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PubMed1996

Speciation and quantitation of underivatized and Ellman's derivatized biological thiols and disulfides by capillary electrophoresis.

Capillary electrophoresis (CE) methods have been developed for the speciation and quantitation of thiols and disulfides of biological interest, including the endogenous compounds glutathione, glutathione disulfide, cysteine, cystine, homocysteine, and homocystine and the therapeutic agents penicillamine, penicillamine disulfide, N-acetylcysteine, and captopril. Good speciation and quantitation were achieved for the underivatized thiols and disulfides using a detection wavelength of 200 nm; detection limits were in the range 20-90 microM (1-4 pmol) using a 50-microns-diameter capillary. To achieve lower detection limits, thiols were derivatized with the thiol-specific probe molecule, 5,5'-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent). Good speciation and quantitation were achieved for the Ellman's derivatized thiols using a detection wavelength of 357 nm; detection limits were in the range 5-50 microM (0.03-0.3 pmol) using a 25-microns-diameter capillary. Both the underivatized and derivatized methods were applied to the determination of glutathione in human erythrocytes. Glutathione concentrations of 2-3 mM were obtained for the erythrocyte samples analyzed, with good agreement between results obtained by the two methods.

Captopril↗
PubMed2002

Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds.

A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available-selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins.

Animals↗
PubMed1993

Comparison of the disulfide bond arrangements of human recombinant and bovine brain heparin binding neurite-promoting factors.

Heparin binding neurite-promoting factor (HBNF) is a highly basic 136 amino acid protein containing 10 cysteine residues. We have determined the redox status and the disulfide arrangement of the cysteine residues in HBNF from bovine brain and refolded human recombinant protein produced in E. coli. Our data indicate that all 10 cysteines are involved in disulfide bond formation. The disulfide linkages of human recombinant and bovine brain HBNF, as determined after proteolytic digestions of the non-reduced proteins by peptide mapping and sequence analysis are: Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109. Thus, recombinant HBNF has the same disulfide arrangement as the native brain-derived protein.

Amino Acid Sequence↗
PubMed1993

Intermolecular disulfide linkages are not required for transthyretin amyloid fibril formation in vitro.

The role of intermolecular disulfide linkages in transthyretin (TTR) amyloid fibril formation was investigated by comparing wild type TTR to Cys-10-Ala TTR which is incapable of disulfide formation. The Cys-10-Ala variant exhibits quaternary structural stability equal to the wild type protein under acidic denaturing conditions. Both Cys-10-Ala and wild type TTR were converted into amyloid fibrils by partial acid denaturation. There was no evidence of intermolecular disulfide formation in the case of wild type amyloid fibrils. These results are inconsistent with a recently proposed model stressing the importance of intermolecular disulfide linkages in TTR amyloid fibril formation, but are consistent with a model relying on noncovalent quaternary contacts made possible through an acid-mediated conformational change.

Alanine↗
PubMed1994

Stabilization of beta-ribbon structures in peptides using disulfide bonds.

The effect of a disulfide crosslink between two peptide chains on the stability of beta-ribbon secondary structures formed by these peptides has been investigated. Based on structural principles, we hypothesized that introduction of an unstrained disulfide crosslink at appropriate locations on two peptide chains should have a stabilizing effect on the beta-ribbon structure formed by these two peptide chains. To test this hypothesis, we designed and synthesized two sets of 9-residue peptides incorporating cysteine in one and (S)-alpha-amino-epsilon-mercaptohexanoic acid in the other. Comparison of the CD data clearly show that the dimer containing a disulfide bond between the longer sidechains of (S)-alpha-amino-epsilon-mercaptohexanoic acid shows dramatically higher beta-ribbon character as compared to the dimer with cystine disulfide bond, thus validating our structural hypothesis.

Amino Acid Sequence↗
PubMed1996

Enhancement of thermostability and catalytic efficiency of AprP, an alkaline protease from Pseudomonas sp., by the introduction of a disulfide bond.

