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Antioxidants & Redox Signaling Jul 2014Modification of cysteine thiols dramatically affects protein function and stability. Hence, the abilities to quantify specific protein sulfhydryl groups within complex... (Review)
Review
SIGNIFICANCE
Modification of cysteine thiols dramatically affects protein function and stability. Hence, the abilities to quantify specific protein sulfhydryl groups within complex biological samples and map disulfide bond structures are crucial to gaining greater insights into how proteins operate in human health and disease.
RECENT ADVANCES
Many different molecular probes are now commercially available to label and track cysteine residues at great sensitivity. Coupled with mass spectrometry, stable isotope-labeled sulfhydryl-specific reagents can provide previously unprecedented molecular insights into the dynamics of cysteine modification. Likewise, the combined application of modern mass spectrometers with improved sample preparation techniques and novel data mining algorithms is beginning to routinize the analysis of complex protein disulfide structures.
CRITICAL ISSUES
Proper application of these modern tools and techniques, however, still requires fundamental understanding of sulfhydryl chemistry as well as the assumptions that accompany sample preparation and underlie effective data interpretation.
FUTURE DIRECTIONS
The continued development of tools, technical approaches, and corresponding data processing algorithms will, undoubtedly, facilitate site-specific protein sulfhydryl quantification and disulfide structure analysis from within complex biological mixtures with ever-improving accuracy and sensitivity. Fully routinizing disulfide structure analysis will require an equal but balanced focus on sample preparation and corresponding mass spectral dataset reproducibility.
Topics: Cysteine; Disulfides; Humans; Mass Spectrometry; Molecular Probes; Oxidation-Reduction; Protein Conformation; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 24383618
DOI: 10.1089/ars.2013.5559 -
The Journal of General Physiology May 2018The muscle acetylcholine (ACh) receptor transduces a chemical into an electrical signal, but the efficiency of transduction, or efficacy, depends on the particular...
The muscle acetylcholine (ACh) receptor transduces a chemical into an electrical signal, but the efficiency of transduction, or efficacy, depends on the particular agonist. It is often presumed that full and partial agonists elicit the same structural changes after occupancy of their binding sites but with differing speed and efficiency. In this study, we tested the alternative hypothesis that full and partial agonists elicit distinct structural changes. To probe structural changes, we substituted cysteines for pairs of residues that are juxtaposed in the three-dimensional structure and recorded agonist-elicited single-channel currents before and after the addition of an oxidizing reagent. The results revealed multiple cysteine pairs for which agonist-elicited channel opening changes after oxidative cross-linking. Moreover, we found that the identity of the agonist determined whether cross-linking affects channel opening. For the αD97C/αY127C pair at the principal face of the subunit, cross-linking markedly suppressed channel opening by full but not partial agonists. Conversely, for the αD97C/αK125C pair, cross-linking impaired channel opening by the weak agonist choline but not other full or partial agonists. For the αT51C/αK125C pair, cross-linking enhanced channel opening by the full agonist ACh but not other full or partial agonists. At the complementary face of the subunit, cross-linking between pairs within the same β hairpin suppressed channel opening by ACh, whereas cross-linking between pairs from adjacent β hairpins was without effect for all agonists. In each case, the effects of cross-linking were reversed after addition of a reducing reagent, and receptors with single cysteine substitutions remained unaltered after addition of either oxidizing or reducing reagents. These findings show that, in the course of opening the receptor channel, different agonists elicit distinct structural changes.
Topics: Amino Acid Substitution; Cell Line; Cross-Linking Reagents; Cysteine; Dithiothreitol; Humans; Hydrogen Peroxide; Ion Channel Gating; Receptors, Cholinergic; Sulfhydryl Reagents
PubMed: 29680816
DOI: 10.1085/jgp.201711881 -
Kidney International May 1991The present study was designed to address the reactivity and accessibility of the particular class of sulfhydryl groups involved in the regulatory process of renin...
The present study was designed to address the reactivity and accessibility of the particular class of sulfhydryl groups involved in the regulatory process of renin secretion. Both mercurial (such as P-chloromercuriphenyl sulfonate [PCMPS] and non-mercurial sulfhydryl reagents (for example, 6,6-dithiodinicotinic acid [DTDN]), which very slowly penetrate the cell membrane of intact cells, stimulated renin secretion. The membrane permeant sulfhydryl reagent N-ethylmaleimide had no effect on renin secretion but its membrane impermeant derivative, stilbene maleimide, strongly stimulated secretion. Furthermore, disulfide reducing agents such as dithiothreitol (DTT) had no effect on renin secretion at low concentrations, but strongly inhibited it at high concentrations. Several reagents which are known to primarily deplete cellular reduced glutathione were without effect on renin secretion. The stimulation of renin secretion by PCMPS was rapid in onset, and prevented and reversed by DTT and L-cysteine. Furthermore, the maximal stimulatory effect of PCMPS was not additive to that by diuretics with sulfhydryl reactivity (such as, ethacrynic acid and mersalyl). The stimulatory effect of PCMPS was not affected by diuretics which lack sulfhydryl reactivity (such as, bumetanide and furosemide). These results suggest that sulfhydryl reagents of both with and without diuretic activity stimulate renin secretion by reacting with specific class of sulfhydryl groups which are readily accessible from the extracellular compartment. In addition, these results provide further support the possibility that a sulfhydryl-disulfide interchange in the membrane may play a regulatory role in the renin secretory process.
