Assignment of the disulfide bonds of huwentoxin-II by Edman degradation sequencing and stepwise thiol modification.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.     

PEGylation of native disulfide bonds in proteins

PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.     

Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat alpha-lactalbumin

Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein. The latter phenomenon is most clearly observed on fluorescence measurements at low temperatures (near 3 degrees C). The photolysis induces some dimerization and oligomerization, but most GLA molecules remain monomeric. To obtain more information about the reaction products, the illuminated protein is treated with iodoacetamide to label the free thiol groups, it is fragmented with trypsin, and the fragments are analyzed by mass spectrometry. Via this approach, we observe that the cleavage of disulfide bonds is restricted to Cys6-Cys120 and Cys73-Cys91 bonds. The photolytic cleavage of either of these disulfide bonds results in the formation of a single free thiol, a phenomenon restricted to Cys120 and Cys91, respectively. We also found indications that a thioether linkage is formed between Cys73 and Trp60. The alkylsulfenylation of Trp60 presumably results from a combination of primary thiyl and tryptyl radicals.     

Disulfide bonds are generated by quinone reduction

The chemistry of disulfide exchange in biological systems is well studied. However, very little information is available concerning the actual origin of disulfide bonds. Here we show that DsbB, a protein required for disulfide bond formation in vivo, uses the oxidizing power of quinones to generate disulfides de novo. This is a novel catalytic activity, which to our knowledge has not yet been described. This catalytic activity is apparently the major source of disulfides in vivo. We developed a new assay to characterize further this previously undescribed enzymatic activity, and we show that quinones get reduced during the course of the reaction. DsbB contains a single high affinity quinone-binding site. We reconstitute oxidative folding in vitro in the presence of the following components that are necessary in vivo: DsbA, DsbB, and quinone. We show that the oxidative refolding of ribonuclease A is catalyzed by this system in a quinone-dependent manner. The disulfide isomerase DsbC is required to regain ribonuclease activity suggesting that the DsbA-DsbB system introduces at least some non-native disulfide bonds. We show that the oxidative and isomerase systems are kinetically isolated in vitro. This helps explain how the cell avoids oxidative inactivation of the disulfide isomerization pathway.     

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Dithiothreitol (DTT), a disulfide reducing agent, diminished the specific binding of [³H] dopamine to partially purified calf striatal membranes (P₂) but did not have an effect on [³H] spiroperidol binding. The thiol reagents, p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM) and iodoacetamide (IA), were also tested for inhibitory effects on agonist and antagonist binding to the dopamine receptor. PCMB inhibited both [³H] dopamine and [³H] spiroperidol binding by changing the affinity (K_d) and the number of binding sites (B_max) for both of these ligands. This effect of PCMB was reversed by the addition of DTT. NEM inhibited binding to the dopamine agonist site but not to the antagonist site, while IA was ineffective on either site. These results indicate that a DTT-reducible disulfide bond may be an essential component for agonist binding to the dopamine receptor. Furthermore, the experiments with PCMB, NEM and IA suggest that the exposure of thiol groups in the dopamine receptor may play an important role in agonist and antagonist binding.     

Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.     

Enhancement of stability of immobilized glucose oxidase by modification of free thiols generated by reducing disulfide bonds and using additives

Stability of glucose oxidase (GOD) immobilized with lysozyme has been considerably enhanced by modification of free thiols generated by reducing disulfide bonds using beta-mercaptoethanol and N-ethylmaleimide in conjunction with additives like antibiotics and salts. Thermal stability of immobilized GOD was quantified by means of the transition temperature, Tm and the operational stability by half-life t1/2 at 70 degrees C. Modification of the free thiols in the enzyme coupled with the presence of kanamycin, NaCl, and K2SO4, led to increase in Tm, to 80, 82 and 84 degrees C (compared to 75 degrees C in control) and t1/2 by 7.7-, 11- and 22-fold, respectively, indicating that this method can be effectively used for enhancing the stability of enzymes.     

