The use of 3-bromopropionic acid for the determination of protein thiol groups.
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ABSTRACT:
SUBMITTER: Bradbury AF
PROVIDER: S-EPMC1177524 | biostudies-other | 1973 Apr
REPOSITORIES: biostudies-other
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Project description:1. The inhibitory effects of iodoacetate, iodoacetamide, p-hydroxymercuribenzoate and sarkomycin were studied in a partially purified preparation of deoxyribonucleic acid nucleotidyltransferase from Landschutz ascites-tumour cells. All of these agents inhibited the activity of the enzyme, and it was shown that the inhibition exerted by the last three compounds obeyed non-competitive kinetics. 2. Inclusion of glutathione or 2-mercaptoethanol in the enzyme assays did not prevent the inhibition by iodoacetate or iodoacetamide, but did prevent inhibition by p-hydroxymercuribenzoate. Inhibition by sarkomycin could be partially prevented by glutathione or 2-mercaptoethanol. 3. The enzyme fraction also catalysed incorporation in the presence of only one triphosphate (thymidine 5'-triphosphate), and the limited incorporation observed in these circumstances was more resistant to the inhibitory action of iodoacetamide and p-hydroxy-mercuribenzoate than was the standard nucleotidyltransferase reaction (four triphosphates present). Levels of inhibition imposed on the standard reaction were achieved in the limited incorporation reaction with 2.5-fold higher concentrations of the two inhibitors. 4. The addition of certain bivalent cations to the standard system resulted in severe inhibition of the reaction: Zn(2+) ions (10mum) gave 50% inhibition; ethylenediaminetetra-acetate (0.4mm) in the reaction mixture gave essentially complete protection against this inhibitory effect of Zn(2+) ions. 5. Deoxyribonucleic acid-nucleotidyltransferase fractions prepared in the presence of a thiol and ethylenediaminetetra-acetate could be stored without loss of activity for 2 months at 0 degrees or for 1 year at -70 degrees .
| S-EPMC1214347 | biostudies-other
Project description:2-Nitro-5-thiocyanatobenzoic acid has been proposed as a reagent for converting thiol groups in proteins into their S-cyano derivatives. Evidence was obtained for formation of both the S-cyano derivative and the mixed disulphide derivative. Formation of the S-cyano derivative can be promoted by addition of excess of CN-to the reaction mixture.
| S-EPMC1164054 | biostudies-other
Project description:Hydrogen peroxide (H2O2) functions as a second messenger that can activate cell proliferation through chemoselective oxidation of cysteine residues in signaling proteins. The connection between H2O2 signaling, thiol oxidation, and activation of growth pathways has emerged as fertile ground for the development of strategies for cancer treatment. Central to achieving this goal is the development of tools and assays that facilitate characterization of the molecular events associated with tumorigenesis and evaluation of patient response to therapy. Here we report on the development of an immunochemical method for detecting sulfenic acid, the initial oxidation product that results when a thiolate reacts with H2O2. For this approach, the sulfenic acid is derivatized with a chemical tag to generate a unique epitope for recognition. The elicited antibody is exquisitely specific, context-independent, and capable of visualizing sulfenic acid formation in cells. Applying this approach to several systems, including cancer cell lines, shows it can be used to monitor differences in thiol redox status and reveals a diverse pattern of sulfenic acid modifications across different subtypes of breast tumors. These studies demonstrate a general strategy for producing antibodies against a specific oxidation state of cysteine and show the utility of these reagents for profiling thiol oxidation associated with pathological conditions such as breast cancer.
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Project description:Indirect evidence suggests that oxidative stress may play a role in the pathogenesis of inherited muscular dystrophy, but the significance and precise extent of this contribution is poorly understood. Compared with normal muscle, significantly higher contents of glutathione, glutathione disulphide, protein-glutathione mixed disulphides and protein carbonyl groups, and significantly lower contents of free protein thiol groups, were found in pectoralis major muscle of genetically dystrophic chickens (the muscle affected by this disease) at 4 weeks of age. Other tissues did not show such marked disease-related differences. Interestingly, the protein pool in normal, but not dystrophic, pectoralis major muscle was relatively less oxidized in relation to the glutathione pool as compared with other tissues studied. The mechanisms by which this unique relationship between the thiol pools is maintained remain unknown. Although the physiological consequences of the increased content of protein carbonyl groups and the altered thiol pools in dystrophic muscle are not clear, the changes evident at such a young age are consistent with the occurrence of oxidative stress and may reflect significant damage to cellular proteins in this disease.
| S-EPMC1138677 | biostudies-other
Project description:Cysteine is arguably the best-studied biological amino acid, whose thiol group frequently participates in catalysis or ligand binding by proteins. Still, cysteine's unusual biological distribution has remained mysterious, being strikingly underrepresented in transmembrane domains and on accessible protein surfaces, particularly in aerobic life forms ("cysteine anomaly"). Noting that lipophilic thiols have been used for decades as radical chain transfer agents in polymer chemistry, we speculated that the rapid formation of thiyl radicals in hydrophobic phases might provide a rationale for the cysteine anomaly. Hence, we have investigated the effects of dodecylthiol and related compounds in isolated biomembranes, cultivated human cells and whole animals (C. elegans). We have found that lipophilic thiols at micromolar concentrations were efficient accelerators, but not inducers of lipid peroxidation, catalyzed fatty acid isomerization to trans-fatty acids, and evoked a massive cellular stress response related to protein and DNA damage. These effects were specific for lipophilic thiols and were absent with thioethers, alcohols or hydrophilic compounds. Catalytic chain transfer activity by thiyl radicals appears to have deeply influenced the structural biology of life as reflected in the cysteine anomaly. Chain transfer agents represent a novel class of biological cytotoxins that selectively accelerate oxidative damage in vivo.
