Effects of Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor and Human Parathyroid Hormone Fragment 13-34

No HeadingPurpose. The effects and mechanisms of different antioxidants, methionine, glutathione, acetylcysteine, and ascorbic acid (AscH₂), on the oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone fragment 13-34 (hPTH 13-34) by hydrogen peroxide (H₂O₂) were quantified and analyzed.Methods. The rates of oxidation of methionine residues in G-CSF were determined by peptide mapping analyses, and the oxidation of methionine residue in hPTH 13-34 was quantified by reverse-phase HPLC.Results. At pH 4.5, free methionine reduces, glutathione and acetylcysteine have no obvious effect on, and AscH₂ promotes the rates of oxidation of methionine residues in G-CSF. The H₂O₂-induced oxidation rate constants for free methionine, acetylcysteine, and glutathione at pH 4.5 were measured to be 32.07, 1.00, and 1.63 M^-1h^-1, respectively, while the oxidation rate constant for Met¹, the most readily oxidizable methionine residue in G-CSF, is 13.95 M^–1h^–1. Therefore, the different effects of free methionine, acetylcysteine, and glutathione on the rates of oxidation of methionine residues in G-CSF are consistent with their different reactivity toward oxidation by H₂O₂. By using hPTH 13-34, the effect of AscH₂ on the H₂O₂-induced oxidation of methionine residue was quantified, and the mechanisms involved were proposed. Because of the presence of trace transition metal ions in solution, at low concentrations, AscH₂ is prone to be a prooxidant, increasing the hydroxyl radical (OH) production rate via Fenton-type reactions. In addition to peroxide oxidation, these radicals lead to the degradation of hPTH 13-34 to smaller peptide fragments. At high concentrations, AscH₂ tends to act as an OH scavenger. EDTA inhibits OH production and thus eliminates the degradation of hPTH 13-34 by forming complexes with transition metal ions. However, the rate of oxidation of the methionine residue in hPTH 13-34 increases as the concentration of AscH₂ is increased from 0 to 200 mM, and the reason for this is still not clear.Conclusions. Our results demonstrate that free methionine is an effective antioxidant to protect G-CSF against methionine oxidation at pH 4.5. Acetylcysteine and glutathione are not effective antioxidants at pH 4.5. Their oxidation rates at different pH values imply that they would be much more effective antioxidants than free methionine at alkaline conditions. AscH₂ is a powerful electron donor. It acts as a prooxidant in the conditions in this study and is unlikely to prevent oxidation by H₂O₂ in protein formulation, whether or not EDTA is present.     

The Solid State Oxidation of Methionine Containing Peptide: A Preliminary Study Using Time of Flight Secondary Ion Mass Spectrometry

Purpose. A surface sensitive mass spectrometric technique: Time ofFlight Secondary Ion Mass Spectrometry (ToF-SIMS) was introducedto study the solid state instability of a methionine containing peptidecaused by the oxidation of the methionine residue. Methods. The oxidation of a neuropeptide Methinonine-Enkephalin(ME) in air and under UV acceleration was studied by ToF-SIMS. Results. The apparent oxidation rate is defined by the peak ratio ofoxidized molecular ion over unoxidized molecular ion. ME is oxidizedat a faster rate to its sulfoxide derivative in the UV accelerated oxidationenvironment than in lab air. The calibration curve for evaluating theionization probability ratio of the oxidized deprotonated molecular iondivided by the unoxidized deprotonated molecular ion was obtained.This could be used to extract the real oxidation rate of ME in thesolid state. Conclusions. The preliminary results showed that ToF-SIMS with simplesample handling, fast data acquisition, together with excellentsurface sensitivity and detection limit could be an applicable and convenienttool to study peptide reactions in the solid state such as oxidationand deamidation process.     

