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PubMed1987

Disulfide inhibition of copper-catalyzed oxidation of ascorbic acid: spectrophotometric evidence for accumulation of a stable complex.

Copper-catalyzed oxidation of ascorbic acid was retarded in the presence of the biological disulfide compounds cystine and oxidized glutathione. The evidence suggested that this effect was due to the formation of a stable complex involving the copper ion, the disulfide compound, and ascorbic acid or a derivative formed during the oxidative process. This indicated that less copper was available for the formation of oxygen complexes which are not as stable as the disulfide complexes. Ellman's reagent (Nbs2) was reduced when it was substituted for the biological disulfides or when added, with EDTA, to solutions in which ascorbic acid, copper ion, and the biological disulfides had been allowed to interact. The complex formed with cystine was detected at 360 nm but the glutathione complex was not detected at this wavelength. It is proposed that disruption of cystine or glutathione complexes by EDTA results in formation of 2,3-diketogulonic acid which acts as a reductant of Ellman's reagent.

Ascorbic Acid↗

PubMed1987

Sulfhydryl oxidase-catalyzed formation of disulfide bonds in reduced ribonuclease.

Sulfhydryl oxidase isolated from bovine skim milk membrane vesicles catalyzes de novo formation of disulfide bonds with the substrates cysteine, cysteine-containing peptides, and reduced proteins using molecular oxygen as the electron acceptor. Initial rates for sulfhydryl oxidase-catalyzed oxidation of reduced ribonuclease exhibited typical Michaelis-Menten kinetics at low substrate concentrations. Substrate inhibition of the oxidative activity was observed at ribonuclease concentrations greater than 40 microM, similar to that observed with reduced glutathione or other small thiol substrates. The inhibition was more pronounced when ribonuclease activity was used to monitor the rates, presumably due to concentration-dependent formation of nonnative disulfide bonds. Thus, a maximum in the rate of regain of ribonuclease activity was observed at a 40 microM concentration, while optimum recovery was observed at 30 microM. The Michaelis constant obtained with reduced ribonuclease is 17.4 microM which corresponds to a sulfhydryl concentration of 0.14 mM, a value that compares favorably with the best small thiol substrate, reduced glutathione. Disulfide-containing intermediates in the oxidation pathway, as determined by ion-exchange chromatography of alkylated reaction mixtures, appeared to be similar for air oxidation and enzyme-catalyzed oxidation of the protein. The pH optimum, tissue location, and kinetic characteristics of sulfhydryl oxidase are compatible with a suggested physiological function of direct catalysis of disulfide bond formation in secretory proteins or indirect participation through provision of oxidized glutathione for protein disulfide-isomerase-catalyzed thiol/disulfide interchange.

Animals↗

PubMed1987

Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis.

Phosphocellulose-purified tubulin has been shown to form a characteristic "ladder" of nonmicrotubular aggregates during nondenaturing gel electrophoresis (J. J. Correia and R. C. Williams, Jr. (1985) Arch. Biochem. Biophys. 239, 120-129). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of alpha- and beta-subunits of tubulin. Omission of beta-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, alpha- or beta-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of beta-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included greater than 75% alcohol, excess H2O2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in alpha beta-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide-crosslinked tubulin aggregates.

Chromatography, Ion Exchange↗

PubMed1984

Characterization of an essential disulfide bond associated with the active site of the renal brush-border membrane D-glucose transporter.

In a previous report (J. Biol. Chem. 258 (1983) 3565-3570) we have demonstrated that the disulfide-reducing agent dithiothreitol has two effects on the sodium-dependent outer cortical brush border membrane D-glucose transporter; the first results in a reversible increase in the affinity of the transporter for the non-transported competitive inhibitor phlorizin, while the second results in a partially reversible loss of phlorizin binding and glucose-transport activity. Evidence was presented that both of these effects are the result of the reduction of disulfide bonds on the transport molecule. In the present paper we extend our observations on the inactivation of the transporter by dithiothreitol. We provide evidence here (i) that the inactivation of the transporter by dithiothreitol is independent of the effect of the reducing agent on the affinity of the transporter, (ii) that this inactivation process is first-order in dithiothreitol and thus presumably due to the reduction of a single disulfide bond essential to the functioning of the transporter. (iii) that it is the reduction of this disulfide bond and not some subsequent conformational or other change in the transporter which results in its inactivation, (iv) that phlorizin and substrates of the transporter provide protection against inactivation by dithiothreitol and that the degree of protection provided correlates well with the known specificity and phlorizin-binding properties of the transporter, and (iv) that the reactivity of the transporter with dithiothreitol is pH-dependent, decreasing with increasing pH over the pH range 6.5-8.5. We conclude that this site of action of dithiothreitol is a single essential disulfide bond intimately associated with the glucose-binding site on the transport molecule.

