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1.
Philip R.P. Salacinski Charles McLean Jane E.C. Sykes Vicky V. Clement-Jones Philip J. Lowry 《Analytical biochemistry》1981,117(1):136-146
A new, commercially available oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) was compared with chloramine-T and solid-phase lactoperoxidase in the radioiodination of proteins, glycoproteins, and peptides. A method for performing low-level iodinations is described and was used to determine maximum 125I incorporation. Iodinated proteins were purified on analytical gel filtration columns and peptides by reverse-phase high-performance liquid chromatography. Both methods were designed to analyze the tracers for the presence of aggregate and breakdown products caused by the iodination. All tracers prepared were tested in antibody dilution and dose-response curves in their respective radioimmunoassays. Results indicate that Iodogen can be used for a wide range of proteins and peptides, can permit theoretical iodine incorporation with minimal oxidation damage, and can produce tracer stable for up to 3 months. 相似文献
2.
Selective oxidation of methionine residues in proteins. 总被引:7,自引:0,他引:7
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). 相似文献
3.
《Biochimica et Biophysica Acta.Protein Structure》1977,490(2):497-505
The rate of deiodination of radioiodinated proteins varies with the method of iodination. To elucidate differences in the iodinated protein labeled by various methods, we have hydrolyzed fibrinogen and several small peptides iodinated by the iodine monochloride, chloramine-T, electrolytic and enzymatic methods. Under conditions of either acidic or basic proteolysis, extensive deiodination occurred and the major product was I−. When a protease of Streptomyces griseus was used, radioiodinated fibrinogen and other polypeptides were degraded to single iodinated amino acid residues and only a small yield of I−. The iodinated amino acids resulting from proteolysis were separated by ion-exchange chromatography. The iodine monochloride and enzymatic methods yielded largely iodotyrosine with small amounts of other iodinated amino acids. The chloramine-T product spectrum varied with the chloramine-T:protein ratio, whereas the electrolytic method yield was a complex function of the reaction conditions. The different methods of iodination lead to some differences in the site of iodination which correlate with stability of the protein-iodine bond. 相似文献
4.
The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed. 相似文献
5.
Solid-phase methodology has previously been applied to labeling of proteins and peptide hormones used in immunoassay with the aid of enzyme sorbent. In this publication a method based on the use of a new carrier-copolymer of maleic anhydride and butanediol divinylether is introduced. As a model, bovine serum albumin (BSA) was labeled using three different procedures: Chemical, with chloramine-T as oxidizing agent: enzymatic, in a liquid phase with lactoperoxidase (LP) and horseradish peroxidase (HRP); and enzymatic, in a solid phase with maleic anhydride butanediol divinylether-copolymer as the carrier of lactoperoxidase and horseradish peroxidase.The lactoperoxidase-mediated iodinating activity in both the liquid and solid phases was similar (incorporating 47 and 39% for the total 125iodine added, 1 ), while HRP was more efficient in a liquid (11%) than in a solid phase (3%).Although the specific activity of the BSA labeled with chloramine-T was highest, this 125I-labeled BSA was badly degraded during iodination. However, in either liquid or solid phase enzymatic iodinations, no degradation of the protein could be observed.Peptide hormones, luteinizing hormone, follicle-stimulating hormone and angiotensin II, iodinated with lactoperoxidase or lactoperoxidase sorbent for radioimmunoassays reacted better than peptide hormones iodinated with chemical oxidants and remained unaltered during storage. 相似文献
6.
Both internal and external proteins in vesicular stomatitis virus were labeled when intact virions were iodinated with 50 μm iodide; however, only the surface proteins were labeled when the same procedure was carried out at low iodide concentrations (below 0.5 μm). This result together with similar observations reported earlier with another enveloped virus, Rous-associated virus-61 (RAV-61), suggest that viral envelopes provide a barrier to iodination by chloramine-T at low, but not at high, iodide concentrations. By monitoring the permeability of the RAV-61 envelope to successive iodinations and to iodination in the presence of chaotropic thiocyanate ions, it was shown that the permeability of the viral envelope was not altered at the higher concentrations of iodide. Further results support the hypothesis that iodination mediated by chloramine-T inolves two different iodinating species: (a) a membrane impermeable one, possibly “iodamine-T,” which predominates at low iodide concentrations, and (b) a membrane permeable species, possibly molecular iodine, which predominates at high concentrations of iodide. These results reinforce the proposal that the chloramine-T procedure is a useful method for specifically labeling surface proteins of lipid-enveloped structures. 相似文献
7.
