of oxygen.

In the presence of Cu(II), the -SH group is still involved in the biological action of the molecule participating both in the one-electron reduction of Cu(II) with formation of CpSSCp, and in Cu(I) chelation. This conclusion is supported by the Raman spectroscopic data which allow to identify the CpSH sites involved in the copper complex at different pH.

These results suggest that CpSH may potentially inhibit oxidative damage both through free radical scavenging and metal chelation. Considering the low CpSH concentration in vivo, the metal chelation mechanism, more than the direct radical scavenging, could play the major role in moderating the toxicological effects of free radicals.     

Tetracycline derivatives and ceftriaxone, a cephalosporin antibiotic, protect neurons against apoptosis induced by ionizing radiation

DNA damage induced by low doses of ionizing radiation causes apoptosis, which is partially mediated via the generation of free radicals. Both free radicals and apoptosis are involved in the majority of brain diseases, including stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Because previous studies have shown that tetracycline derivatives doxycycline and minocycline have anti-inflammatory effects and are protective against brain ischemia, we studied whether minocycline and doxycycline or ceftriaxone, a cephalosporin antibiotic with the potential to inhibit excitotoxicity, protect neurons against ionizing radiation in primary cortical cultures. A single dose of 1 Gy significantly increased lactate dehydrogenase release, induced DNA fragmentation in neurons and triggered microglial proliferation. Treatment with minocycline (20 nM), doxycycline (20 nM) and ceftriaxone (1 microM) significantly reduced irradiation-induced lactate dehydrogenase release and DNA fragmentation. The most efficient protection was achieved by minocycline treatment, which also inhibited the irradiation-induced increase in microglial cell number. Our results suggest that some tetracycline derivatives, such as doxycycline and minocycline, and ceftriaxone, a cephalosporin derivative, protect neurons against apoptotic death.     

Mechanism of DNA strand breakage induced by photosensitized tetracycline-Cu(II) complex

Tetracyclines (TCs) in combination with Cu(II) ions exhibited significant DNA damaging potential vis a vis tetracyclines per se. Interaction of tetracyclines with DNA resulted in alkylation at N-7 and N-3 positions of adenine and guanine bases, and caused destabilization of DNA secondary structure. Significant release of acid-soluble nucleotides from tetracycline-modified DNA upon incubation with S(1) nuclease ascertained the formation of single stranded regions in the DNA. Also, the treatment of tetracycline-modified DNA with 0.1 and 0.5M NaOH resulted in 62 and 76% hydrolysis compared to untreated control. Comparative alkaline hydrolysis of DNA modified with tetracycline derivatives showed differential DNA damaging ability in the order as DOTC > DMTC > TC > OTC > CTC. Addition of Cu(II) invariably augmented the extent of tetracycline-induced DNA damage. The alkaline unwinding assay clearly demonstrated the formation of approximately six strand breaks per unit DNA at 1:10 DNA nucleotide/TC molar ratio in the presence of 0.1mM Cu(II) ions. At a similar Cu(II) concentration, a progressive transformation of covalently closed circular (CCC) (form-I) plasmid pBR322 DNA to forms-II and -III was noticed with increasing tetracycline concentrations. The results obtained with the free-radical quenchers viz. mannitol, thiourea, sodium benzoate and superoxide dismutase (SOD) suggested the involvement of reactive oxygen species in the DNA strand breakage. It is concluded that the tetracycline-Cu(II)-induced DNA damage occurs due to (i) significant binding of tetracycline and Cu(II) with DNA, (ii) methyl group transfer from tetracycline to the putative sites on nitrogenous bases, and (iii) metal ion catalyzed free-radical generation in close vicinity of DNA backbone upon tetracycline photosensitization. Albeit, the DNA alkylation and strand cleavage are repairable lesions, but any defect in the critical repair pathway may augment the damage accumulation and mutagenesis.     

Mechanism of site-specific DNA damage induced by methylhydrazines in the presence of copper(II) or manganese(III).