Animals↗

PubMed1989

Evidence for oligomeric forms of transducins alpha subunit: formation of intermolecular alpha-alpha disulfide linkages.

Transducin, the retinal G-protein, is a heterotrimeric protein composed of alpha, beta and gamma subunits. Intermolecular disulfide linkages between the alpha-subunits of transducin molecules are spontaneously formed when the purified G-protein is placed in a non-reducing buffer system. The beta and gamma subunits do not participate in the intermolecular disulfide bridge formation. The alpha-alpha subunit disulfide bonds result in the inhibition of transducin activation by bleached rhodopsin which is restored by reducing the disulfides with dithiothreitol. The trapping of oligomers by disulfide bond formation provides physical evidence for specific intermolecular interactions between alpha-subunits of transducin.

Animals↗

PubMed1988

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation.

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.

Animals↗

PubMed1988

Destabilization of lens protein conformation by glutathione mixed disulfide.

Mixed disulfide between lens crystallin and glutathione has been observed in human cataracts and could be formed in vitro by thiol-disulfide exchange reaction. The glutathionyl crystallins have been reported to become partially unfolded. The present paper reports the conformational destabilization by the mixed disulfide formation in calf alpha- and gamma-II crystallin. The conformational stability was studied by the denaturants urea and guanidine hydrochloride (Gdn-HCl), and by proteolytic degradation. The denaturation curves of both urea and Gdn-HCl shift to lower denaturant concentration for crystallins of glutathione mixed disulfide. The decrease in conformational stability is estimated to be 0.22- and 0.92 kcal mol-1 for modified alpha- and gamma-II crystallin, respectively. Proteolytic digestion also shows a faster rate of degradation for the modified crystallins. These results indicate that mixed disulfide destabilizes the crystallin conformation. The destabilization may make crystallins more susceptible to changes as observed in aging lenses.

Animals↗

PubMed1989

Identification of thiol groups and a disulfide crosslink site in bovine myelin proteolipid protein.

The existence of disulfide crosslinks limits the number of possible folded structures a protein can assume. Thus localization of disulfide and thiol groups is a key to understanding the conformation and orientation of myelin proteolipid protein (PLP) in the myelin membrane. [14C]Carboxamidomethylated PLP was fragmented with chymotrypsin, and the resulting mixture was partially separated by reversed-phase HPLC. Purified 14C-labeled peptides and a disulfide containing peptide were characterized by amino acid analysis. These experiments showed that Cys-32 and Cys-34 are free thiols, and are presumably on the interior of the cell or within the membrane bilayer, and that Cys-200 and Cys-219 are joined by a disulfide bond, and are probably located on the extracellular face of the membrane. Sequence analysis experiments indicate that Cys-5, Cys-6 and Cys-9 are linked by disulfides, probably to other parts of the protein on the extracellular face of the membrane.

Amino Acids↗

PubMed1992

Thermal motions of surface alpha-helices in the D-galactose chemosensory receptor. Detection by disulfide trapping.