A previously reported method for iodination of the tyrosine moiety of oxidation-sensitive biomolecules was found to cause unacceptable damage to biomolecules containing thiols and thioether groups. This was due to the oxidation of the sulfur-containing residues by molecular iodine (I(2)). To selectively iodinate the tyrosine moiety with minimum oxidation to the sulfur functionality, studies of the kinetics of the reactions between I-(3) and various amino acids and small peptides at various pH values in phosphate buffer were undertaken. Within the pH range studied (5.5-8.2), the results showed that the iodination reaction is strongly catalyzed by hydroxide ions, whereas the oxidation of the sulfur group was insensitive to pH. The results also showed that both reactions are strongly catalyzed by HPO-(4) ion. In a complex molecule, such as methionine-enkephalin, oxidation of the methionine residue (undesirable reaction) proceeds in parallel with iodination of the tyrosine residue (desirable reaction). If such a molecule was iodinated in 0.01 M phosphate buffer at pH values above 7.5, the iodination reaction would proceed much more rapidly than the oxidation reaction, resulting in a high yield of iodinated substrate with little oxidative damage. 相似文献
8.
Carol Dziadik Turner Steven B. Chernoff Alvin Taurog Allen B. Rawitch 《Archives of biochemistry and biophysics》1983,222(1):245-258
The distribution of iodine among the polypeptides of human goiter thyroglobulin (Tg) was examined. Tg was iodinated in vitro with 131I to levels of 2 to 84 gram atoms (g.a.)/mol using thyroid peroxidase (TPO) or a chemical iodination system. The samples were reduced, alkylated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two low-molecular-weight peptides appeared preferentially in radioautograms of the sodium dodecyl sulfate (SDS) gels of TPO-iodinated samples. Iodination of these peptides increased sharply in the TPO-treated Tg as the level of total iodine/ molecule rose. Radioiodine was incorporated into these same gel regions in the chemically treated Tg, but only after much higher levels of total iodination were reached. Differences in iodoamino acid distribution were also noted between the chemically and enzymatically iodinated thyroglobulins. In the chemically iodinated samples, little thyroxine (T4) was synthesized, even at high iodine levels. In the TPO-treated samples only small amounts of T4 were seen below 14 g.a. total I/mol, while at or above that level of iodination T4 formation increased sharply. To examine the coupling process, Tg was chemically iodinated, excess I? removed, and the samples treated with TPO and a H2O2-generating system in the absence of iodide. Radioautograms obtained from SDS-polyacrylamide gels of reduced and alkylated protein from such coupling assays showed an increase in the level of iodine in the low-molecular-weight peptides after TPO treatment. Thyroxine production also increased with TPO treatment. The addition of free DIT (a known coupling enhancer) to the [131I]Tg/TPO incubation increased both the production of T4 and the amount of iodine in the smaller polypeptides. Two-dimensional maps prepared from CNBr-digested TG showed differences between the coupled and uncoupled samples. Our observations confirm the importance of the lowmolecular-weight peptides derived from Tg in thyroid hormone synthesis. At total iodine levels above 14 g.a./mol Tg in enzymatically treated samples there is selective incorporation of iodine into both the low-molecular-weight polypeptides and into thyroid hormone. 相似文献
9.
A technique for radioactive labeling by iodination of sensitive biological material that preserves the functional activity of the samples to a greater extent than the standard iodination methods is presented. The reaction is carried out keeping in separate phases the substrate and the iodine-generating system. Chloramine-T in the presence of water and Cl? ions generates Cl2 on a piece of filter paper kept close to the surface of the solution containing the substrate and the 125I?. The Cl2 molecules diffuse from the paper, enter the solution, and, reacting with the iodide ions, generate iodine that modifies the aromatic rings of the sample. Protein A, lysozyme, and ribosomes treated under these iodination conditions are much less affected in their activity than when iodinated by the standard chloramine-T method or the iodogen system. In addition, this technique, which we have called “two phases” system, seems to act preferentially on the surface of the structures as shown by studying the iodination pattern of the ribosomal proteins. 相似文献
10.
