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Acta Medica Okayama Apr 19832-Mercaptoethanol increases the optical density of assay solutions at wavelengths between 280 to 400 nm, and therefore interferes with the measurement of protein...
2-Mercaptoethanol increases the optical density of assay solutions at wavelengths between 280 to 400 nm, and therefore interferes with the measurement of protein concentration by the microbiuret method. Protein concentration can be determined in the presence of 2-mercaptoethanol up to 6 mM by modification of the method as follows: after the precipitation of protein by trichloroacetic acid in the presence of deoxycholate, the precipitate is resolubilized with NaOH solution. Dithiothreitol interfered with the protein determinations could by made in the presence of 4 mM of dithiothreitol with the modified microbiuret method. This modified method is time-saving and more reliable than other methods for protein determination, such as Lowry's method, in the presence of sulfhydryl reagents.
Topics: Proteins; Spectrophotometry, Ultraviolet; Sulfhydryl Reagents
PubMed: 6869062
DOI: 10.18926/AMO/32414 -
Biomedical Chromatography : BMC Jul 2003Thiol-containing compounds pose bioanalytical challenge in several dimensions due to extreme reactivity of the sulfhydryl group. The development of robust bioanalytical... (Review)
Review
Thiol-containing compounds pose bioanalytical challenge in several dimensions due to extreme reactivity of the sulfhydryl group. The development of robust bioanalytical methodology for thiol groups should address the aspects of adequate stabilization in the biological matrix and selectivity considerations. In this context, availability of plethora of thiol reagents provides ample opportunity to achieve the above goals. However, several considerations need to be factored into the decision-making of a suitable scheme for the thiol drugs under investigation. This review provides some critical insights to many such considerations that may be vital for development and validation of methods for thiol-containing drugs.
Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Humans; Molecular Structure; Pharmaceutical Preparations; Substrate Specificity; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 12884392
DOI: 10.1002/bmc.256 -
Science (New York, N.Y.) Oct 1981Ethanol induces hemorrhagic gastric erosions and causes a dose-dependent decrease in the concentration of nonprotein sulfhydryl compounds in rat gastric mucosa....
Ethanol induces hemorrhagic gastric erosions and causes a dose-dependent decrease in the concentration of nonprotein sulfhydryl compounds in rat gastric mucosa. Sulfhydryl-containing drugs protect rats from ethanol-induced gastric erosions, whereas sulfhydryl blocking agents counteract the mucosal cytoprotective effect of prostaglandin F2 beta. These observations suggest that endogenous nonprotein sulfhydryls may mediate prostaglandin-induced gastric cytoprotection and that sulfhydryl drugs may have potential for preventing or treating hemorrhagic gastric erosions.
Topics: Animals; Ethanol; Gastric Mucosa; Prostaglandins; Prostaglandins F; Rats; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 7280691
DOI: 10.1126/science.7280691 -
Chemico-biological Interactions May 1980The effect of sulfhydryl reagents on phagocytosis and concomitant enzyme release and on ionophore A 23187 + Ca2+-induced exocytosis in rabbit polymorphonuclear...
The effect of sulfhydryl reagents on phagocytosis and concomitant enzyme release and on ionophore A 23187 + Ca2+-induced exocytosis in rabbit polymorphonuclear leukocytes (PMN's) was studied. Membrane-penetrating sulfhydryl reagents such as cytochalasin A and N-naphthylmaleimide in micromolar concentrations inhibit both phagocytosis and exocytosis. Poorly penetrating reagents such as p-chloromercuribenzene sulfonate (pCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), inhibit only in high concentrations (pCMBS), or they are ineffective as inhibitors (DTNB). Inhibition by pCMBS is not reversed by glutathione or dithiothreitol; this suggests that some pCMBS probably enters the cell. Specific intracellular sulfhydryl compounds appear to be essential in the cellular apparatus involved in phagocytosis and exocytosis; various possibilities are considered. A concentration of N-naphthylmaleimide which completely inhibits phagocytosis and exocytosis leaves cellular ATPase activity intact.
Topics: 4-Chloromercuribenzenesulfonate; Cytochalasins; Exocytosis; Glutathione; Ionophores; Maleimides; Muramidase; Neutrophils; Phagocytosis; Sulfhydryl Reagents
PubMed: 6248254
DOI: 10.1016/0009-2797(80)90121-0 -
Antioxidants & Redox Signaling Aug 2001Recent results on the oxidation of cysteine residues that bind zinc in transcription factors and their analogous peptides and in related proteins and model systems are... (Review)
Review
Recent results on the oxidation of cysteine residues that bind zinc in transcription factors and their analogous peptides and in related proteins and model systems are reviewed. Two classes of oxidants, the transition metals and dioxygen, hydrogen peroxide, and related species, are considered, and the role of metal ions in suppressing or enhancing Cys oxidation is a major focus. Cysteines in the zinc-bound structures of transcription factors are less susceptible to oxidation than in the metal-free form, and this appears to correlate with reduced accessibility of the thiolates to oxidants. Substitution of other metal ions for Zn(II) increases the rate of Cys oxidation, apparently through increased oxidant accessibility. Reactions that result in reversible or irreversible oxidation of these zinc-binding cysteines under biological conditions are identified in the context of deleterious implications for gene expression.
