Identification of modified tryptophan residues in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein

Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu(2+), treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu(2+)-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu(2+)-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu(2+)-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu(2+)-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.     

Structural analysis of gelsolin using synchrotron protein footprinting

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.     

Broad-spectrum antioxidant peptides derived from His residue-containing sequences present in human paraoxonase 1

Hydroxyl or peroxyl radicals and hypochlorous acid (HOCl) are known to cause the oxidation of lipoproteins. Here, we examined Cu²⁺-binding property of paraoxonase 1 (PON1), and antioxidant actions of peptides, resembling His residue-containing sequences in PON1, against oxidations by Cu²⁺, peroxyl radicals or HOCl. When Cu²⁺-binding property of PON1 was examined spectrophotometrically, the maximal Cu²⁺ binding was achieved at 1:1 molar ratio of PON1: Cu²⁺. Additionally, Cu²⁺-catalyzed oxidative inactivation of PON1 was prevented by Ca²⁺-depleted PON1 at 1:1 ratio, but not diethylpyrocarbonate (DEPC)-modified PON1, suggesting the participation of His residue in Cu²⁺-binding. When His-containing peptides were examined for antioxidant actions, those with either His residue at N-terminal position 2 or 3, or His-Pro sequence at C-terminal remarkably prevented Cu²⁺-mediated low density lipoprotein (LDL) oxidation and PON1 inactivation. Especially, FHKALY, FHKY or NHP efficiently prevented Cu²⁺-induced LDL oxidation (24 h), indicating a tight binding of Cu²⁺ by peptides. In support of this, the peptide/Cu²⁺ complexes exhibited a superoxide-scavenging activity. Separately, in oxidations by 2,2'-azobis-2-amidinopropane hydrochloride or HOCl, the presence of Tyrosine (Tyr) or Cysteine (Cys) residue markedly enhanced antioxidant action of His-containing peptides. These results indicate that His-containing peptides with Tys or Cys residues correspond to broad spectrum antioxidants in oxidation models employing Cu²⁺, 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) or HOCl.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.     

Serum level of IgG autoantibodies against oxidized low density lipoproteins and lag-phase of serum oxidation in coronary heart disease--inverse correlation.

High affinity IgG autoantibodies (ABs) against oxLDLs and lag-phase of serum oxidation were tested in patients with coronary heart disease (CHD). Fifty one (37 M/14 F) patients with CHD defined as Q-wave myocardial infarction and/or stenosis of more than 50% and 51 (34 M/17 F) healthy blood donors as controls participated in this study. LDLs were isolated by gradient ultracentrifugation and oxidized with CuSO4. The modified LDLs (oxLDLs) or native LDLs (nLDLs) were used as antigens in an enzyme immunoassay (ELISA) to detect IgG ABs in both groups. The serum was oxidized by CuSO4 and the oxidation was monitored spectrophotometrically at lambda = 234 nm to follow the formation of conjugated diens. The lag-phase (in minutes) is the interval between the addition of CuSO4 to the serum and the beginning of extensive oxidation (increasing absorbance at 234 nm). The concentrations of total cholesterol, triglycerides, HDL-cholesterol, apo-A and apo-B were measured as well. The mean level of ABs against oxLDLs (expressed as optical density units) was 0.590 +/- 0.330 in CHD-patients vs 0.244 +/- 0.200 in controls (p < 0.001). The lag-phase in minutes was 47.00 +/- 27.19 in CHD-patients and 80.23 +/- 26.30 in controls (p < 0.001). A negative correlation between ABs levels and lag-phase was established in CHD-patients (r = -0.69, p < 0.001) and controls (r = -0.62, p < 0.001). A poor correlation was established between ABs levels or lag-phase, on one hand, and other measured parameters. In conclusion, the lag-phase of serum oxidation by Cu2+ could be informative for LDL susceptibility to modification and the extent of consequent humoral immune response.     

Identification and characterization of dominant helper T-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2(b)), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2(k)), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2(d)) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines.     

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.     

Method to site-specifically identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoliquid chromatography Fourier transform mass spectrometry

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. This presents a significant advancement over current methods, combining strengths of current methods and adding the abilities to multiplex, quantitate, and probe more modified amino acids. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N',N' ',N'-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. O-ECAT can be loaded with a variety of metals, which yields the ability to generate mass pairs and multiplex multiple samples. The O-ECAT moiety also serves as a handle for identification, quantitation, and affinity purification. After proteolysis, the AOD-tagged peptides are affinity purified and analyzed by nanoLC-FTICR-MS (nanoliquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry), which provides high specificity in extracting coeluting AOD mass pairs with a unique mass difference and allows relative quantitation based on isotopic ratios. Using this methodology, we have quantified and mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation both at the protein and amino acid levels. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic, and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure, validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities toward oxidation, independent of amino acid residue. This novel methodology not only has the ability to identify and quantitate oxidized proteins but also yields site-specific quantitation on a variety of individual amino acids. We expect to extend this methodology to study disease-related oxidation.     