In the presence of hydrogen peroxide, the heme protein lactoperoxidase is able to oxidize thiocyanate and iodide to hypothiocyanite, reactive iodine species, and the inter(pseudo)halogen cyanogen iodide. The killing efficiency of these oxidants and of the lactoperoxidase-H2O2-SCN?/I? system was investigated on the bioluminescent Escherichia coli K12 strain that allows time-resolved determination of cell viability. Among the tested oxidants, cyanogen iodide was most efficient in killing E. coli, followed by reactive iodine species and hypothiocyanite. Thereby, the killing activity of the LPO-H2O2-SCN?/I? system was greatly enhanced in comparison to the sole application of iodide when I? was applied in two- to twenty-fold excess over SCN?. Further evidence for the contribution of cyanogen iodide in killing of E. coli was obtained by applying methionine. This amino acid disturbed the killing of E. coli mediated by reactive iodine species (partial inhibition) and cyanogen iodide (total inhibition), but not by hypothiocyanite. Changes in luminescence of E. coli cells correlate with measurements of colony forming units after incubation of cells with the LPO-H2O2-SCN?/I? system or with cyanogen iodide. Taken together, these results are important for the future optimization of the use of lactoperoxidase in biotechnological applications. 相似文献
11.
Rates of oxidation of α-tocopherol by the hydroxyl- and superoxide free radicals were measured. The radicals were produced in known yields by radiolysis of aqueous solutions with gamma rays. Two main systems were used to dissolve the tocopherol; micelles, made up from charged and uncharged amphiphiles, and membranes made from dimyristyl phosphatidylcholine which could be charged by addition of stearyl amine or dicetyl phosphate. The HO. radicals were efficient oxidants of α-tocopherol in all systems, with up to 83% of radicals generated in micelle and 32% in membrane suspensions initiating the oxidation. The HO radical was an even more effective oxidant, but when most of it was in the O form at neutral or alkaline pH, the oxidation rates became low. Tocopherol held in positively charged micelles or membranes was oxidized at a higher rate by the O than in uncharged or negative particles. Possible biological significance of these results is discussed. 相似文献
12.
Jerzy Silberring Fred Nyberg 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,562(1-2)
A simple and unambiguous method for the detection of the amino acids tyrosine and methionine in peptide structures has been developed. The procedure, which was applied in studies of opioid peptides, is based on continuous-flow fast atom bombardment mass spectrometry (CF-FAB-MS) following chemical modification of the residue to be analyzed. Thus, for the detection of tyrosine, modification reactions such as acetylation or non-radioactive iodination were performed prior to analysis by CF-FAB-MS. O-Acetylation of the tyrosine residue with N-acetylimidazole was accompanied by a shift of 42 Da in the molecular mass of the peptide under investigation. This modification was reversed by treatment with hydroxylamine hydrochloride. Incorporation of iodine resulted in a molecular weight shift of 126 Da per iodine atom. Methionine residues were detected in methionine-enkephalin-containing peptides following S-oxidation with hydrogen peroxide. The procedures described may have a wide application in peptide chemistry, particularly for the identification of peptide fragments containing the above residues, e.g. in studies of processing or degradation of the enkephalins or other neuropeptides (e.g. endorphins and tachykinins). 相似文献
13.
Hao Zhang 《Archives of biochemistry and biophysics》2009,484(2):134-618
Recent reports suggest that intramolecular electron transfer reactions can profoundly affect the site and specificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects of methionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H2O2 and NO2− and peroxynitrite (ONOO−) or ONOO− and bicarbonate (HCO3−) in model peptides, tyrosylmethionine (YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides were analyzed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediates were identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown (Zhang et al., J. Biol. Chem. 280 (2005) 40684-40698) that oxidation and nitration of tyrosyl residue was inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show that methionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighboring tyrosine residue in the presence of ONOO− (or ONOOCO2−) or MPO/H2O2/NO2− system. Nitration of tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO− (formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF, YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteine residues have been attributed to differences in the direction of intramolecular electron transfer in these peptides. Further support for the interpretation was obtained by steady-state radiolysis and photolysis experiments. Potential implications of the intramolecular electron transfer mechanism in mediating selective nitration of protein tyrosyl groups are discussed. 相似文献
14.