The D-galactose chemosensory receptor of Escherichia coli is a .32 kDa globular protein possessing two distinct structural domains, each organized in an alpha/beta folding motif. Helices I and X lie at adjacent approximately parallel positions on the surface of the N-terminal domain, near the hinge region. In order to analyze the relative thermal motions of these two helices, the present study utilizes a generalizable disulfide trapping approach: first, site-directed mutagenesis is used to place a pair of cysteine residues at locations of interest on the protein surface, then disulfide bond formation is used to trap intramolecular cysteine-cysteine collisions resulting from thermal motions. Specifically, four engineered di-cysteine receptors have been constructed, each possessing one cysteine at position 26 on helix I, and a second cysteine at varying positions on helix X. A fifth control receptor possesses one cysteine at position 26, and a second on the opposite surface of the molecule. These surface cysteine substitutions have little or no effect on the measurable receptor parameters as judged by ligand binding equilibria and kinetics, protein stability, and 19F nuclear magnetic resonance, indicating that the engineered receptors are useful probes of native backbone dynamics. Spatial and kinetic features of backbone motions have been investigated by measuring intramolecular disulfide formation rates for cysteine pairs in the fully liganded receptor. The resulting rates decrease monotonically with increasing distance between cysteines in the crystal structure, while no disulfide formation is observed for the control pair unless the molecule is unfolded. The minimum translational amplitudes of the observed backbone motions range from 4.5 to 15.2 A, and the minimum rotational amplitudes are as large as 35 degrees. For each motion the rate of intramolecular sulfhydryl-sulfhydryl collision has been estimated from the measured rate of disulfide formation: the 4.5 and 15.2 A translations yield approximately 10(4) and approximately 10 collisions s-1 molecule-1, respectively. These collision rates, which are faster than ligand dissociation, likely underestimate the actual motional frequencies since only an undetermined fraction of the total motions yield collisions. The simplest plausible trajectory capable of producing such collisions is a rate-limiting translation of one or both helices along their long axes, coupled with minor helix rotations. When sugar is removed from the receptor, a substantial increase in backbone dynamics is observed, indicating the presence of new long-range backbone trajectories. Overall, the results suggest that internal motions in proteins may have larger amplitudes than previously observed.

Calcium-Binding Proteins↗

PubMed1992

Disulfide bonds are essential for the stability of the Sindbis virus envelope.

Sindbis virus is a membrane-containing virus which has two glycoproteins organized in an icosahedral lattice. Protein-protein associations have been identified which participate in the formation of the icosahedron and these associations are stabilized by intramolecular disulfide bridges (Anthony, R. P., and Brown, D. T., 1990, J. Virol. 65, 1187-1194). The present study further examines the role of disulfides in the structure and function of Sindbis virus by following the effect of dithiothreitol on the protease sensitivity of envelope proteins as well as the electron microscopic appearance and infectivity of Sindbis virus. Treatment of isolated virus with 5 mM dithiothreitol for 6 hr causes a marked increase in trypsin sensitivity of both E1 and E2, profound morphological alterations in the viral envelope, increased susceptibility of the nucleocapsid to RNase, and 95% loss of infectivity. These effects are greatly enhanced and accelerated when treatment with DTT is preceded by a brief exposure of the virus to pH 5.3, suggesting that acid-induced conformational changes render structurally critical disulfides more accessible to reductive cleavage by DTT. When compared to other manipulations known to change the conformation of the viral envelope, such as heating to 51 or 60 degrees or exposure to acid pH, only the exposure to DTT with or without prior acid treatment caused marked structural changes correlated with a loss of infectivity. These data provide electron microscopic and functional evidence that intact disulfide bonds are critical for the stability of the virus envelope and suggest that the cleavage of critical disulfide(s) may play a role in the process of virus infection.

Animals↗

PubMed1995

Predicted disulfide-bonded structures for three uniquely related proteins of Plasmodium falciparum, Pfs230, Pfs48/45 and Pf12.

Pfs230 is a surface protein of the gametes of Plasmodium falciparum and has been demonstrated to be a target of malaria transmission-blocking antibodies; it is an important candidate antigen for a transmission-blocking vaccine. The target epitopes of transmission-blocking antibodies against Pfs230 are almost all reduction sensitive suggesting that disulfide bonds are critical for folding the native molecule. Following the cloning of the Pfs230 gene attempts are now underway to express subunits of the protein for use in vaccine trials. It will be important to understand the disulfide-bond structure of the Pfs230 to achieve this goal. In this paper we present a model for this structure based on the observation that the Pfs230 molecule contains a series of regularly repeated cysteine-containing motifs. Four such motifs have been identified, together with a fifth cysteineless motif, which occur in the same relative order, with regular alternating omission of specific motifs, 14 times throughout the length of the protein. Each of the 14 sets of motifs contains an even number of cysteine residues (2, 4 or 6). We postulate that each set folds into a separate disulfide-bonded domain in which corresponding pairs of cysteines form an equivalent disulfide bond in every such domain. The postulated bonding arrangements in the different domains are mutually confirmatory throughout the sequence of Pfs230. We have identified two other malaria proteins, Pfs48/45 and Pf12, which share the same arrangements of motifs and conform to the same disulfide-bond structure proposed for Pfs230; no other proteins in the sequence data base share these characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

The positions of the disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa.