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Project description:Activation of the fourth component of complement (C4) by C1s results in the generation of a reactive acyl group, able to react with putrescine, and in the release of a free thiol group that cannot be detected in the native haemolytically active molecule. Both the reactive acyl group and the free thiol group have been shown to reside in C4d, a fragment of the alpha'-chain of C4b derived from digestion of the molecule with the control proteins C3b inactivator and C4-binding protein. Peptides derived from CNBr digestion of [1,4-14C]putrescine-labelled and iodo(2-14C]acetic acid-labelled C4d have been obtained and used to establish a continuous sequence of 88 residues from the N-terminus of the molecule. The thiol and reactive acyl groups are contained in an octapeptide that shows near identity with the equivalent sequences reported for alpha 2-macroglobulin and C3. Other adjacent short sections also show homology of sequence between the three proteins, and it is highly likely that they contribute to the overall structure that gives a unique reactivity to the thiol ester bond postulated to exist in the native forms of the three proteins.
| S-EPMC1163379 | biostudies-other
Project description:A physics-based method aimed at determining protein structures by using NOE-derived distances together with observed and computed 13C chemical shifts is proposed. The approach makes use of 13Calpha chemical shifts, computed at the density functional level of theory, to obtain torsional constraints for all backbone and side-chain torsional angles without making a priori use of the occupancy of any region of the Ramachandran map by the amino acid residues. The torsional constraints are not fixed but are changed dynamically in each step of the procedure, following an iterative self-consistent approach intended to identify a set of conformations for which the computed 13Calpha chemical shifts match the experimental ones. A test is carried out on a 76-amino acid, all-alpha-helical protein; namely, the Bacillus subtilis acyl carrier protein. It is shown that, starting from randomly generated conformations, the final protein models are more accurate than an existing NMR-derived structure model of this protein, in terms of both the agreement between predicted and observed 13Calpha chemical shifts and some stereochemical quality indicators, and of similar accuracy as one of the protein models solved at a high level of resolution. The results provide evidence that this methodology can be used not only for structure determination but also for additional protein structure refinement of NMR-derived models deposited in the Protein Data Bank.
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Project description:The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-18F-fluoropropane-1-thiol and 2-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (18F-fluoro-PEG4 thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix222/K2CO3 conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-18F-fluoropropyl) benzothioate and 18F-fluoro-PEG4 benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37-47% and 28-35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15?min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]2), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting 18F-fluoride ranged from 6 to 22% in a synthesis time of about 90?min. 18F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]2 maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. 18F-fluoro-PEG4-S-E[c(RGDfK)]2 displayed a somewhat favorable pharmacokinetics compared to 18F-fluoropropyl-S-E[c(RGDfK)]2. Bone uptake was low for both indicating in vivo stability.
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Project description:Peroxynitrite mediates the oxidation of the thiol group of both cysteine and glutathione. This process is associated with oxygen consumption. At acidic pH and a cysteine/peroxynitrite molar ratio of < or = 1.2, there was a single fast phase of oxygen consumption, which increased with increasing concentrations of both cysteine and oxygen. At higher molar ratios the profile of oxygen consumption became biphasic, with a fast phase (phase I) that decreased with increasing cysteine concentration, followed by a slow phase (phase II) whose rate of oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5,5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismutase also inhibited oxygen consumption in phase I, while catalase added during phase II decreased the rate of oxygen consumption. For both cysteine and glutathione, oxygen consumption in phase I was maximal at neutral to acidic pH: in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-phenylnitrone indicated that the yield of thiyl radical adducts had a pH profile comparable with that found for oxygen consumption. The apparent second-order rate constants for the reactions of peroxynitrite with cysteine and glutathione were 1290 +/- 30 M-1.S-1 and 281 +/- 6 M-1.S-1 respectively at pH 5.75 and 37 degrees C. These results are consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction of the peroxynitrite anion with the protonated thiol group, in a second-order process likely to involve a two-electron oxidation, and (b) the reaction of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reaction.
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Project description:Normal human immunoglobulin G (IgG) was treated with radioactive N-ethylmaleimide in the absence of a reducing agent but in the presence of a denaturing solvent. The sites of reaction were determined by isolation of the radioactive peptides from thermolytic digests of the heavy and light chains. Six radioactive peptides were purified from the digest of the light chain and ten from that of the heavy chain. When sequenced, all the peptides were identical with peptides that would be predicted for the half-cystine residues involved in disulphide bonds. The specific radioactivity of the peptides indicated that the proportion of the half-cystine residues in the reduced form varied from 0.57 to 2.54%. These results indicate that the disulphide bonds of IgG are not completely oxidized. The estimate of 0.2mol of SH group/mol of IgG (Cecil & Stevenson, 1965; Luks & Connell, 1968) can be accounted for if 0.8% of every half-cystine residue of the intrachain bonds was in the reduced form. Variable cysteine residues as have been described in several myeloma proteins must occur extremely infrequently among the immunoglobulins.
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