Peroxidase deficiency of nickel-transformed hamster cells correlates with their increased resistance to cytotoxicity of peroxides

Using a procedure aimed at isolation of genes that are inactivated during nickel-induced carcinogenesis in Chinese hamster cells, a homolog of genes encoding human and mouse heme containing peroxidases has been cloned. Northern blot analysis of normal cultured fibroblasts and two nickel-transformed cell lines confirmed that this gene was expressed in normal but not in transformed cells. Nickel-transformed cells also tested negative for peroxidase activity using a sensitive fluorescence assay. Cultured embryo cells or fibroblasts that express peroxidase activity and their nickel-transformed peroxidase-deficient counterparts were employed to investigate the role of peroxidase-catalyzed processes in cytotoxicity induced by tert-butyl hydroperoxide or cumene hydroperoxide. It has been found that peroxidase-deficient cells were significantly more resistant to cytotoxic effect of these compounds suggesting that cytotoxic effect of hydroperoxides may be mediated in part by free radicals generated in the course of peroxidase-catalyzed reactions.     

Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins

Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10-25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at alpha-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.     

Heme spin states and peroxide-induced radical species in prostaglandin H synthase

We have examined the optical, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectra of pure ovine prostaglandin H synthase in its resting (ferric) and ferrous states and after addition of hydrogen peroxide or 15-hydroperoxyeicosatetraenoic acid. In resting synthase, the distribution of heme between high- and low-spin forms was temperature-dependent: 20% of the heme was low-spin at room temperature whereas 50% was low-spin at 12 K. Two histidine residues were coordinated to the heme iron in the low-spin species. Anaerobic reduction of the synthase with dithionite produced a high-spin ferrous species that had no EPR signals. Upon reaction with the resting synthase, both hydroperoxides quickly generated intense (20-40% of the synthase heme) and complex EPR signals around g = 2 that were accompanied by corresponding decreases in the intensity of the signals from ferric heme at g = 3 and g = 6. The signal generated by HOOH had a doublet at g = 2.003, split by 22 G, superimposed on a broad component with a peak at g = 2.085 and a trough at g = 1.95. The lipid hydroperoxide generated a singlet at g = 2.003, with a linewidth of 25 G, superimposed on a broad background with a peak at g = 2.095 and a trough around g = 1.9. These EPR signals induced by hydroperoxide may reflect synthase heme in the ferryl state complexed with a free radical derived from hydroperoxide or fragments of hydroperoxide.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

Lipid oxidation in biological membranes. Electron transfer proteins as initiators of lipid autoxidation.

Biological lipid autoxidation has been studied in a model system composed of sonicated phospholipids as substrate and electron transfer proteins found in membranes as possible catalysts. Heme compounds, flavoproteins, and iron-sulfur proteins were examined for their ability to initiate lipid autoxidation. Among many heme compounds tested, the most active were hematin ?microperoxidase ? methemoglobin > cytochrome c. With fresh preparations of phospholipids, reaction rates (nanomoles of oxygen/minute nanomoles of heme) ranged from 5 (cytochrome c) to 350 (hematin). Only the oxidized heme compounds were active as catalysts. Reduced heme compounds, flavoproteins and riboflavin were inactive. In the presence of heme compounds, aged preparations of sonicated phospholipids were much more rapidly oxidized than fresh preparations. They also had a higher content of fatty acid hydroperoxides as judged from their characteristic diene absorption peak at 234 nm. This observation agrees with the postulated mechanism of lipid autoxidation by heme compounds, namely, homolytic scission of preformed fatty acid hydroperoxides. Iron-sulfur proteins were also active as initiators of lipid autoxidation when destabilized in the presence of an appropriate iron chelator (o-phenanthroline or 2,2′-bipyridine) or a chaotropic ion. Oxygen uptake rates (nanomoles of oxygen/minute × milligrams of protein) varied from about 200 for an iron-sulfur protein isolated from complex I to about 5500 for Clostridium pasteurianum ferredoxin. However, per nanomole of labile sulfide, the rates for all active iron-sulfur proteins were 4–7 nmol of oxygen/min × nmol of labile sulfide.Superoxide-generating systems did not initiate lipid autoxidation, nor did erythrocuprein inhibit the autoxidations induced by heme compounds or ferredoxin. However, lipid oxidations induced by two other iron-sulfur proteins were partially inhibited by erythrocuprein. It is concluded that in the above system Superoxide anion is neither an initiator nor an obligatory intermediate of lipid autoxidation.     

Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis

High levels of NaCN (20 to 250 mM) were required to inhibit cyclooxygenase catalysis and cause extended lag periods (up to 1.6 min), whereas CO failed to inhibit catalysis. This NaCN inhibition was easily overcome by endogenous or exogenous hydroperoxides. Added hydroperoxides acted to eliminate lag periods without undergoing net conversion to other chemical species. In addition, experiments with glutathione peroxidase inhibition showed that hydroperoxides were essential not only in the early phases, but throughout catalysis. In spectrophotometric experiments, NaCN formed a complex with ferriheme cyclooxygenase (Kd = 1.3 mM) and inhibited hydroperoxide interaction with this form of the enzyme. Phenolic antioxidants, only slightly extended lag periods while inhibiting oxygenation rates more than 50%. Low levels of phenol (which is normally stimulatory) or alpha-naphthol when combined with NaCN or glutathione peroxidase (agents which interfere with peroxide activation) resulted in potent synergistic inhibition with long lag times. A mechanism consistent with all of the above properties of cyclooxygenase has been elucidated, Further mechanistic explanation was sought for reaction-catalyzed self-inactivation of cyclooxygenase. This phenomenon could not be explained simply by heme lability, or cyclooxygenase sensitivity to destruction by ambient hydroperoxides, Rather, it appears to involve a destructive reaction intermediate intrinsic to involve a destructive reaction intermediate intrinsic to the cyclooxygenase mechanism.     

Mitochondria induce oxidative stress, generation of reactive oxygen species and redox state unbalance of the eye lens leading to human cataract formation: disruption of redox lens organization by phospholipid hydroperoxides as a common basis for cataract disease

The aging eye appears to be at considerable risk from oxidative stress. Lipid peroxidation (LPO) is one of the mechanisms of cataractogenesis, initiated by enhanced promotion of oxygen free radicals in the eye fluids and tissues and impaired enzymatic and non-enzymatic antioxidant defenses of the crystalline lens. The present study proposes that mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian and human lens epithelial cells and that therapies that protect mitochondria in lens epithelial cells from damage and reduce damaging ROS generation may potentially ameliorate the effects of free radical-induced oxidation that occur in aging ocular tissues and in human cataract diseases. It has been found that rather than complete removal of oxidants by the high levels of protective enzyme activities such as superoxide dismutase (SOD), catalase, lipid peroxidases in transparent lenses, the lens conversely, possess a balance between peroxidants and antioxidants in a way that normal lens tends to generate oxidants diffusing from lenticular tissues, shifting the redox status of the lens to become more oxidizing during both morphogenesis and aging. Release of the oxidants (O(2)(-)·, H(2)O(2) , OH·, and lipid hydroperoxides) by the intact lenses in the absence of respiratory inhibitors indicates that these metabolites are normal physiological products inversely related to the lens life-span potential (maturity of cataract) generated through the metal-ion catalyzed redox-coupled pro-oxidant activation of the lens reductants (ascorbic acid, glutathione). The membrane-bound phospholipid (PL) hydroperoxides escape detoxification by the lens enzymatic reduction. The lens cells containing these species would be vulnerable to peroxidative attack which trigger the PL hydroperoxide-dependent chain propagation of LPO and other damages in membrane (lipid and protein alterations). The increased concentrations of primary LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moiety of the aqueous humor samples obtained from patients with cataract as compared to normal donors. Since LPO is clinically important in many of the pathological effects and aging, new therapeutic modalities, such as patented N-acetylcarnosine prodrug lubricant eye drops, should treat the incessant infliction of damage to the lens cells and biomolecules by reactive lipid peroxides and oxygen species and "refashion" the affected lens membranes in the lack of important metabolic detoxification of PL peroxides. Combined in ophthalmic formulations with N-acetylcarnosine, mitochondria-targeted antioxidants are promising to become investigated as a potential tool for treating a number of ROS-related ocular diseases, including human cataracts.     