Photoaffinity labeling of T4 endonuclease V with a substrate containing a phenyldiazirine derivative.

T4 endonuclease V recognizes thymine photodimers in DNA duplexes and, in a two-step reaction, cleaves the glycosyl linkage of the 5'-side thymidine and the phosphodiester linkage. To determine the amino acid residues responsible for binding thymine photodimers, a photoaffinity reagent, 4-(1-azi-2,2,2-trifluoroethyl)-benzoate, was linked to the aminoalkylphosphonate of a thymine photodimer in a 14-mer duplex. The reactive substrate was treated with the enzyme under UV light (365 nm). The nascent enzyme and the modified enzyme were treated with lysyl endopeptidase, and the peptide maps were compared. Three peptides from the C terminus were found to interact with the reactive oligonucleotide to various extents. The three modified peptides were isolated and analyzed by Edman degradation. The amino acid residues Gly-133, Tyr-129, and Thr-89 were partially linked with the reactive substrate and may be involved in the binding of thymine photodimers.     

Identification and analysis of products formed from phospholipids in the free radical oxidation of human low density lipoproteins

Phospholipids reside in the surface layer of LDLs and constitute approximately 20-25% of the particle by weight. We report a study of the primary products generated from the most abundant molecular species of phosphatidylcholines present in LDL during in vitro free radical oxidations. The 13-hydroperoxides of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and 1-stearoyl-2-linoleoyl-sn-glycero-phosphocholine (SLPC) and the 15-hydroperoxides of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphocholine (SAPC) were found to increase in a time-dependent manner and in significant amounts even in the presence of alpha-tocopherol. Phospholipid alcohols also formed during the course of the oxidations. Early in the LDL oxidations, while alpha-tocopherol was still present, the thermodynamically favored trans,trans products of PLPC and SLPC were found to form in significantly larger quantities than those formed from cholesteryl linoleate. Additionally, quantities of PAPC 11-hydroperoxide (11-OOH) decreased over time relative to PAPC 15-OOH, even while alpha-tocopherol was still present in the oxidation, presumably as a result of further oxidation of PAPC 11-OOH to form cyclic peroxide oxidation products. These results suggest that alpha-tocopherol is more closely associated with the inner cholesteryl ester-rich hydrophobic core of an LDL particle and is not as effective as an antioxidant in the outer phospholipid layer as it is in the lipid core.     

Free radical mechanism of oxidation of uroporphyrinogen in the presence of ferrous iron

Human porphyria cutanea tarda is an unusual consequence of common hepatic disorders such as alcoholic liver disease. Hepatic iron plays a key role in the expression of the metabolic lesions, i.e., defective hepatic decarboxylation of porphyrinogens, catalyzed by uroporphyrinogen decarboxylase. This prompted the present study to determine the in vitro effects of iron on the uroporphyrinogen substrate in the absence and presence of atmospheric oxygen. We observed that (i) unless oxygen is the limiting reactant, autoxidation of ferrous iron and iron-catalyzed oxidation of uroporphyrinogen occurred soon after initiating the reaction at pH 7.4 and 30 degrees C in buffers which are non- or poor chelators of iron; (ii) the rates of uroporphyrinogen oxidation were proportional to the initial concentration of ferrous ion; (iii) about 70% of the oxidations of uroporphyrinogen were accountable due to a free-radical chain reaction pathway involving superoxide radical and hence inhibitable by superoxide dismutase; (iv) uroporphyrinogen could be further oxidized to completion by the hydroxyl radical since the reaction was partially inhibited by both mannitol and catalase which prevent hydroxyl radical production; (v) the oxidizing effects of ferric ion on uroporphyrinogen were none or negligible as compared to those of ferrous ion. Ferric was reduced to ferrous ion in the presence of dithiothreitol. When the ferrous ion thus formed was reoxidized in the presence of atmospheric oxygen, minor but definite oxidations of both uroporphyrinogen and dithiothreitol were observed. The oxidations of Fe2+ and uroporphyrinogen could be blocked by 1,10-phenanthroline, a ferrous iron chelator. The data suggest that ferrous is the reactive form of iron that may contribute to pathogenic development of the disease by irreversibly oxidizing the porphyrinogen substrates to nonmetabolizable porphyrins, which accumulate in porphyric liver.     

Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation

Uptake of cholesterol-containing lipoproteins by macrophages in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. There are a number of possible mechanisms by which macrophages might accumulate cholesterol, and one that has attracted much interest recently involves the uptake of oxidatively modified low density lipoprotein (LDL) via a specific cell surface receptor, termed the scavenger or acetyl-LDL receptor. Previous studies have shown that chemical derivatization of LDL with reagents that result in neutralization of the charge of lysine amino groups also allows recognition by this receptor. As well, it has been shown that oxidation of LDL is accompanied by a decrease in free lysine groups and binding of lipid products to apolipoprotein B. The present studies were done to further characterize the receptor-binding domain on oxidized LDL. It was found that LDL could be modified by incubation with water-soluble products derived from autoxidized unsaturated fatty acids under conditions that inhibited oxidation of the LDL itself. The LDL modified in this way had increased electrophoretic mobility but showed no evidence of the oxidative damage that typifies LDL oxidized by exposure to metal ions. Furthermore, the oxidation product-modified LDL was rapidly degraded by cultured macrophages through the scavenger receptor pathway. Bovine albumin modified by oxidation products also showed greatly accelerated degradation by macrophages. When analyzed by reverse-phase high pressure liquid chromatography, the reactive oxidation products appeared less polar than fatty acids or simple medium-chain aldehydes. When treated with the carbonyl reagent 2,4-dinitrophenylhydrazine, the reactive fractions yielded derivatives, some of which were identified by mass spectrometry as hydrazones of nonenal, heptenal, pentenal, and crotonaldehyde. A series of 2-unsaturated aldehydes (acrolein to 2-nonenal) were all found to modify LDL, but none of these aldehyde-modified LDLs were recognized by the scavenger receptor of macrophages and all were degraded much more slowly by these cells than LDL modified with oxidation products. Furthermore, copper-oxidized LDL had only very slight immunoreactivity toward a panel of antibodies specific for adducts of simple 2-unsaturated aldehydes. Analysis of underivatized autoxidized fatty acids by coupled liquid chromatography/thermospray mass spectrometry revealed compounds with m/z corresponding to M+17, M+31, and 2M+31 in fractions that were capable of modifying LDL. The unoxidized fatty acids showed a dominant peak at M-1. These results indicate that the scavenger receptor of macrophages can recogn     

Differential toxicities of air (mO-LDL) or copper-oxidized LDLs (Cu-LDL) toward endothelial cells.

In vivo low density protein (LDL) oxidation is a progressive phenomenon leading to the presence of minimally and highly oxidized LDLs in the subendothelial arterial space. Oxidized LDLs have been reported to be cytotoxic against endothelial cells. The goal of this study was to determine which of the minimally and highly oxidized LDLs were the most cytotoxic against bovine aortic endothelial cells (BAEC). Both the morphological aspect of the cells themselves, and LDH or MTT tests revealed that mO- or Cu-LDLs had similar cytotoxicity with up to 8 hours of oxidation, showing no relation with the level of LDL oxidation; for longer oxidation times, Cu-LDL cytotoxicity decreased. This phenomenon is linked to their different oxidation kinetics. Moreover, in the initial hours following BAEC incubation with mO- or Cu-LDLs, total cell glutathione dropped, whereas after 16 hours of incubation, highly oxidized Cu-LDL increased the glutathione level in the cell. The biphasic evolution of glutathione concentration corresponds to an autoprotective mechanism of cells against oxidized LDL cytotoxicity. This study suggests that the specific chemical characteristics of the different types of oxidized LDLs should always be precisely described in future assays devoted to studying the biological effects of what are known under the generic term as "oxidized LDLs". This precaution should prevent any confusion in interpreting different studies.     

Effect of N-bromosuccinimide-modification of tyrosine side chains of cardiotoxin II of the Indian cobra on biological activity

The essential role of tyrosine residue(s) of cardiotoxin II in the biological activity of the toxin was evaluated using N-bromosuccinimide. N-bromosuccinimide effected oxidation of the tyrosine residues in cardiotoxin II with enhancement in absorbance at 260 nm. The influence of various solvent media such as acetate-formate buffer (pH 4.0), 0.01 N H₂SO₄ (pH 2.0) and Tris-HCl buffer (pH 8.5) on oxidation of tyrosine residues was exa mined. In comparison with 0.01 N H₂S O₄, acetate-formate buffer could prevent secondary oxidations as revealed by lower consumption of oxidant, N-bromosuccinimide, to achieve oxidation. In Tris-HCl buffer oxidation of tyrosine did not take place effectively. N-iodo-succinimide caused only limited oxidation as evident from minor increase in absorbance at 260 nm. N-chlorosuccinimide was completely ineffective. Oxidation of cardiotoxin II with 3.75 equivalents of N-bromosuccinimide tyrosine residue led to complete loss of lethal activity. However, the derivative retained the ability to protect bacterial protoplasts from lysis in solutions of low tonicity. Unlike cardiotoxin II oxidized with N-chlorosuccinimide (50 equivalents/mol of toxin) which retained lethal activity as well as the ability to protect protoplasts from lysis, performic acid-oxidized toxin had lost both the activities.     