A site-directed mutagenesis in AprP, an alkaline protease isolated from Pseudomonas sp. KFCC 10818 was carried out in order to obtain increased thermostability. Sites for cysteine substitutions to form disulfide bond within AprP were chosen by comparing the sequences with aqualysin I, an alkaline thermostable serine protease whose disulfide bonds seems to be important for its thermostability. Gly199 and Phe236 residues were each replaced with cysteine by site-directed mutagenesis. The G199C/F236C mutant enzyme appeared to form a disulfide bond spontaneously during its expression. It also showed improved kinetic parameters for the hydrolysis of a synthetic peptide substrate at pH 8.5 and 10.5 compared to those of the wild-type enzyme. The half-life of the G199C/F236C mutant was found to be 2 to 4.8 times longer than that of wild-type under various experimental conditions, except when tested under reducing condition, where no significant differences in the half-life of the two types were observed. Therefore, it is concluded that the introduction of the disulfide bond enhanced the thermostability and the catalytic efficiency of the enzyme AprP.

Alkaline Phosphatase↗
PubMed1997

The role of disulfide bond C191-C220 in trypsin and chymotrypsin.

Serine proteases of the chymotrypsin family contain three conserved disulfide bonds: C42-C58, C168-C182, and C191-C220. C191-C220 connects the loops around the substrate binding pocket. Using site directed mutagenesis, cysteines of this disulfide bridge were replaced by alanines in trypsin, in chymotrypsin, and in Tr-->Ch-[S1+L1+L2+Y172W], a mutant trypsin with high chymotrypsin like activity. The functional role of this "active site" disulfide was assessed by comparing the catalytic properties of wild-type and mutant enzymes. Its removal from all three proteases caused a decrease in kcat/KM of two to three orders of magnitude, mainly as a consequence of a dramatic increase in KM. The pH dependence of the activity also changed: the rather wide pH optimum, characteristic of the wild-type enzymes (especially trypsin), narrowed since the pKa in the alkaline region shifted downwards. Results show that C191-C220 is necessary for the high activity of both trypsin and chymotrypsin. By contrast, elimination of this disulfide bridge greatly decreased the specificity of trypsin and of Tr-->Ch-[S1+L1+L2+Y172W], but had no significant change on that of chymotrypsin.

Amino Acid Sequence↗
PubMed1997

The structure of synenkephalin (pro-enkephalin 1-73) is dictated by three disulfide bridges.

Mass spectrometry of fragments produced by limited proteolytic digestion of pro-enkephalin was used to locate the disulfide bridges in synenkephalin (pro-enkephalin 1-73), a domain which contains sorting information for targeting the pro-neuropeptide to the granules of the regulated secretory pathway in neuroendocrine cells. Mass spectrometric analysis was optimized by using chemicals that gave low interference with the ionization and desorption processes, and computer software which simplified the identification of all possible disulfide-linked peptide fragments. Three disulfide bridges between Cys2-Cys24, Cys6-Cys28, and Cys9-Cys41 were identified. Protein conformational prediction of synenkephalin1-42 shows beta-turns which facilitate the formation of these disulfide bonds.

Amino Acid Sequence↗
PubMed1998

Platelet membrane actin may be partially embedded in lipid bilayer and disulfide linked.

When platelet membranes previously treated by 0.6M KI were reacted with 14C-NEM, 9 protein bands including membrane actin were labeled. If KI treated platelet membranes were first reacted with cold NEM, beta-Mercaptoethanol, and 14C-NEM sequentially only three protein bands, one of which was actin, were labeled. These results imply that some of the tightly associated membrane actin thiol groups are free and some of them form disulfide bonds with two other labeled proteins. The candidates that might form disulfide bonds with actin were identified by monoclonal antibody to be GpIIb and/or GpIIIa. Extraction experiments showed that even when the disulfide bonds that link actin to membrane integral protein were first reduced by DTT and then extracted with 0.6M KI, membrane actin still remained tightly associated to the membrane by some other means. Membrane actin could be extracted with 1% octyl glucoside but remained as part of a high-molecular-weight complex. From these results we believe that platelet membrane actin may be partially embedded into the bilayer of the lipid membrane and disulfide linked to membrane integral proteins. It may thus act as a nucleating center for the polymerization of cytosolic actin in the assembly of the cytoskeleton.