Effects of Excipients on the Hydrogen Peroxide-Induced Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor

No HeadingPurpose. The objective of this study was to elucidate the different mechanisms of action of different excipients on the oxidation of Met¹, Met¹²², Met¹²⁷, and Met¹³⁸ in granulocyte colony-stimulating factor (G-CSF) by using hydrogen peroxide as the oxidant.Methods. The oxidation of Met¹, Met¹²⁷, and Met¹³⁸ was quantified by peptide mapping analysis. The oxidation of Met¹²² has biphasic oxidation kinetics with a faster second phase. Therefore, the oxidation of Met¹²² was quantified by two different methods: peptide mapping analysis for the first phase of oxidation and direct reverse-phase HPLC for the second phase of oxidation.Results. The current work reveals that the preferential excluding excipients sorbitol, sucrose, and trehalose, in the concentration range 0–30% (w/v), and the preferential binding excipients urea and guanidine hydrochloride, in the concentration range 0–0.8 M, do not affect the oxidation of methionine residues in G-CSF at pH 4.5. The chelating agents citrate and EDTA have different effects on the rates of oxidation of methionine residues in G-CSF. At low concentrations, citrate decreases the rates, while at high concentrations, citrate increases the rates. EDTA decreases the rates of oxidation of methionine residues in G-CSF, such that its effect becomes more and more as its concentration is increased from 0 to 200 mM. The efficacy of EDTA on the rates of oxidation of the four methionine residues in G-CSF follows the order Met¹²² > Met¹²⁷ > Met¹³⁸ > Met¹.Conclusions. Our results indicate that EDTA can protect the methionine residues in G-CSF against oxidation induced by hydrogen peroxide. The more exposed the methionine residue is, the more difficult it is to be protected by EDTA. The mechanism may be due to the specific ion binding of EDTA to proteins.     

Effects of Polyaminocarboxylate Metal Chelators on Iron-thiolate Induced Oxidation of Methionine- and Histidine-Containing Peptides

Purpose. Site-specific protein oxidation induced by prooxidant/metal/ oxygen has been recognized as one of the major degradation pathways of protein pharmaceuticals. Polyaminocarboxylate (PAC) metal chelators are commonly employed to prevent metal-catalyzed oxidation, for they sequester metals. However, studies have indicated that iron chelates may still be catalytically active due to their specific coordination geometry. The purpose of this study was to investigate how PAC chelators affect prooxidant/metal/oxygen-catalyzed oxidation of peptides containing histidine (His) and methionine (Met). Methods. PACs were applied to a model oxidizing system, dithiothreitol/iron/oxygen, which was shown to promote the oxidation of Met to Met sulfoxide in the two model peptides, GGGMGGG and GHGMGGG. Results. PAC chelators did not suppress the peptide oxidation but significantly changed the product pattern. In particular, the yield of Met sulfoxide dropped significantly, while a number of other products emerged, including oxidation products from the N-terminus and His (if present). Overall, the oxidation became rather non-selective in the presence of PACs. The oxidation kinetics were significantly accelerated by nitrilotriacetate (NTA), ethylenediaminediacetate (HDDA), and ethylenediaminetetraacetate (EDTA), but they were slowed down by ethyl-enebis(oxyethylenenitrilo)tetraacetate (EGTA) and diethylenetriaminepentaacetate (DTPA). Meanwhile the PAC chelators were also observed to undergo degradation. Scavengers of hydrogen peroxide or hydroxyl radicals exerted only partial inhibition on the peptide oxidation. Conclusions. The results of this study are rationalized by the abilities of PAC chelators (i) to extract iron from potential binding sites of the peptides to impair site-specific oxidation, and (ii) to promote the formation of ROS different from the species formed at the peptide metal-binding sites.     