Circular dichroic study of conformational changes in ovalbumin induced by modification of sulfhydryl groups and disulfide reduction

Sulfhydryl groups of ovalbumin were chemically modified under denaturing conditions in the absence and presence of dithiothreitol, and effects on the secondary structure of the protein were investigated by circular dichroic (CD) measurements. The contents of -helix, -structure, and random coil (unordered, nonrepetitive structure) were estimated by simulation of the CD spectra and using the parameters established by Chen et al. The principal findings were these: (1) Modification of the four free sulfhydryl groups [with 5,5-dithiobis(2-nitrobenzoic acid), iodoacetate, or iodoacetamide] caused ovalbumin molecule to unfold partially and to undergo primarily helix-to- structure transition. (2) Cleavage of the disulfide bond did not lead to a further conformational change in the sulfhydryl-modified ovalbumin. (3) The remaining helical structure existed in a destabilized state with increased chain flexibility, as the modified protein was very susceptible to denaturation by guanidine and urea. (4) Further evidence for increased chain flexibility was provided by the finding that sodium dodecyl sulfate (SDS) induced helix formation in the sulfhydryl-modified, but not native, ovalbumin. And (5), since both nonreduced and reduced proteins, with their sulfhydryl groups blocked, displayed similar transitions in solutions of guanidine, urea, and SDS suggested that the single disulfide bond did not physically constrain the ovalbumin molecule.     

Enzyme reduction of disulfide bonds by thioredoxin. The reactivity of disulfide bonds in human choriogonadotropin and its subunits.

The NADPH-dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, alpha and beta, was examined with thioredoxin and thioredoxin reductase from Escherichia coli. With 12 muM thioredoxin and 0.1 muM thioredoxin reductase at pH 7 all disulfide bonds in the alpha subunit could be reduced in 15 min. The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced. Partial reduction of the alpha subunit with thioredoxin followed by S-carboxymethylation with iodol[2-3H]acetic acid and analysis of tryptic peptides indicated that all S-S bonds in the alpha subunit were surface oriented and equally reactive. The usefulness of thioredoxin reduction of disulfide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits.     

Catalysis of thiol/disulfide exchange: single-turnover reduction of protein disulfide-isomerase by glutathione and catalysis of peptide disulfide reduction

Protein disulfide-isomerase, a protein localized to the lumen of the endoplasmic reticulum of eukaryotic cells, catalyzes the posttranslational formation and rearrangement of protein disulfide bonds. As isolated from bovine liver, the enzyme contains 0.8 free sulfhydryl group per mole of protein monomer and 3.1 disulfide bonds. Single-turnover experiments in which the disulfide bonds of the native enzyme are reduced by glutathione reveal three distinct reduction steps corresponding to the sequential reduction of the three disulfide bonds. The fastest disulfide to be reduced undergoes a change in the rate-determining step with increasing GSH concentration from a step which is second-order with respect to GSH concentration to a step which is first-order in GSH concentration. The disulfide which is reduced at an intermediate rate displays kinetics that are first-order in GSH concentration, and the slowest disulfide to be reduced exhibits kinetics which are second-order in GSH concentration. The enzyme catalyzes the steady-state reduction of a disulfide-containing hexapeptide (CYIQNC) by GSH. Initial velocity kinetic experiments are consistent with a sequential addition of the substrates to the enzyme. Saturation behavior is not observed at high levels of both substrates (Km for GSH much greater than 14 mM, Km for CYIQNC much greater than 1 mM). Only one of the three disulfides appears to be kinetically competent in the steady-state reduction of CYIQNC by GSH. The second-order thiol/disulfide exchange reactions catalyzed by the enzyme are 400-6000-fold faster than the corresponding uncatalyzed reactions.     

Detecting native folds in mixtures of proteins that contain disulfide bonds

High-throughput in vitro refolding of proteins that contain disulfide bonds, for which soluble expression is particularly difficult, is severely impeded by the absence of effective methods for detecting their native forms. We demonstrate such a method, which combines mass spectrometry with mild reductions, requires no prior experimentation or knowledge of proteins' physicochemical characteristics, function or activity, and is amenable to automation. These are necessary criteria for structural genomics and proteomics applications.     