Actins↗
PubMed1998

The multi-domain structure of protein disulfide isomerase is essential for high catalytic efficiency.

Protein disulfide isomerase (PDI) catalyzes protein folding linked to disulfide bond formation in secreted proteins. It consists of four major domains, denoted a, b, b' and a'. The a and a' domains each contain an active site motif, -CGHC-, which is directly involved in thiol-disulfide exchange reactions during catalysis. The roles of the b and b' domains and the functional necessity for the multi-domain structure of PDI are unknown. We now demonstrate that full catalytic activity requires the involvement of multiple PDI domains and that the b' domain has a particularly important role in catalysis. Reconstruction of the PDI molecule from the isolated a and a' domains results in a progressive increase in catalytic efficiency as further domains are added. These effects are especially significant in the catalysis of disulfide bond rearrangements in folded substrates, for which all the domains of the protein are required for maximum catalytic efficiency. It is likely that all of the domains of PDI participate in substrate binding interactions and that PDI has evolved its multidomain structure as an adaptation that allows it to catalyze transformations involving difficult conformational changes.

Aprotinin↗
PubMed1998

The nature of antibody heavy chain residue H6 strongly influences the stability of a VH domain lacking the disulfide bridge.

Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles.

Amino Acid Sequence↗
PubMed2001

Crystallization and initial crystallographic analysis of the disulfide bond isomerase DsbC in complex with the alpha domain of the electron transporter DsbD.

The protein disulfide bond isomerase DsbC catalyzes the rearrangement of incorrect disulfide bonds during oxidative protein folding in the periplasm of Escherichia coli. The active site cysteines of DsbC are maintained in the active reduced form by the transmembrane electron transporter DsbD. DsbD obtains electrons from the cytoplasm, transports them across the inner membrane, and passes them onto periplasmic substrates, such as DsbC. The electron transport process involves several thiol disulfide exchange reactions between different classes of thiol oxidoreductase. We were able to trap the final electron transport reaction using active site mutants yielding a stable DsbC-DsbDalpha complex. This disulfide cross-linked complex was purified to homogeneity and crystallized. Dehydration of the tetragonal crystals changed the unit cell dimensions from a approximately b = 73 A, c = 267.5 A to a = b = 68.9 A, c = 230.3 A, reducing the cell volume by 23% and the solvent content from 55 to 41%. Crystal dehydration and cryo-cooling improved the diffraction quality of the crystals from 7 to 2.3 A resolution.

Crystallization↗
PubMed1999

Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is a protein-thiol oxidoreductase that catalyzes the oxidation, reduction and isomerization of protein disulfides. In the endoplasmic reticulum PDI catalyzes both the oxidation and isomerization of disulfides on nascent polypeptides. Under the reducing condition of the cytoplasm, endosomes and cell surface. PDI catalyzes the reduction of protein disulfides. At those locations, PDI has been demonstrated to participate in the regulation of reception function, cell-cell interaction, gene expression, and actin filament polymerization. These activities of PDI will be discussed, as well as its activity as a chaperone and subunit of prolyl 4-hydroxylase and microsomal triglyceride transfer protein.

Abetalipoproteinemia↗
PubMed1997

Analyses of disulfides present in the rubella virus E1 glycoprotein.

The surface of Rubella virus contains the glycoproteins E1 and E2. The E1 protein induces neutralizing antibodies and has been implicated in the process of recognition of cellular receptors. To gain information on the structural organization of the E1 protein we have analyzed the disulfide bonds present within this molecule. The reactivity of the protein with radioactively labeled iodoacetic acid indicates that all 20 cysteine residues present in the ectodomain of the E1 protein are involved in disulfide formation. E1 protein was purified by preparative SDS-PAGE under nonreducing conditions from virus particles grown in tissue culture in the presence of [35S]cysteine. The purified protein was digested with a number of proteases followed by reversed phase high-performance liquid chromatography (HPLC). [35S]cysteine-containing peptides were identified and characterized by N-terminal amino acid sequence determination. These analyses identified the following eight disulfide bridges: C(1)-C(2); C(3)-C(15); C(6)-C(7); C(9)-C(10); C(11)-C(12); C(13)-C(14); C(17)-C(18); and C(19)-C(20). The two disulfide bridges formed by the residues C(4), C(5), C(8), and C(16) have not been identified with certainty, but a likely organization can be derived. The data obtained are discussed in the context of a possible structural and functional organization of the E1 protein.