Heme binding and polymerization by Plasmodium falciparum histidine rich protein II: influence of pH on activity and conformation.

The histidine rich protein II (HRPII) from Plasmodium falciparum has been implicated as a heme polymerase which detoxifies free heme by its polymerization to inactive hemozoin. Histidine-iron center coordination is the dominant mechanism of interaction between the amino acid and heme. The protein also contains aspartate allowing for ionic/coordination interactions between the carboxylate side chain and the heme metal center. The pH profile of heme binding and polymerization shows the possibility of these two types of binding sites being differentiated by pH. Circular dichroism studies of the protein show that pH and heme binding cause a change in conformation above pH 6 implying the involvement of His-His+ transitions. Heme binding at pHs above 6 perturbs HRPII conformation, causing an increase in helicity.     

Heme inhibits the DNA binding properties of the cytoplasmic heme binding protein of Shigella dysenteriae (ShuS)

Heme uptake and utilization by pathogenic bacteria are critical for virulence and disease, since heme and heme proteins are a major source of iron within the host. Although the role of outer membrane heme receptors in this process has been extensively characterized at the genetic and biochemical level, the role of the cytoplasmic heme binding proteins is not yet clear. The Shigella dysenteriae cytoplasmic heme binding protein, ShuS, has previously been shown to promote utilization of heme as an iron source at low to moderate heme concentrations and to protect against heme toxicity at high heme concentrations. Herein, we provide evidence that ShuS of S. dysenteriae sequesters DNA non-sequence-specifically with a binding affinity of 3.6 microM as determined by fluorescence anisotropy studies. The ability to bind DNA was observed to be restricted to the apoprotein only. The molecular mass of the apo-ShuS-DNA complex was estimated to be approximately 700 kDa by size exclusion chromatography. Atomic force microscopy (AFM) revealed that apo-ShuS forms aggregates in the presence of DNA and provides a scaffolding matrix from which DNA is observed to loop outward. The AFM images of apo-ShuS-DNA complexes were strikingly similar to the AFM images of the stress-induced Escherichia coli protein, Dps, when complexed with DNA; however, unlike the Dps protein, ShuS failed to protect DNA against oxidative stress in vitro and in vivo. Since free heme can generate reactive oxygen species which are damaging to cellular DNA, the ability of ShuS to physically sequester DNA may provide a molecular basis for its role in preventing toxicity associated with high heme concentrations.     

Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangement reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from beta-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after beta-scission of an initial alkoxyl radical.     

Radiation-induced peroxidation of small unilamellar vesicles of phosphatidylcholine generated by sonication

The present study was aimed at determining the peroxidation of model membranes constituted of liposomes of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) submitted to hydroxyl free radicals (generated by gamma-radiolysis) attack. Liposomes of PLPC were prepared using the sonication technique, and dynamic light-scattering (DLS) measurements allowed characterization of the liposomal dispersions. Irradiation damages in sonication-generated liposomes were assessed by monitoring several oxidation products, such as conjugated dienes (by means of UV--visible spectrophotometry) and hydroperoxides (using reverse phase high-performance liquid chromatography (HPLC) associated with chemiluminescence detection). It has been shown that three different families of hydroperoxides are formed: the first one (at low radiation doses) results from HO. attack on the linoleyl chain of PLPC, giving phosphatidylcholine hydroperoxides possessing a conjugated dienic structure; the two others (at high radiation doses) are obtained by the secondary HO. attack on the primary hydroperoxide family. The quantification of these products associated with the comparison of their radiation-dose-dependent formation has provided valuable information concerning the mechanisms of their formation. Analysis by HPLC -- mass spectrometry has confirmed the presence of hydroperoxides and underlined various other products, like chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl chain residues. Structural assignment proposals of some oxidation products have been proposed.     