Oxidation of Human Insulin-like Growth Factor I in Formulation Studies: Kinetics of Methionine Oxidation in Aqueous Solution and in Solid State

Purpose. The aim of this work was to study the kinetics of oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I)¹ in aqueous solution and in the solid state by the aid of quantification of oxygen. Methods. The oxidized form of hIGF-I was characterized by tryptic peptide analysis, RP-HPLC and FAB-MS and quantified by RP-HPLC. The oxygen content was quantified polarographically by a Clark-type electrode. Results. Second-order kinetics with respect to amount of protein and dissolved oxygen was found to be appropriate for the oxidation of methionine in hIGF-I. The rate constants ranged from 1 to 280 M^–1 month^–l and had an activation energy of 95 (+/–4) kJ/mole. Light exposure, storage temperature and oxygen content were found to have a considerable impact on the oxidation rates. No significant difference in reaction rates was found for the oxidation of hIGF-I in aqueous solution or in the solid state. A method for decreasing the oxygen content in aqueous solution without purging is described. Conclusions. Polarographic quantification of dissolved oxygen makes it possible to establish the kinetics for oxidation of proteins. The oxidation of methionine in hIGF-I appears to follow second-order kinetics.     

Chemical Pathways of Peptide Degradation. VIII. Oxidation of Methionine in Small Model Peptides by Prooxidant/Transition Metal Ion Systems: Influence of Selective Scavengers for Reactive Oxygen Intermediates

In the presence of oxygen, Fe(III), and an appropriate electron donor (e.g. ascorbic acid, dithiothreitol), the oxidation of methionine residues to methionine sulfoxides in small model peptides can be induced. It is shown in this study that these oxidations can be retarded by catalase in a pH-dependent manner, by some hydroxyl radical scavengers, and by azide. In contrast, superoxide dismutase has only a minimal effect, indicating that the superoxide radical does not contribute significantly to the oxidation of the methionine residue. The experimental results can be interpreted by invoking hydrogen peroxide as the major oxidizing species at pH 7, whereas the involvement of free hydroxyl radicals seems to be negligible. Other reactive oxygen intermediates such as iron-bound hydroperoxy, or site-specifically generated reactive oxygen species may be actively involved in the oxidation of methionine residues at pH > 7.     

Oxidative Degradation of Antiflammin 2

Purpose. To study the oxidation of the methionine residue of antiflammin 2 (HDMNKVLDL, AF2) as a function of pH, buffer concentration, ionic strength, and temperature using different concentrations of hydrogen peroxide and to determine the accessibility of methionine residue to oxidation. Methods. Reversed-phase high-performance liquid chromatography (RPHPLC) was used as the main analytical method in determining the oxidation rates of AF2. Calibration curves for AF2 and the oxidation product, methionine sulfoxide of AF2 (Met(O)-3-AF2), were constructed for each measurement using standard materials. Fast Atom Bombardment Mass Spectroscopy (FABMS) was used to characterize the product. Results. Met(O)-3-AF2 was the only oxidation product detected at pH 3.0 to 8.0. The oxidation rates were independent of buffer concentrations, ionic strength, and pH from 3.0 to 7.0. However, there was an acceleration of the rates at basic pHs, and small amounts of degradation products other than Met(O)-3-AF2 were observed in this alkaline region. Conclusions. Oxidation of methionine in AF2 does not cause the biological inactivation reported by other laboratories since this drug is relatively stable under neutral conditions in the absence of oxiding agent.     

Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides

Aspartate and asparagine residues in polypeptides are subject to nonenzymatic reactions that lead to deamidation, isomerization, peptide bond cleavage and racemization. Much of this reactivity is due to the propensity for the initial formation of a cyclic succinimide intermediate. We have been interested in determining the effect of the side chains of neighboring histidine and cysteine residues in facilitating these reactions, particularly in the possibility that they can act as general acids and bases. In this study, we found little or no effect of histidine residues preceding an asparagine residue in hexapeptides derived from the sequence of adrenocorticotropic hormone, while a histidine residue preceding an aspartic acid residue was found to increase the rate of succinimide formation 8- to 11-fold. The presence of a histidine residue following either an asparagine or aspartic acid residue did not effect the rate of succinimide formation by peptide-bond nitrogen attack, but did increase the rate of the competing side-chain nitrogen attack leading to cleavage in the asparaginyl-containing peptide. We found that the effect of a cysteine residue following an asparagine or aspartic acid residue was in general similar to that of a serine residue, although the cleavage reaction appeared to be enhanced. These results suggest that His-Asp sequences may be particularly labile to spontaneous degradation in proteins and peptides, possibly owing to the ability of the histidine residue to facilitate succinimide formation by protonating the OH^? leaving group on the side chain carboxylic acid of the aspartic acid residue. Finally, we have also utilized these results, along with previously accumulated data on succinimide formation in related peptides, to correlate the rate of succinimide formation with the predicted acidity of the peptide bond nitrogen atom that is involved in the initial nucleophilic attack. © Munksgaard 1995.     