Topics: 4-Chloromercuribenzenesulfonate; Animals; Cell Membrane; Ethylmaleimide; Kidney Cortex; Rabbits; Renin; Sulfhydryl Reagents
PubMed: 1648645
DOI: 10.1038/ki.1991.109 -
Journal of Biochemistry May 1988The effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively...
The effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively studied by using a newly developed HPLC assay method. This HPLC method directly measures the product (p-aminobenzoate, PAB), and is not interfered with by sulfhydryl-reactive agents. Further, because the substrate solution of this HPLC assay method contains only substrate (biotin 4-amidobenzoate) and phosphate buffer, accurate studies on the effects of sulfhydryl blocking reagents on the purified enzyme could be performed. Biotinidase activities in human sera (n = 83) were always enhanced by 2-mercaptoethanol (ME). The optimum concentration was found to be 1 mM. The degree of activation was variable (100 to 400% of the original) depending on the serum sample. Sulfhydryl blocking reagents such as organic mercurials were tested on fresh serum and purified enzyme. Mercuric agents were found to inhibit the activity of fresh serum and purified enzyme at 0.05 and 0.005 mM, respectively. Sulfhydryl alkylating agents, N-ethylmaleimide (NEM) and dithiobis(2-nitro)benzoic acid (DTNB), inhibited 100 and 64% of the activity of the purified enzyme at 0.1 and 1.0 mM, respectively. However, lower concentrations (less than 5 nM) of organic mercurials and mercuric ion exhibited a slight enhancement (20-30%) of the activity of the purified enzyme. These results indicate the presence of an essential sulfhydryl residue at the active center. The enzyme contains 2.5 sulfhydryls per molecule, as determined by using Ellman's assay method. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) did not inhibit the enzyme activity at 0.05 mM or higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Amidohydrolases; Biotinidase; Chromatography, High Pressure Liquid; Enzyme Activation; Humans; In Vitro Techniques; Mercaptoethanol; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 3182746
DOI: 10.1093/oxfordjournals.jbchem.a122345 -
Bulletin of the World Health... 1981In vitro cultivation of Treponema pallidum would facilitate many different aspects of syphilis research. This review summarizes developments in this field that have been... (Review)
Review
In vitro cultivation of Treponema pallidum would facilitate many different aspects of syphilis research. This review summarizes developments in this field that have been published since 1975. Findings are discussed in terms of treponemes and the oxygen question, treponemal metabolism involving proteins, nucleic acids, and fatty acids, and treponemal interaction with tissue culture cells. Suggested future approaches and potential problem areas pertinent to successful cultivation are discussed.
Topics: Animals; Bacterial Proteins; Culture Techniques; DNA, Bacterial; Oxygen Consumption; RNA, Bacterial; Rabbits; Sulfhydryl Reagents; Treponema pallidum
PubMed: 6172213
DOI: No ID Found -
Current Protocols in Protein Science May 2001The reagents and methods for purification of the denaturants guanidine hydrochloride (guanidine-HCl) and urea are described. Sulfhydryl reagents (reducing agents) and...
The reagents and methods for purification of the denaturants guanidine hydrochloride (guanidine-HCl) and urea are described. Sulfhydryl reagents (reducing agents) and "oxido-shuffling" (or oxidative regeneration) systems are also discussed.
Topics: Dithiothreitol; Glutathione; Guanidine; Mercaptoethanol; Oxidants; Protein Denaturation; Protein Folding; Reducing Agents; Sulfhydryl Reagents; Urea
PubMed: 18429069
DOI: 10.1002/0471140864.psa03as00 -
Biochimica Et Biophysica Acta.... Jan 2018Cardiovascular dysfunction and organ damage are hallmarks of sepsis and septic shock. Protein S-nitrosylation by nitric oxide has been described as an important modifier...