The positions of the interchain and intrachain disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa were determined. The lectin (Mr 140,000) is composed of the same subunit (Mr 22,000) which is cross-linked by disulfide bonds to form a dimer. Intact lectin yielded two fragments, CB1 and CB2, by cleavage with cyanogen bromide. One intrachain and two interchain disulfide bonds were identified as Cys-53-Cys-61, Cys-14-Cys-50' and Cys-50-Cys-14', respectively, by enzymatic digestion and Edman degradation of CB1. Two intrachain disulfide bonds were determined as Cys-78-Cys-168 and Cys-144-Cys-160 by enzymatic digestion of CB2. The two intrachain disulfide bonds are well conserved through all invertebrate lectins and calcium-dependent animal lectins. S-Carboxamidomethylated lectin was digested with Staphylococcus aureus V8 proteinase and separated by reversed-phase HPLC. Glycopeptides were detected by the 4-N,N-dimethylamino-4'-azobenzene sulfonyl hyrazide method. Sequence analyses of the glycopeptides showed that a carbohydrate chain attached to Asn-39.

Amino Acid Sequence↗

PubMed1993

A new mixed disulfide species in human cataractous and aged lenses.

The process of ageing in the normal human eye lens is unique among tissues due to the absence of turnover in the structural proteins. These proteins accumulate a variety of modifications throughout their lifetime. Significantly, the cysteine residues are subject to disulfide formation with the low molecular weight thiol compounds present in the lens. It has been shown that accumulation of glutathione and cysteine mixed disulfides in the proteins of normal human lens is a function of age. In this report a third mixed disulfide species gamma-glutamylcysteine (gamma-Glu-Cys), has been identified by comparison with standards which were produced through two distinct methods. This new mixed disulfide is only prominent in old lenses (> 60 years) and cataractous lenses. In these situations its level may approach those of cysteine mixed disulfide. The appearance of gamma-Glu-Cys may be coincident with biochemical abnormalities preceding cataract formation. This protein modification may be a result of changes in the GSH biosynthetic pathway within the lens.

Adult↗

PubMed1995

Synthesis and biochemical evaluation of RNA containing an intrahelical disulfide crosslink.

BACKGROUND: Several factors impede the elucidation of RNA structure and function by X-ray and NMR methods, including the complexity of folded RNA motifs, the tendency of RNA to aggregate, and its ability to fold into multiple isomeric structures. The ability to constrain the process of RNA folding to give a single, homogeneous product would assist these investigations. We therefore set out to develop a synthetic procedure for the site-specific insertion of a disulfide crosslink into oligoribonucleotides. We also examined the ability of a crosslinked species to serve as a substrate for ricin, an RNA glycosylase. RESULTS: A convertible nucleoside derivative (C) suitable for the site-specific introduction of N4-alkylcytidine residues into RNA has been developed. The corresponding C phosphoramidite was employed in the synthesis of an 8-mer oligonucleotide, 5'-CGGA-GACG-3', which was then efficiently converted to an 8-mer containing two S-protected N4-(2-thioethyl)C residues. Upon deprotection and air oxidation, the 8-mer efficiently formed an intramolecular disulfide bond, yielding a GAGA tetraloop presented on a two-base-pair CpG disulfide crosslinked ministem. We show that this ministem-loop is an excellent substrate for ricin. Control 8-mers lacking the disulfide crosslink were substantially poorer substrates for ricin. CONCLUSIONS: The nucleoside chemistry described here should be generally useful for the site-specific introduction of a range of non-native functional groups into RNA. We have used this chemistry to constrain an RNA ministem through introduction of an intrahelical disulfide crosslink. That this tetraloop substrate linked to a two base-pair ministem is efficiently processed by ricin is clear evidence that ricin makes all of its energetically favorable contacts to the extreme end of the stem-loop structure, and that the two base pairs of the stem abutting the loop remain intact during recognition and processing by ricin.