52-kDa Ro/SSA epitopes preferentially recognized by antibodies from mothers of children with neonatal lupus and congenital heart block

Neonatal lupus erythematosus is a rare disorder caused by the transplacental passage of maternal autoantibodies. The 52-kDa Ro/SSA antigen (Ro52) ribonucleoprotein represents an antigenic target strongly associated with the autoimmune response in mothers whose children have neonatal lupus and cardiac conduction disturbances, mainly congenital heart block. The objective of this study was to identify putative Ro52/60-kDa Ro/SSA antigen (Ro60) epitopes associated with neonatal lupus and congenital heart block. The reactivity of IgG antibodies present in the sera from mothers with systemic lupus erythematosus and Sj?gren's syndrome and in the sera from asymptomatic mothers (a longitudinal study of 192 samples from 66 subjects) was investigated by ELISA using Ro52, Ro60 and 48-kDa La/SSB antigen proteins, as well as 45 synthetic peptides, 13-24 residues long, of Ro52/Ro60 proteins. One to 19 samples collected before, during and after pregnancy were available for each mother. Forty-three disease controls selected randomly and normal sera were tested in parallel. Although no differences were found between Sj?gren's syndrome and asymptomatic mothers of group I, who had at least one infant with neonatal lupus, and of group II, who had healthy babies only, significant differences were observed between lupus mothers from both groups. In the former group of lupus mothers, a significantly higher frequency of antibodies to Ro52 peptides 107-122 and 277-292 was observed. Between 18 and 30 weeks of gestation, the period of risk, there was clearly an elevated level of antibodies reacting with Ro52 peptides 1-13, 277-292 and 365-382. Antibodies to Ro52 peptide 365-382 have been shown previously to cross-react with residues 165-185 of the heart 5-HT4 serotoninergic receptor, and might be pathologically important. The level of these Ro52 antibody subsets decreased at the end of pregnancy and after delivery. IgG antibodies to Ro52 peptides 1-13, 107-122, 277-292 and 365-382 may therefore represent important biomarkers to predict a complication in pregnant lupus women with Ro52 antibodies.     

Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Trp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin E1 in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404W, F-413W, F-443W, F-484W, and W-495 peptides exhibited small and/or insignificant REES changes (ΔREES) whereas W-424, F-431W, and Y-507W channel peptides possessed modest REES changes (3 nm≤ΔREES≤7 nm). In contrast, wild-type, Y-367W, W-460, Y-478W, and I-499W channel peptides showed large ΔREES values upon membrane binding (7 nm<ΔREES≤12 nm). The REES data for the membrane-bound structure of the colicin E1 channel peptide proved consistent with previous data for the topology of the closed channel state, which lends further credence to the currently proposed channel model. In conclusion, the REES method provides another source of topological data for assignment of the bilayer location for Trp residues within membrane-associated proteins; however, it also requires careful interpretation of spectral data in combination with structural information on the proteins being investigated.     

Effects of oxidation on copper-binding properties of Aβ1-16 peptide: A pulse radiolysis study

Abstract

The reaction of hydroxyl radicals (^?OH) with Aβ1-16 peptide was carried out using pulse radiolysis to understand the effect of oxidation of peptide on its copper-binding properties. This reaction produced oxidized, dimeric and trimeric Aβ1-16 peptide species. The formation of these products was established with the help of fluorescence spectroscopy and mass spectrometry. The mass spectral data indicate that the major site of oxidation is at His6, while the site for dimerization is at Tyr10. Diethyl pyrocarbonate-treated Aβ1-16 peptide did not produce any trimeric species upon oxidation with ^?OH. The quantitative chemical modification studies indicated that one of the three histidine residues is covalently modified during pulse radiolysis. The copper-binding studies of the oxidized peptide revealed that it has similar copper-binding properties as the unoxidized peptide. Further, the cytotoxicity studies point out that both oxidized and unoxidized Aβ1-16 peptide are equally efficient in producing free radicals in presence of copper and ascorbate that resulted in comparable cell death.     

Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Trp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin E1 in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404 W, F-413 W, F-443 W, F-484 W, and W-495 peptides exhibited small and/or insignificant REES changes (Delta REES) whereas W-424, F-431 W, and Y-507 W channel peptides possessed modest REES changes (3 nm< or = Delta REES< or = 7 nm). In contrast, wild-type, Y-367 W, W-460, Y-478 W, and I-499 W channel peptides showed large Delta REES values upon membrane binding (7 nm< Delta REES< or =12 nm). The REES data for the membrane-bound structure of the colicin E1 channel peptide proved consistent with previous data for the topology of the closed channel state, which lends further credence to the currently proposed channel model. In conclusion, the REES method provides another source of topological data for assignment of the bilayer location for Trp residues within membrane-associated proteins; however, it also requires careful interpretation of spectral data in combination with structural information on the proteins being investigated.