Further studies on the thiol groups of human haemoglobin.
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ABSTRACT:
SUBMITTER: Adler A
PROVIDER: S-EPMC1270182 | biostudies-other | 1966 Dec
REPOSITORIES: biostudies-other
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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.
| S-EPMC1166115 | biostudies-other
Project description:1. alpha(A)-Chain sub-units have been isolated from neutral haemolysates containing certain unstable beta-chain haemoglobin variants. They are identical, in electrophoretic properties, tryptic peptide analyses and their ability to recombine with Hb-beta(A) (4) at neutral pH, with the alpha(A)-chain sub-units isolated from Hb-A at acid pH. Abnormal alpha(G)-chain sub-units have been prepared from haemolysates containing the alpha-chain variant Hb-G. 2. alpha(A)-Chain sub-units prepared by either method are eluted from Sephadex at low haemoglobin concentration as monomer sub-units. The elution volume, sedimentation coefficient and apparent molecular weight are, however, concentration-dependent, suggesting an equilibrium between monomers and higher polymers with the former as the predominant species. Elevated temperatures cause the alpha(A)-chain sub-unit to aggregate and denature. 3. The product of the reaction between the alpha(A)-chain sub-unit and Hb-beta(A) (4) has been characterized as Hb-A. Only Hb-A and Hb-G can be detected as the products when a mixture of alpha(A)- and alpha(G)-chain sub-units reacts with Hb-beta(A) (4). The absence of the species alpha(A)alpha(G)beta(A) (2) can be explained in terms of the rapid equilibrium between whole and half haemoglobin molecules and the reassortment of alphabeta sub-units during the separation process.
| S-EPMC1270193 | biostudies-other
Project description:1. The proportion of thiol groups in the total thiol+disulphide of histone extracts from fertilized eggs from Echinus and Psammechinus was increased during periods of structural alterations in the nucleus. 2. The probable start of DNA synthesis in the fertilized eggs coincided with periods of maximum thiol content. 3. Histone extracts from rat liver and regenerating liver were predominantly in the thiol form and no significant variations could be detected during the first 30hr. after partial hepatectomy. 4. An assay system was developed to follow the phosphorylation believed to be associated with the arginine-rich histone F3. Phosphorylation increased by 50% at 1 and 2hr. after partial hepatectomy. The phosphate content also increased during the period of DNA synthesis. 5. The increased phosphorylation found 1hr. after partial hepatectomy was not prevented by actinomycin or prior irradiation. 6. The phosphate content of histone F1 was very high in livers from foetal rats and declined in neonatal rats similarly to the decline in DNA synthesis.
| S-EPMC1187644 | biostudies-other
Project description:A variety of alkenes substituted by electron-withdrawing groups serve as competent electrophiles for the stereoselective, intramolecular nucleophilic addition of sulfonimidoyl carbanions to form benzothiazines. This reaction generally proceeds with complete stereoselectivity within the limits of our detection. In some cases, benzothiazine formation occurs in a single pot at relatively high temperatures during N-arylation of the simple sulfoximine used in this study. Yet, the process occurs with the same direction and extent of stereoselectivity as that seen when the Michael addition is performed at very low temperatures.
| S-EPMC2692266 | biostudies-literature
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: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.
| S-EPMC7365990 | biostudies-literature
Project description:Rhnull human erythrocytes lack all the antigens of the Rhesus blood-group system and are associated with mild chronic haemolytic anaemia. These erythrocytes have an abnormal shape and increased osmotic fragility. Labelling studies with the impermeant maleimide N-maleoylmethionine [35S]sulphone show that Rhnull erythrocytes lack two extracellular thiol-group-containing membrane components of apparent mol.wts. 32 000 and 34 000. Immunoprecipitation with mouse monoclonal antibody R6A (which reacts with all normal erythrocytes, but fails to react with Rhnull erythrocytes) specifically precipitates the 34 000-mol.wt. component from normal erythrocytes. Similar studies with human anti-Rh(D) serum shows that this antibody reacts with the 32 000-mol.wt. component. The results suggest that the R6A-binding polypeptide and the Rh(D) polypeptide may be involved in the maintenance of the shape and viability of the human erythrocyte.
| S-EPMC1152118 | biostudies-other
Project description:1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.
| S-EPMC1178058 | biostudies-other
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.
| S-EPMC1218173 | biostudies-other
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.
| S-EPMC6983705 | biostudies-literature