Cardiovascular dysfunction and organ damage are hallmarks of sepsis and septic shock. Protein S-nitrosylation by nitric oxide has been described as an important modifier of protein function. We studied whether protein nitrosylation/denitrosylation would impact positively in hemodynamic parameters of septic rats. Polymicrobial sepsis was induced by cecal ligation and puncture. Female Wistar rats were treated with increasing doses of DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] 30min before or 4 or 12h after sepsis induction. Twenty-four hours after surgery the following data was obtained: aorta response to phenylephrine, mean arterial pressure, vascular reactivity to phenylephrine, biochemical markers of organ damage, survival and aorta protein nitrosylation profile. Sepsis substantially decreases blood pressure and the response of aorta rings and of blood pressure to phenylephrine, as well as increased plasma levels of organ damage markers, mortality of 60% and S-nitrosylation of aorta proteins increased during sepsis. Treatment with DTNB 12h after septic shock induction reversed the loss of response of aorta rings and blood pressure to vasoconstrictors, reduced organ damage and protein nitrosylation and increased survival to 80%. Increases in protein S-nitrosylation are related to cardiovascular dysfunction and multiple organ injury during sepsis. Treatment of rats with DTNB reduced the excessive protein S-nitrosylation, including that in calcium-dependent potassium channels (BK), reversed the cardiovascular dysfunction, improved markers of organ dysfunction and glycemic profile and substantially reduced mortality. Since all these beneficial consequences were attained even if DTNB was administered after septic shock onset, protein (de)nitrosylation may be a suitable target for sepsis treatment.
Topics: Animals; Arterial Pressure; Denitrification; Disease Models, Animal; Dithionitrobenzoic Acid; Female; Nitrosation; Nitrosative Stress; Rats; Rats, Wistar; Shock, Septic; Sulfhydryl Reagents; Treatment Outcome
PubMed: 29111468
DOI: 10.1016/j.bbadis.2017.10.029 -
Dental Materials : Official Publication... Apr 2014Thiol- and allyl-functionalized siloxane oligomers are synthesized and evaluated for use as a radical-mediated, rapid set elastomeric dental impression material....
OBJECTIVES
Thiol- and allyl-functionalized siloxane oligomers are synthesized and evaluated for use as a radical-mediated, rapid set elastomeric dental impression material. Thiol-ene siloxane formulations are crosslinked using a redox-initiated polymerization scheme, and the mechanical properties of the thiol-ene network are manipulated through the incorporation of varying degrees of plasticizer and kaolin filler. Formulations with medium and light body consistencies are further evaluated for their ability to accurately replicate features on both the gross and microscopic levels. We hypothesize that thiol-ene functionalized siloxane systems will exhibit faster setting times and greater detail reproduction than commercially available polyvinylsiloxane (PVS) materials of comparable consistencies.
METHODS
Thiol-ene functionalized siloxane mixtures formulated with varying levels of redox initiators, plasticizer, and kaolin filler are made and evaluated for their polymerization speed (FTIR), consistency (ISO4823.9.2), and surface energy (goniometer). Feature replication is evaluated quantitatively by SEM. The Tg, storage modulus, and creep behavior are determined by DMA.
RESULTS
Increasing redox initiation rate increases the polymerization rate but at high levels also limits working time. Combining 0.86 wt% oxidizing agent with up to 5 wt% plasticizer gave a working time of 3 min and a setting time of 2 min. The selected medium and light body thiol-ene formulations also achieved greater qualitative detail reproduction than the commercial material and reproduced micrometer patterns with 98% accuracy.
SIGNIFICANCE
Improving detail reproduction and setting speed is a primary focus of dental impression material design and synthesis. Radical-mediated polymerizations, particularly thiol-ene reactions, are recognized for their speed, reduced shrinkage, and 'click' nature.
Topics: Allyl Compounds; Cross-Linking Reagents; Dental Impression Materials; Elastomers; Kaolin; Materials Testing; Polymerization; Polymers; Siloxanes; Sulfhydryl Reagents
PubMed: 24553250
DOI: 10.1016/j.dental.2014.01.011 -
Methods in Cell Biology 2010Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule... (Review)
Review
Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule polymerization. 5',5'-Dithiobis(2-nitrobenzoate) (DTNB) reacts with free and accessible sulfhydryls and stoichiometrically produces a detectable product, which allows an exact measurement of reacted thiols. Since binding of small ligands may alter conformational dynamics, it may also affect the reactivity of thiols on tubulin. Differences in DTNB reactivity with thiols upon ligand binding can therefore be used to deduce binding characteristics. We will describe two methods that use tubulin cysteine reactivity with DTNB in the presence of drug to define ligand-binding characteristics.
Topics: Animals; Drug Evaluation, Preclinical; Humans; Ligands; Protein Binding; Sulfhydryl Compounds; Sulfhydryl Reagents; Tubulin; Tubulin Modulators
PubMed: 20466146
DOI: 10.1016/S0091-679X(10)95021-8 -
The Journal of Biological Chemistry Jul 1991Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed...
Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.
Topics: Alanine; Animals; Ethylmaleimide; Glutaminase; Glutamine; Hydroxybutyrate Dehydrogenase; Intracellular Membranes; Kidney Cortex; Kinetics; Mitochondria; Submitochondrial Particles; Sulfhydryl Reagents; Swine
PubMed: 2071598
DOI: No ID Found