Oxidation of Recombinant Human Interleukin-2 by Potassium Peroxodisulfate

Purpose. The oxidation of recombinant human interleukin-2 (rhIL-2) by potassium peroxodisulfate (KPS) with or without N,N,N,N-tetramethylethylenediamine (TEMED), which are used for the preparation of dextran-based hydrogels, was investigated. Methods. The oxidation of (derivatives of) methionine, tryptophan, histidine and tyrosine, as well as rhIL-2 was investigated. Both the oxidation kinetics (RP-HPLC) and the nature of the oxidation products (mass spectrometry) were studied as a function of the KPS and TEMED concentration, and the presence of a competitive antioxidant, methionine. Results. Under conditions relevant for the preparation of rhIL-2 loaded hydrogels, only methionine and tryptophan derivatives were susceptible to oxidation by KPS. The oxidation of these compounds was inhibited once TEMED was present, suggesting that the peroxodisulfate anion, rather than the radicals formed in the presence of TEMED, is the oxidative species. KPS only induced oxidation of the four methionines present in rhIL-2, whereas the tryptophan residue remained unaffected. The radicals, formed after KPS decomposition by TEMED, induced some dimerization of rhIL-2. The oxidation of rhIL-2 could be substantially reduced by the addition of methionine, or by pre-incubation of KPS with TEMED. Conclusions. Only the methionine residues in rhIL-2 are oxidized by KPS. The extent of oxidation can be minimized by a proper selection of the reaction conditions.     

Isolation and Characterization of a Monomethioninesulfoxide Variant of Interferon α-2b

Purpose. To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon -2b. Methods. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. Results. The rHuIFN -2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN -2b molecule. The far-UV CD spectra showed a slight loss of -helical content and an increase in the -sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon -2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. Conclusions. A monomethioninesulfoxide rHuIFN -2b variant was found to be present in the rHuIFN -2b bulk drug substance in solution. The Met¹¹¹ residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met¹¹¹ residue did not seem to have a detectable effect on the biological activity of the molecule.     

Metal-Catalyzed Oxidation of Brain-Derived Neurotrophic Factor (BDNF): Analytical Challenges for the Identification of Modified Sites

Purpose. We examined the metal-catalyzed oxidation of brain-derivedneurotrophic factor (BDNF) using the Cu(II)/ascorbate/O₂ modeloxidative system. Methods. Electrospray ionization mass spectrometry, peptide mappingand amino acid analysis were utilized to determine the nature of thecovalent modification induced by the metal-catalyzed oxidative system.Additionally, analytical ultracentrifugation, the Bradford assay, circulardichroism and ANSA dye-binding were used to determine the natureof any conformational changes induced by the oxidation. Results. Exposure of BDNF to the Cu(II)/ascorbate/O₂ system led tothe modification of ca. 35% of Met⁹² to its sulfoxide, and to subsequentconformational changes. The proteolytic digestion procedure wassensitive to this conformational change, and was unable to detect themodification. Chemical digestion with CNBr, however, was not sensitive tothis change, and allowed for the identification of the site ofmodification. Conclusions. The modification of Met⁹² to its sulfoxide rendered theoxidized BDNF inaccessible to proteolytic digestion, due toconformational changes associated with the oxidation.     