Catalysis↗

PubMed2005

Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study.

We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in alpha-conotoxin SII, of which approximately 30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. alpha-Conotoxin SII was shown to possess 3-8, 2-18, and 4-14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS). This method achieved high yields of the differentially reduced disulfide bonded intermediates and economic use of reduced peptide. Intermediates were alkylated with either N-phenylmaleimide or 4-vinylpyridine. The resulting alkyl products were assigned by ESMS and their alkyl positions sequentially identified via conventional Edman degradation. The methodology described allows a more efficient, rapid, and reliable assignment of disulfide bond connectivity in synthetic and native cysteine-rich peptides.

Amino Acid Sequence↗

PubMed2005

Recovery and purification of highly aggregation-prone disulfide-containing peptides: application to islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is a 37-residue pancreatic hormone. It is responsible for the formation of islet amyloid in vivo and is very insoluble and aggregation-prone in vitro, particularly at basic pH. The peptide contains a disulfide bridge between residues two and seven and an amidated C terminus. There is no reported expression system for the production of amidated IAPP. The peptide is difficult to synthesize and formation of the disulfide by traditional methods is problematic. We have found that the use of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or dimethyl sulfoxide (DMSO) significantly improves disulfide formation and purification of highly aggregation-prone IAPP sequences. The use of these organic solvents increases the solubility of the hydrophobic peptides, avoids the use of aqueous basic solutions, and eliminates the need for continuous stirring during oxidation to form the Cys-2 to Cys-7 disulfide bridge. Elimination of the stirring step and basic solution helps to reduce aggregation and allows for more consistent high-performance liquid chromatography (HPLC) retention times. Formation of the intramolecular disulfide using DMSO was found to be the most effective method for IAPP oxidation, reducing the reaction time from 24 to 5 h. Aggregated IAPP can be resolubilized by HFIP or DMSO and recovered by HPLC with very good yield.

Amino Acid Sequence↗

PubMed2005

Biocompatibility and stability of disulfide-crosslinked hyaluronan films.

Hyaluronan (HA) can be chemically modified to engineer robust materials with pre-selected mechanical properties and resorption rates that can be dictated by the intended clinical use. Disulfide-crosslinked HA films were prepared by air oxidation of thiol-modified HA, followed by treatment with 0.3% hydrogen peroxide. The degradation of the disulfide-crosslinked films in vitro was very slow (<10% in 7 days) in buffer alone and shorter (t1/2=3-5 days) in the presence of hyaluronidase (HAse). The cytocompatibility of the disulfide-crosslinked HA films was determined using two separate conditions: (i) in vitro culture of mouse fibroblasts in indirect contract with the films, and (ii) in vitro culture of fibroblasts directly on films coated with poly d-lysine. Excellent cytocompatibility was observed in murine fibroblasts that were cultured in indirect contact with thiolated HA films. Although cells were unable to attach and spread on thiolated HA films, pre-coating the thiolated HA films with poly D-lysine resulted in attachment and spreading equivalent to that observed on polystyrene. Rates of resorption in vivo were obtained by subcutaneous implantation of disulfide-crosslinked HA films into the backs of Wistar rats. Biocompatibility in vivo was determined in both subcutaneous flank and peritoneal cavity implantation of the films in Wistar rats. The disulfide-crosslinked HA films were less than 30% resorbed after 42 days in vivo, and histochemical and cytochemical analysis indicated that the films were well-tolerated with mild inflammatory response at both sites of implantation.

Absorbable Implants↗

PubMed2005

A disulfide relay system in mitochondria.

In this issue of Cell, show that there is a disulfide relay system in the intermembrane space (IMS) of mitochondria that is comprised of the proteins Mia40 and Erv1. This disulfide relay system promotes the import and oxidative folding of proteins. Oxidized Mia40 traps newly imported proteins through mixed disulfide bridges. Subsequent isomerization of these disulfide bridges allows the imported protein to be folded in the IMS. The reduced Mia40 generated is then reoxidized by the sulfhydryl oxidase Erv1, promoting the next round of disulfide exchange. The new work clarifies the molecular function of Mia40 and reveals Mia40 to be the first physiological substrate for the FAD-linked Erv1.

Animals↗