H.Brian Dunford Isobel M. Ralston 《Biochemical and biophysical research communications》1983,116(2):639-643
Existing data contain proof that the iodinating species of tyrosine and its derivatives contained in mixtures of iodine and iodide is hypoiodous acid, HOI. It appears likely that the peroxidase-catalyzed iodination reaction with hydrogen peroxide, tyrosine or a tyrosine derivative and either iodide or iodine as substrates involves enzyme-activated HOI. 相似文献
15.
Hsiu-Ping Li Benjamin Daniel Danielle Creeley Russell Grandbois Saijin Zhang Chen Xu Yi-Fang Ho Kathy A. Schwehr Daniel I. Kaplan Peter H. Santschi Colleen M. Hansel Chris M. Yeager 《Applied and environmental microbiology》2014,80(9):2693-2699
The release of radioactive iodine (i.e., iodine-129 and iodine-131) from nuclear reprocessing facilities is a potential threat to human health. The fate and transport of iodine are determined primarily by its redox status, but processes that affect iodine oxidation states in the environment are poorly characterized. Given the difficulty in removing electrons from iodide (I−), naturally occurring iodide oxidation processes require strong oxidants, such as Mn oxides or microbial enzymes. In this study, we examine iodide oxidation by a marine bacterium, Roseobacter sp. AzwK-3b, which promotes Mn(II) oxidation by catalyzing the production of extracellular superoxide (O2−). In the absence of Mn2+, Roseobacter sp. AzwK-3b cultures oxidized ∼90% of the provided iodide (10 μM) within 6 days, whereas in the presence of Mn(II), iodide oxidation occurred only after Mn(IV) formation ceased. Iodide oxidation was not observed during incubations in spent medium or with whole cells under anaerobic conditions or following heat treatment (boiling). Furthermore, iodide oxidation was significantly inhibited in the presence of superoxide dismutase and diphenylene iodonium (a general inhibitor of NADH oxidoreductases). In contrast, the addition of exogenous NADH enhanced iodide oxidation. Taken together, the results indicate that iodide oxidation was mediated primarily by extracellular superoxide generated by Roseobacter sp. AzwK-3b and not by the Mn oxides formed by this organism. Considering that extracellular superoxide formation is a widespread phenomenon among marine and terrestrial bacteria, this could represent an important pathway for iodide oxidation in some environments. 相似文献
16.
Oxidation of proteins in rat heart and lungs by polymorphonuclear leukocyte oxidants 总被引:2,自引:0,他引:2
Henry Fliss 《Molecular and cellular biochemistry》1988,84(2):177-188
Summary The ability of the polymorphonuclear leukocyte (PMN) oxidants, hypochlorous acid (HOC1) and hydrogen peroxide (H2O2), to oxidize proteins in rat heart and lung tissues was investigated. Cardiac myocytes, heart tissue slices, isolated perfused hearts, and lung tissue slices, were treated with HOCI and H2O2 and the extent of methionine and cysteine oxidation was determined in the cellular proteins. Cardiac tissues were found to be highly susceptible to oxidation by physiological concentrations of HOCl. For example, in isolated hearts perfused for 60 min with 100 M HOCI, approximately 18010 of the methionine and 2801o of the cysteine residues were oxidized. Lung tissues, unlike those of the heart, were resistant to physiological concentrations of HOCI, showing no oxidation of proteins. HOCI was much more effective than H2O2 in oxidizing proteins, suggesting that HOCI may be the most reactive oxidant produced by activated PMN. These studies show that PMN oxidants, in particular HOC I, can cause significant oxidation of proteins in target tissues, and may therefore constitute a primary cause of tissue injury at sites of inflammation. In addition, these studies show that different tissues may have varying susceptibilities to PMN oxidants. 相似文献
17.
A new procedure is described for the radioiodination of proteins with sulfhydryl groups essential for their biological activity. Aniline is iodinated with 125I-labeled sodium iodide in the presence of chloramine-T, the product separated by solvent extraction, diazotized and coupled to protein. 相似文献
18.