The Kinetics of Relaxin Oxidation by Hydrogen Peroxide

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10–14 kcal mol^–1, with an entropy of activation of the order of –30 cal K^–1 mol^–1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.     

ANALOGS OF AMANIN

Iie³-amaninamide (3-R) and its diastereomeric sulfoxide (3-S) are obtained by oxidation of the bicyclic thioether peptide 2 by hydrogen peroxide in acetic acid. 2 was prepared by an intramolecular Savige-Fontana reaction of the linear octapeptide tert.-butylester 4 whose N-terminal Boc-Hpi residue on treatment with TFA loses the Boc group and reacts under thioether formation with the released cysteine-SH. The concomitantly deprotected carboxyl terminus is coupled intramolecularly with the free amino group of the secocompound 5 using the MA or DCCI method, thus forming the homodetic peptide ring. Compounds 3-R and 3-S agree very well with analog samples in chiroptical behavior. Thioether 2 and sulfoxide 3-R exert 50% inhibition of RNA polymerase II (or B) from Drosophila melanogaster in 10–⁶ M solution whereas K_i of 3-S is about five times higher.     

Comparison Between Light Induced and Chemically Induced Oxidation of rhVEGF

Purpose. The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. Methods. A liquid formulation of rhVEGF was exposed to fluorescent light (2 × 10⁴ lux for up to 4 weeks), hydrogen peroxide (H₂O₂), or t-butylhydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. Results. Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78,81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%, possibly due to the role of Met18 in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H₂O₂, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H₂O₂ and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. Conclusions. Methionine residues with exposed surface areas greater than 65 Ų and sulfur surface areas greater than 16 Ų were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H₂O₂ to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 Ų, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.     

New method for routine production of L‐[methyl‐11C]methionine: in loop synthesis

A fast, clean and reproducible method for the manufacture of the radiotracer L‐[methyl‐¹¹C]methionine is reported. The reaction at room temperature of the non‐radioactive precursor L‐homocysteine (1 mg solution in ethanol/water 50/50) with [¹¹C]CH₃I in an HPLC loop led to the formation of the desired radiotracer with a high radiochemical yield (38.4±4.1% end of synthesis) in a short production time (12 min). Radiochemical purity of the final radiotracer was 99.9±0.05%. Specific activities in the range 11–45 GBq/µmol were obtained. The presence of the undesired enantiomer (D‐[methyl‐¹¹C]methionine) was not detected in any of the cases. Copyright © 2008 John Wiley & Sons, Ltd.     

Oxidation of Recombinant Human Parathyroid Hormone: Effect of Oxidized Position on the Biological Activity

Purpose. To determine the oxidation products of recombinant human parathyroid hormone (rhPTH) treated with H₂O₂, the amino acid residue oxidized, and the biological activity of the oxidation products. Methods. Oxidized residues were determined by CNBr cleavage, trypsin digestion and subsequent fast atom bombardment mass spectrometry. The biological activity of each oxidized rhPTH was examined in rat osteosarcoma cell adenylate cyclase assay. Results. Three oxidized products were isolated, namely, Met at position 8 (Met8) sulfoxide, Met at position 18 (Met 18) sulfoxide and both positions Met sulfoxide. It appears that the Met8 and Met 18 oxidized forms are intermediates in the generation of the Met doubly oxidized form. All oxidized forms possessed reduced biological activity, more so for oxidation at Met8 than at Met 18. Conclusions. The region around Met8 is important for the activity of the parathyroid hormone.     