Amino Acid Sequence↗
PubMed1977

Disulfide bonds: key to wheat protein functionality.

Disulfide bonds in wheat proteins are major factors that determine the properties of the proteins and their functionality in wheat flour. The gliadin proteins contain mostly intramolecular disulfide bonds. In contrast, the high-molecular-weight glutenins are formed by disulfide linkages of several diverse polypeptide chains which have been separated and characterized. The linkage of these proteins in a fairly linear array contributes to the unique viscoelastic properties of glutenin. The glutenin has been separated into two fractions differing in molecular weight. The amount of highest molecular weight component is correlated with the rheological behavior of the flours from different wheat varieties. Various oxidizing and reducing agents are widely used to alter the functional behavior of wheat proteins by the action on sulfhydryl and disulfide groups.

Amino Acids↗
PubMed1993

An isoform of protein disulfide isomerase is expressed in the developing acrosome of spermatids during rat spermiogenesis and is transported into the nucleus of mature spermatids and epididymal spermatozoa.

We have purified an isoform of protein disulfide isomerase (EC 5.3.4.1) from rat liver, and raised a specific antibody against the purified protein in rabbit. Immunohistochemical studies using this antibody on rat testis sections, at both light and electron microscopic levels, showed a specific localization of the isoform of protein disulfide isomerase in the developing acrosome of the spermatids. The protein was transferred to the acrosomic vesicle from the Golgi apparatus at late Golgi phase, and remained present in the acrosome of spermatids during cap phase, acrosome phase, and maturation phase. In addition to the acrosome, the protein appeared in the nucleus of spermatids during maturation phase, and was localized in the nucleus of epididymal spermatozoa. By immunoblot analysis, almost all of the isoform of protein disulfide isomerase in the testis was found to be extractable by an isotonic buffer. On the contrary, detergent extraction was required for complete solubilization of the protein in the epididymis. These results suggest that the isoform of protein disulfide isomerase is a new intra-acrosomal soluble protein, and that the protein begins to enter the nucleus of mature spermatids in the testis and tightly binds to the nuclear components in epididymal spermatozoa.

Acrosome↗
PubMed1980

Abundance of protein-bound sulfhydryl and disulfide groups at chromosomal nucleolus organizing regions: a cytochemical study on the selective silver staining of NORs.

Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.

Cells, Cultured↗
PubMed1994

Mortality of Dutch workers exposed to carbon disulfide.

Epidemiological studies carried out in Great Britain, Scandinavia and the United States indicate that workers exposed to carbon disulfide are at an increased risk for cardiovascular disease (CVD) mortality, and in particular for ischemic heart disease (IHD) mortality. In the epidemiological study reported here a retrospective cohort design was used. The total study population consisted of 3322 workers from a Dutch viscose textile plant who had all been employed for at least half a year between 1 January 1947 and 1 January 1980. Only production and maintenance workers were selected for the study. A group of 1434 workers who had been exposed to carbon disulfide was identified from the files available at the plant. The remaining 1888 workers who had not been exposed to carbon disulfide were used as a reference group. The total study population was followed for mortality until 1 January 1988. For the 762 workers who had died before that date, the causes of death were checked at the Central Bureau of Statistics. The results show a slightly but significantly increased risk for CVD mortality (CVD-specific SMR of 115), despite the observation of the so-called healthy worker effect and the statistical uncertainties inherent to this type of study. The results are in accordance with those reported by other investigators. The results indicate that exposure to relatively low levels of carbon disulfide increases the risk of CVD mortality.

Carbon Disulfide↗