DNA damage induced by methylhydrazines (monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine) in the presence of metal ions was investigated by a DNA sequencing technique. 1,2-Dimethylhydrazine plus Mn(III) caused DNA cleavage at every nucleotide without marked site specificity. ESR-spin-trapping experiments showed that the hydroxyl free radical (.OH) is generated during the Mn(III)-catalyzed autoxidation of 1,2-dimethylhydrazine. DNA damage and .OH generation were inhibited by .OH scavengers and superoxide dismutase, but not by catalase. The results suggest that 1,2-dimethylhydrazine plus Mn(III) generates .OH, not via H2O2, and that .OH causes DNA damage. In the presence of Cu(II), DNA cleavage was caused by the three methylhydrazines frequently at thymine residues, especially of the GTC sequence. The order of Cu(II)-mediated DNA damage (1,2-dimethylhydrazine greater than monomethylhydrazine approximately 1,1-dimethylhydrazine) was not correlated with the order of methyl free radical (.CH3) generation during Cu(II)-catalyzed autoxidation (monomethylhydrazine greater than 1,1-dimethylhydrazine much greater than 1,2-dimethylhydrazine). Catalase and bathocuproine, a Cu(I)-specific chelating agent, inhibited DNA damage while catalase did not inhibit the .CH3 generation. The order of DNA damage was correlated with the order of ratio of H2O2 production to O2 consumption observed during Cu(II)-catalyzed autoxidation of methylhydrazines. These results suggest that the Cu(I)-peroxide complex rather than the .CH3 plays a more important role in methylhydrazine plus Cu(II)-induced DNA damage.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

Radical-Induced Damage to Bovine Serum Albumin: Role of the Cysteine Residue

The reactions of cerium(IV) and the hydroxyl radical [generated from iron(ii)/H₂O₂] with bovine serum albumin (BSA) have been investigated by EPR spin trapping. With the former reagent a protein-derived thiyl radical is selectively generated; this has been characterized via the anisotropic EPR spectra observed on reaction of this radical with the spin trap DMPO. Blocking of the thiol group results in the loss of this species and the detection of a peroxyl radical, believed to be formed by reaction of oxygen with initially-generated, but undetected, carbon-centred radicals from aromatic amino acids. Experiments with a second spin trap (DBNBS) confirm the formation of these carbon-centred species and suggest that damage can be transferred from the thiol group to carbon sites in the protein. A similar transfer pathway can be observed when hydroxyl radicals react with BSA.

Further experiments demonstrate that the reverse process can also occur: when hydroxyl radicals react with BSA, the thiol group appears to act as a radical sink and protects the protein from denaturation and fragmentation through the transfer of damage from a carbon site to the thiol group. Thiol-blocked BSA is shown to be more susceptible to damage than the native protein in both direct EPR experiments and enzyme digestion studies. Oxygen has a similar effect, with more rapid fragmentation detected in its presence than its absence.     

The effects of chelating agents on radical generation in alkaline peroxide systems, and the relevance to substrate damage

A spin-trapping EPR technique has been employed to explore the generation of hydroxyl radicals from reactions between a series of first row transition metal ions and aqueous hydrogen peroxide at pH 10, and with a range of chelating agents (EDTA, DTPMP and the readily biodegradable ligands S,S-EDDS and IDS). In the absence of these chelating agents only Cu(II) generates a significant level of hydroxyl radicals; in their presence with Cu(II) EDTA and IDS give similar behaviour whereas EDDS and DTPMP inhibit hydroxyl radical generation. For Fe(II), EDTA, DTPMP and IDS significantly enhance ^√OH production under these conditions whereas EDDS does not. Results from model cellulose damage experiments broadly confirm the findings for copper, though experiments with Fe(II) lead to somewhat contrasting results. Our findings are discussed in terms of binding constants and implications for alkaline peroxygen bleaching systems.     

Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1

Glycation, the nonenzymatic reaction between protein amino groups and reducing sugars, induces protein damage that has been linked to several pathological conditions, especially diabetes, and general aging. Here we describe the direct identification of a protein-bound free radical formed during early glycation of histone H1 in vitro. Earlier EPR analysis of thermal browning reactions between free amino acids and reducing sugars has implicated the sugar fragmentation product glycolaldehyde in the generation of a 1,4-disubstituted pyrazinium free radical cation. In order to evaluate the potential formation of this radical in vivo, the early glycation of BSA, lysozyme, and histone H1 by several sugars (D-glucose, D-ribose, ADP-ribose, glycolaldehyde) under conditions of physiological pH and temperature was examined by EPR. The pyrazinium free radical cation was identified on histone H1 glycated by glycolaldehyde (g = 2.00539, aN = 8.01 [2N], aH = 5.26 [4H], aH = 2.72 [4H]), or ADP-ribose. Reaction of glycoaldehyde with poly-L-lysine produced an identical signal, whereas reaction with BSA or lysozyme produced only a minor unresolved singlet signal. In the absence of oxygen the signal was stable over several days. Our results raise the possibility that pyrazinium radicals may form during glycation of histone H1 in vivo.