Characterization and comparison of leuprolide degradation profiles in water and dimethyl sulfoxide

Abstract: The effect of solvent on the rate of leuprolide degradation and on the structure of the degradation products was explored. Leuprolide solutions (370 mg/mL) were prepared in water and dimethyl sulfoxide (DMSO) for delivery in DUROS^TM osmotic implants. Both solvent systems demonstrated better than 90% stability after 1 year at 37°C, where the DMSO formulation afforded better stability than the aqueous formulation and was used in subsequent clinical trials. The rate of leuprolide degradation in DMSO was also observed to accelerate with increasing moisture content, indicating that the aprotic solvent minimized chemical degradation. Interestingly, leuprolide degradation products varied with formulation vehicle. The proportions of leuprolide degradation products observed to form in water and DMSO at 37°C were hydrolysis > aggregation > isomerization > oxidation and aggregation > oxidation > hydrolysis > isomerization, respectively. Specifically, more N-terminal hydrolysis and acetylation were observed under aqueous conditions, and increased Trp oxidation and Ser β-elimination were seen under non-aqueous conditions. Furthermore, the major chemical degradation pathway changed with temperature in the DMSO formulation (decreasing oxidation with increasing temperature), but not in the aqueous formulation.     

高效液相色谱法同时测定甲硫氨酸维生素B1注射液两组分含量

目的探讨用高效液相色谱法（HPLC法）同时测定甲硫氨酸维生素B。注射液中甲硫氨酸和维生素B1的含量。方法采用氨基键舍柱，流动相为乙腈-水（50：50），检测波长为220nm。结果平均回收率及RSD甲硫氨酸分别为99.5％和0．9％，维生素B1分别为99．4％和1.2％。结论高效液相色谱法简便、准确，适用于该产品的质量分析检验。     

Effect of Ascorbate on Adriamycin-Fe3+–Induced Lipid Peroxidation and DNA Damage

Adriamycin-Fe³⁺ -induced lipid peroxidation was enhanced by ascorbate at low concentrations. High concentrations of ascorbate also enhanced the peroxidation reaction, but only at an early stage. The initial rate of peroxidation depended upon the ratio of adriamycin-Fe²⁺/adriamycin-Fe³⁺ and the maximum rate was observed at the ratio of 1:1. These results suggest that the adriamycin-Fe³⁺ -induced lipid peroxidation may be initiated by an adriamycin-Fe²⁺–oxygen-adriamycin-Fe³⁺ complex. Ascorbate also promoted bathophenanthroline-Fe²⁺ formation from adriamycin-Fe³⁺ in a dose-dependent manner. It seems likely that ascorbate influences the peroxidation reaction via the reduction of adriamycin-Fe³⁺. During the interaction of adriamycin-Fe³⁺ with ascorbate, deoxyribose was not degraded, suggesting that hydroxyl radical formation did not occur. In contrast, plasmid PM2 DNA was readily damaged during the interaction of adriamycin-Fe³⁺ with ascorbate. Catalase, mannitol and dimethylsulfoxide prevented DNA damage. No DNA damage occured when the reaction was run under nitrogen gas, indicating that oxygen is involved.     

Effect of oxidation of β-amyloid precursor protein on its β-secretase cleavage. A model study with synthetic peptides and candidate β-secretases

As the amyloidogenic processing of β-amyloid precursor protein (βAPP) proceeds under conditions of oxidative stress, the methionine-596 residue at the β-secretase cleavage point is likely in an oxidized state. In the present work, possible consequences of the oxidation of Met-596 for the generation of the N-terminus of amyloid p protein were modeled using synthetic peptide substrates, matching 587-606 sequence fragment of βAPP and containing either intact methionine or methionine sulfoxide. Patterns and rates for the cleavage of these substrates by purified mast cell chymase, cathepsin G, cathepsin D, matrix metallopro-teinase-3 and neutrophil elastase, were compared. Only the three first proteases, all previously suggested as candidate β-secretases, preferentially cleaved the “intact” substrate after Met-596. For chymase and cathepsin G, the specificity of this cleavage increased upon a shift from optimal alkaline pH to acidic pH, which is also more compatible with the plausible intracellular localization of amyloidogenic βAPP processing. The substitution of methionine sulfoxide for methionine in the substrate slowed down the cleavage rate for all the enzymes tested, by a factor of 6-15. This was associated with shifts of cleavage preferences to points of minor importance for the “intact” peptide, suggesting a specific resistance of the peptide bond after MetSO-596 against proteolysis.