Hemopexin in the human retina: Protection of the retina against heme-mediated toxicity

The existence of the blood-retinal barrier means that proteins that protect the retina from damage by reactive oxygen species must either be made locally or specifically transported across the barrier cells; however, such transepithelial transport does not seem to occur. Among the circulatory proteins that protect against iron-catalyzed production of free radicals are apo-transferrin, which binds ferric iron and has previously been shown to be made by cells of the neural retina (Davis and Hunt, 1993, J. Cell Physiol., 156:280–285), and the extracellular antioxidant, apo-hemopexin, which binds free heme (iron-protoporphyrin IX). Since hemorrhage and heme release can be important contributing factors in retinal disease, evidence of a hemopexin-based retinal protection system was sought. The human retina has been shown to contain apo-hemopexin which is probably synthesized locally since its mRNA can be detected in retinal tissue dissected from human donor eyes. It is likely that the retina contains a mechanism for the degradation of hemopexin-bound heme since the blood-retinal barrier also precludes the exit of heme-hemopexin from the retina. Retinal pigment epithelial cells have been found to bind and internalize heme-hemopexin in a temperature-dependent, saturable, and specific manner, analogous to the receptor-mediated endocytic system of hepatoma cells. Moreover, the binding of heme-hemopexin to the cells stimulates the expression of heme oxygenase-1, metallothionein-1, and ferritin. © 1996 Wiley-Liss, Inc.     

Heme inhibits human neutrophil apoptosis: involvement of phosphoinositide 3-kinase, MAPK, and NF-kappaB

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1-50 microM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-kappa B translocation reversed heme antiapoptotic effect. NF-kappa B (p65 subunit) nuclear translocation and I kappa B degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.     

Inhibition of heme sequestration of histidine‐rich protein 2 using multiple epitope‐targeted peptides

Plasmodium falciparum is the most lethal species of malaria. In infected human red blood cells, P. falciparum digests hemoglobin as a nutrient source, liberating cytotoxic free heme in the process. Sequestration and subsequent conversion of this byproduct into hemozoin, an inert biocrystalline heme aggregate, plays a key role in parasite survival. Hemozoin has been a longstanding target of antimalarials such as chloroquine (CQ), which inhibit the biocrystallization of free heme. In this study, we explore heme‐binding interactions with histidine‐rich‐protein 2 (HRP2), a known malarial biomarker and purported player in free heme sequestration. HRP2 is notoriously challenging to target due to its highly repetitious sequence and irregular secondary structure. We started with three protein‐catalyzed capture agents (PCCs) developed against epitopes of HRP2, inclusive of heme‐binding motifs, and explored their ability to inhibit heme:HRP2 complex formation. Cocktails of the individual PCCs exhibit an inhibitory potency similar to CQ, while a covalently linked structure built from two separate PCCs provided considerably increased inhibition relative to CQ. Epitope‐targeted disruption of heme:HRP2 binding is a novel approach towards disrupting P. falciparum‐related hemozoin formation.     

Bio-markers of lipid peroxidation in vivo: hydroxyoctadecadienoic acid and hydroxycholesterol

The biological role of lipid peroxidation products has continued to receive a great deal of attention not only for the elucidation of pathological mechanisms but also for their practical application to clinical use as bio-markers. In the last fifty years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. In this decade, F2-isoprostanes from arachidonates and neuroprostanes from docosahexanoates have been proposed as bio-markers. Although these markers are formed by a free radical-mediated oxidation, the yields from the parent lipids are minimal. Compared to these markers, hydroperoxy octadecadienoates (HPODE) from linoleates and oxysterols from cholesterols are yielded by much simpler mechanisms from more abundant parent lipids in vivo. Recently, the method in which both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as total hydroxyoctadecadienoic acid (tHODE) and 7-hydroxycholesterol (t7-OHCh), respectively, was proposed. The concentrations of tHODE and t7-OHCh determined by GC-MS analysis from physiological samples were much higher than that of 8-iso-prostagrandin F(2alpha). In addition to this advantage, hydrogen-donor activity of antioxidants in vivo could be determined by the isomeric-ratio of HODE (9- and 13-(Z,E)-HODE/9- and 13-(E,E)-HODE).     

Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme.

An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric-biliverdin IX alpha in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH-cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH-cytochrome P450 reductase is greatly retarded after the oxy-heme complex formation. The optical absorption spectra of the heme-enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue-shifted and relatively strong CT bands of the high-spin component in the ferric form spectrum. The heme-enzyme complex shows the acid-base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, H mu O.     

Interactions of plasmalogens and their diacyl analogs with singlet oxygen in selected model systems

Plasmalogens are phospholipids containing a vinyl-ether linkage at the sn-1 position of the glycerophospholipid backbone. Despite being quite abundant in humans, the biological role of plasmalogens remains speculative. It has been postulated that plasmalogens are physiological antioxidants with the vinyl-ether functionality serving as a sacrificial trap for free radicals and singlet oxygen. However, no quantitative data on the efficiency of plasmalogens at scavenging these reactive species are available. In this study, rate constants of quenching of singlet oxygen, generated by photosensitized energy transfer, by several plasmalogens and, for comparison, by their diacyl analogs were determined by time-resolved detection of phosphorescence at 1270nm. Relative rates of the interactions of singlet oxygen with plasmalogens and other lipids, in solution and in liposomal membranes, were measured by electron paramagnetic resonance oximetry and product analysis using HPLC-EC detection of cholesterol hydroperoxides and iodometric assay of lipid hydroperoxides. The results show that singlet oxygen interacts with plasmalogens significantly faster than with the other lipids, with the corresponding rate constants being 1 to 2 orders of magnitude greater. The quenching of singlet oxygen by plasmalogens is mostly reactive in nature and results from its preferential interaction with the vinyl-ether bond. The data suggest that plasmalogens could protect unsaturated membrane lipids against oxidation induced by singlet oxygen, providing that the oxidation products are not excessively cytotoxic.     

Chemiluminescent assay of lipid hydroperoxides

The addition of luminol plus a catalyst such as peroxidase or a heme prosthetic group to a solution containing a small quantity of lipid hydroperoxides results in a flash of chemiluminescence, the intensity of which is a function of the hydroperoxide concentrations. Various protocols for lipid hydroperoxide assays have been described and we have studied conditions to increase their sensitivity and specificity. Plasma lipid hydroperoxide determinations require an extraction, since compounds present in plasma interfere with light emission. Moreover, the sensitivity of the assay is by the presence of hydrogen peroxide in the medium, which causes high background values. Catalase does not act on lipid hydroperoxides and can be used to eliminate hydrogen peroxide from the reaction medium. The determination requires a blank tube in which hydroperoxides are destroyed by incubating the sample with haematin plus ascorbate. The increase in the chemiluminescence of the assay tube caused by the presence of lipid hydroperoxides is then compared to the value obtained for an internal standard.     

Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy

Low-temperature electron paramagnetic resonance (EPR) spectroscopy, circular dichroism and two-component redox titration have previously provided evidence for two different ascorbate-reducible heme centers in cytochrome b(561) present in chromaffin granule membranes. These species have now been observed by room and liquid nitrogen temperature absorption spectroscopy. The visualization of these heme centers becomes possible as a consequence of utilizing chromaffin granule membranes prepared by a mild procedure. Additionally, a new redox center, not reducible by ascorbate, was discovered by both EPR and absorption spectroscopy. It constitutes about 15% of the heme absorbance of chromaffin membranes at 561 nm and has EPR characteristics of a well-organized highly axial low-spin heme center (thus making it unlikely that it is a denatured species). This species is either an alternative form of one of the hemes of cytochrome b(561) that has a very low redox potential or a b-type cytochrome distinct from b(561).