We have estimated the number of sites on each protein of the 30 S ribosome which are accessible to chemical iodination. First, the total number of iodinatable sites was determined for the intact 30 S ribosome. The proteins were extracted, separated and the relative distribution of iodine in each protein determined. This distribution of iodine divided into the total sites per ribosome gave an estimate of the number of sites per individual protein.Second, the iodinated proteins were purified and their trypsin digestion products separated. The number of radioactive peptides was taken as a measure of the number of sites on that protein open to the iodination reaction. The number of iodinatable sites for each protein was found to be radically different by the two methods. In almost all cases, the number of unique, radioactively labeled peptides, derived from a given 30 S protein, far exceeded the total incorporation into that protein. We suggest that the best explanation for this unexpected discrepancy is that the 30 S ribosome population we used in these experiments is heterogeneous in its topography.In addition we have compared the topography by the chemical iodination procedure for ribosomes in two different conformations: active and inactive (see Zamir et al., 1971). We have found very little change in the chemical reactivity of the proteins when the ribosomes are in the two different conformations. The most notable changes involve proteins S10, and especially . 相似文献
19.
Role of ubiquitin conformations in the specificity of protein degradation: iodinated derivatives with altered conformations and activities 总被引:1,自引:0,他引:1
Three iodinated derivatives of ubiquitin have been synthesized and these derivatives have been characterized in the ubiquitin-dependent protein degradation system. With chloramine-T as the oxidant, a derivative containing monoiodotyrosine is formed in the presence of 1 M KI and a derivative containing diiodotyrosine is produced in the presence of 1 mM KI. These derivatives exhibit phenolate ionizations at pH 9.2 and 7.9 with absorbance maxima at 305 and 314 nm, respectively. In addition to modification of the tyrosine residue, these conditions lead to the oxidation of the single methionine residue and iodination of the single histidine residue [M.J. Cox, R. Shapira, and K.D. Wilkinson (1986) Anal. Biochem. 154, 345-352]. Iodination of ubiquitin under these conditions renders the protein sensitive to hydrolysis by trypsin and results in an enhanced susceptibility to alcohol-induced helix formation. When the derivatives are tested in the ATP: pyrophosphate exchange reaction catalyzed by the ubiquitin adenylating enzyme, they are found to exhibit activity comparable to the native protein. When these derivatives are tested for the ability to act as a cofactor in the ubiquitin-dependent protein degradation system, they are both found to support a rate of protein degradation that is twice that of native ubiquitin. At high concentrations of derivatives, the rate of protein degradation is inhibited, while the steady state level of conjugates increases. Thus, the free derivatives inhibit the protease portion of the reaction, but are fully active in the activation and conjugation portions of the reaction. With iodine as the modification reagent, monoiodination of tyrosine is the predominant reaction. This derivative exhibits activity similar to native ubiquitin. Thus, it appears that modification of the histidine residue is responsible for the increased activity of the more highly iodinated derivatives. The enzymes of the system must recognize different portions of the ubiquitin structure, or different conformations of ubiquitin that are affected by the iodination of the histidine residue. These results suggest a conformational change of the ubiquitin molecule may be important in determining the rate and specificity of proteolysis. 相似文献
20.
Min-Fu Tsan 《Journal of cellular physiology》1982,111(1):49-54
The myeloperoxidase-mediated oxidation of methionine was studied using a purified canine myeloperoxidase preparation. The system required the simultaneous presence of myeloperoxidase, H2O2, and a halide anion. When 0.1 mM H2O2 was used, the system has a pH optimum of approximately pH 5–5.5. Bromide and iodide were much more effective than chloride in the myeloperoxidase-mediated oxidation of methionine. Horseradish peroxidase was unable to oxidize methionine whether chloride or iodide was used. In contrast, lactoperoxidase oxidized methionine in the presence of iodide but not chloride. Based on studies of (1) the effect of various inhibitors and singlet oxygen quenchers, as well as (2) the effect of D2O on the oxidation of methionine, by the myeloperoxidase system, OCl?, or methylene blue, it was shown that the oxidation of methionine by the myeloperoxidase system was not mediated by OCl? or 1O2. The mechanism of the myeloperoxidase-mediated oxidation of methionine remains unclear. However, it may be one mechanism by which the myeloperoxidase system damage microorganisms. 相似文献