Topics: Amino Acid Sequence; Antioxidants; Binding Sites; Cobalt; Cysteine; Gene Expression Regulation; Hydrogen Peroxide; Iron; Metalloproteins; Metallothionein; Models, Molecular; Molecular Sequence Data; Nickel; Oxidants; Oxidation-Reduction; Oxygen; Protein Conformation; Structure-Activity Relationship; Sulfhydryl Reagents; Transcription Factors; Zinc; Zinc Fingers
PubMed: 11554444
DOI: 10.1089/15230860152542925 -
ACS Chemical Biology Aug 2017The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of...
The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.
Topics: Cell Line, Tumor; Cell Membrane; Drug Discovery; Humans; Mass Spectrometry; Models, Biological; Molecular Structure; Phenols; Sulfhydryl Reagents; Sulfones; Tetrazoles
PubMed: 28687042
DOI: 10.1021/acschembio.7b00444 -
Analytical Biochemistry Mar 1987Human lactate dehydrogenase isozymes, LDH-1 and LDH-5, were inactivated at 25 degrees C and pH 7.5 by N-alkylmaleimides of varying chain length, and by fluorescein...
Human lactate dehydrogenase isozymes, LDH-1 and LDH-5, were inactivated at 25 degrees C and pH 7.5 by N-alkylmaleimides of varying chain length, and by fluorescein mercuric acetate. Second-order rate constants for the inactivation of LDH-5 by N-alkylmaleimides increased with increasing chain length of the maleimide derivative while essentially no chain-length effect was observed in the inactivation of LDH-1. Both isozymes were effectively inactivated by low concentrations of fluorescein mercuric acetate, and in both cases saturation kinetics were observed. Dissociation constants obtained from double-reciprocal plotting methods indicated a twofold better binding of fluorescein mercuric acetate to LDH-1. Protection from fluorescein mercuric acetate by NAD was observed with both enzymes.
Topics: Fluoresceins; Humans; Isoenzymes; Kinetics; L-Lactate Dehydrogenase; Organomercury Compounds; Structure-Activity Relationship; Sulfhydryl Reagents
PubMed: 3578792
DOI: 10.1016/0003-2697(87)90449-0 -
European Journal of Biochemistry Mar 1995The interaction of sulfhydryl reagents with the carnitine carrier of rat liver mitochondria was studied in detail in proteoliposomes. The addition of N-ethylmaleimide,...
Probing the active site of the reconstituted carnitine carrier from rat liver mitochondria with sulfhydryl reagents. A cysteine residue is localized in or near the substrate binding site.
The interaction of sulfhydryl reagents with the carnitine carrier of rat liver mitochondria was studied in detail in proteoliposomes. The addition of N-ethylmaleimide, mercurials at low concentrations, Cu(2+)-phenanthroline and diamide modified a single sulfhydryl group (the class II group) that is involved in transport function. The treatment of the inhibited protein with 1,4-dithioerythritol led to full recovery of carnitine exchange except for N-ethylmaleimide. Evidence is provided that the addition of carnitine to the carrier blocks the interaction of the sulfhydryl reagents with the protein. This result strongly suggests that the critical cysteine residue is localized in, or near, the substrate binding site. Interaction of other cysteine residues in the carrier protein with high concentrations of mercurials modified another class of sulfhydryl groups (the class I group) that are not directly involved in carnitine transport. The oxidized and reduced forms of the carnitine carrier show slightly different molecular masses on SDS/PAGE. Disulfide bridge(s) induced by Cu(2+)-phenanthroline and diamide are present in a single polypeptide part of the protein and induced no disulfide bridges between two polypeptide chains.
Topics: Animals; Binding Sites; Carnitine; Carrier Proteins; Cysteine; Disulfides; Mitochondria, Liver; Molecular Weight; Oxidation-Reduction; Phenanthrolines; Rats; Sulfhydryl Reagents
PubMed: 7705339
DOI: No ID Found -
Inhibition of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi by sulfhydryl reagents.Comparative Biochemistry and... 19791. The NADP-linked glutamate dehydrogenase purified from epimastigotes of Trypanosoma cruzi was strongly, but not completely, inhibited by sulfhydryl reagents, in the...
1. The NADP-linked glutamate dehydrogenase purified from epimastigotes of Trypanosoma cruzi was strongly, but not completely, inhibited by sulfhydryl reagents, in the presence of Tris-HCl or phosphate buffers. 2. The enzyme modified by preincubation with o-iodosobenzoate had a kinetic behaviour different from that shown by the enzyme modified with other inhibitors, such as N-ethylmaleimide or p-chloromercuribenzoate. 3. The inhibition by o-iodosobenzoate was additive with the inhibition by the other reagents tested. 4. It is suggested that two or more different sulfhydryl groups, placed probably near the active site, are involved in these effects.
Topics: Animals; Glutamate Dehydrogenase; Kinetics; Sulfhydryl Reagents; Trypanosoma cruzi
PubMed: 400963
DOI: 10.1016/0305-0491(79)90059-2 -
Science (New York, N.Y.) Jul 1946
Topics: Heart; Humans; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 21064784
DOI: No ID Found