The native metastable fold of C1-inhibitor is stabilized by disulfide bonds

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.     

Determination of reduced disulfide groups in monoclonal antibodies

Reduction of disulfide bonds to sulfhydryl groups for direct radiolabeling of antibodies for immunoscintigraphic and therapeutic applications continues to be of considerable interest. Sensitive spectrophotometric methods have been evaluated that will enable investigators to determine submicrogram quantities of cysteine units produced, for the assurance of controlled reduction. One method, which generates a cysteine-ninhydrin complex (520 nm), has a molar extinction coefficient of 30 250 and can determine 0.04 micrograms/ml cysteine units with an absorbance of 0.01. The method has been applied to determine the quantity of cysteine groups produced by the reduction of an immunoglobin G antibody with five different reducing agents in normal to five times the previously determined optimal molar ratios. The quantities of cysteine units produced from the controlled reduction from 240 micrograms immunoglobin G ranged from 0.073 +/- 0.01 to 1.07 +/- 0.04 micrograms, which were merely 0.54 +/- 0.08% to 7.9 +/- 0.28% of the total available disulfide groups in the protein.     

Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis

A general method by polyacrylamide gel electrophoresis for the determination of sulfhydryls and disulfides in a protein was developed. The method included a two-step alkylation procedure: the first step consisted of alkylation of the sulfhydryl groups with iodoacetic acid in the presence and absence of 8 M urea; the second step consisted of alkylation of the disulfide groups with iodoacetamide after reduction with a thiol. By high-pH urea gel electrophoresis, all the half-cystine residues in a protein could be categorized into three states: reactive sulfhydryls, nonreactive sulfhydryls, and disulfide bonded. The particular advantage of the method is that the states of half-cystines in different protein species can be analyzed independently both in isolated protein and in biological translation systems.     

Selective reduction of the disulfide bonds of ovine placental lactogen

Reduction and carbamidomethylation of two of the three disulfide bridges of ovine placental lactogen was accomplished by the use of 20-fold molar excess of dithiothreitol over protein disulfide content. The derivative retained its binding capacity to somatogenic as well as lactogenic rat liver receptors, although the latter was somewhat diminished. The two disulfide bonds exposed to the reducing agent are those located near the carboxy- and amino-terminus, while the larger loop remained intact after reduction. This behaviour is similar to that of bovine growth hormone, where the larger loop was also more resistant to reduction.     

Donor substrate specificity and thiol reduction of glutathione disulfide peroxidase.

By isolation of a mixed disulfide product of glutathione and cysteine, glutathione peroxidase was shown to be highly specific for only one donor substrate. Using the coupled assay of NADPH and yeast glutatione reductase, which is highly specific for flutathione disulfide, it was shown that the apparent inhibition of glutathione peroxidase by mercaptoethanol can be described kinetically and that it is competitive with glutathione. Also, when limiting amounts of hydroperoxide were present in the reaction mixture with mercaptoethanol or cysteine, the total amount of glutathione disulfide produced decreased as compared with that in a reaction mixture without mercaptoethanol or cysteine. This finding is consistent with enzymatic formation of mixed disulfides. Data presented suggest that the selenium in glutathione peroxidase was oxidized to a seleninic acid in the absence of glutathione. These results can be explained by a mechanism for glutathione peroxidase wherein the selenium atom is the only atom in the enzyme that undergoes oxidation reduction.     

Superoxide involvement in the reduction of disulfide bonds of wheat gel proteins

A soluble epoxide hydrase which catalyzes the hydration of 9,10-epoxypalmitic acid has been partially purified from cell-free preparations from $\text{Bacillus}\text{megaterium}$ ATCC 14581. The hydrase can be cleanly separated from a soluble cytochrome P-450-dependent monooxygenase complex, previously demonstrated in this bacterium, that can catalyze the epoxidation of palmitoleic acid.