Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.     

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.     

Formation of disulfide bonds in proteins and peptides

For many proteins and peptides, disulfide bridges are prerequisite for their proper biological function. Many commercialized proteins are crosslinked by disulfide bridges that increase their resistance to destructive effects of extreme environment used in industrial processes or protect protein-based therapeutics from rapid proteolytic degradation. Manufacturing of these products must take into account oxidative refolding--a formation of native disulfide bonds by specific pairs of cysteines located throughout a sequence of linear protein. This review describes basic and practical aspects of oxidative folding that should be considered while designing and optimizing manufacturing of proteins using chemical synthesis, semi-synthesis and a recombinant expression.     

Intramolecular versus intermolecular disulfide bonds in prion proteins

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.     

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.     

Identification of plasma proteins containing sulfite-reactive disulfide bonds

Plasma protein S-sulfonate compounds (RS-SO-3) have previously been shown to form, presumably by sulfitolysis of disulfide bonds, as a result of exposure to sulfite. In the investigations reported here, we identify two proteins in rabbit plasma, namely albumin and plasma fibronectin, which contain reactive sites for S-sulfonate formation. Separation and identification of these proteins following in vitro and in vivo exposure to sulfite was accomplished primarily by column chromatographic and electrophoretic techniques. In addition, the structure of presumed S-sulfonate groups was confirmed by the identification of cysteinyl-S-sulfonate residues in protein hydrolysates generated by enzymatic digestion. The molar ratio of RS-SO-3 in both albumin and plasma fibronectin was less than one. Data from our experiments suggest that the mixed disulfide site of non- mercaptalbumin is the reactive site for S-sulfonate formation. The site(s) of formation within the plasma fibronectin molecule was not investigated. The possible physiological significance of disulfide sulfitolysis of albumin and plasma fibronectin is discussed.     

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.     

Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.     

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.     

The role of disulfide bonds in maintaining the gel structure of bronchial mucus.

Solubilization of the gel phase of sputum by reduction with dithiothreitol and alkylation with iodoacetamide resulted in different gel filtration patterns when sputa from different patients were examined. Two extreme types of of behavior were identified; in one the glycoproteins were completely excluded from Sepharose 4B, and in the other all the glycoproteins penetrated the gel matrix to a certain extent. Pronase digestion of the products of reduction and alkylation of the former resulted in a gel filtration pattern similar to that obtained by reduction and alkylation alone in the latter. The disulfide bonds cleaved by dithiothreitol were labeled by reaction with [1-¹⁴C]iodoacetamide and the glycoproteins isolated. Pronase digestion of the labeled glycoproteins revealed that, although most of the cysteine residues occurred in peptide regions cleaved by Pronase, some were situated in resistant peptide regions. Structures are proposed for the bronchial glycoproteins isolated from the two extreme types of sputum. These structures consist of a glycoprotein subunit, resistant to Pronase and attached by covalent bonds to a “naked” peptide region. Whereas the glycoprotein subunits are similar in both types of sputum, the “naked” peptide is a continuous peptide chain in one type but a discontinuous peptide chain in the other.     

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.     

Barnacle cement proteins. Importance of disulfide bonds in their insolubility

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 m dithiothreitol at 60 degrees C in a 7 m guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific expression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.     

Intermolecular disulfide bonds link specific high-molecular-weight glutenin subunits in wheat endosperm.

A detergent wash extracted soluble proteins from wheat flour, leaving a residue enriched with insoluble glutenin aggregates. Digestion of this residue with endoproteinase Lys-C, which showed a limited specificity for glutenin subunits, produced several peptides with apparent molecular weights close to those of intact high-molecular-weight glutenin subunits. N-terminal sequencing indicated that the isolated peptides were composed of high-molecular-weight glutenin subunit fragments joined by an intermolecular disulfide bond. In two of these peptides, only two components were found, one from an x-type subunit and the other from a y-type subunit. The isolated peptides all contained at least one x-type C-terminal region and one y-type N-terminal region, suggesting a specific orientation to the intermolecular disulfide linkage.     

Partial reduction of disulfide bonds in the hormone-specific subunits of TSH and LH.

Partial reduction at pH 7.0 of the hormone specific (β) subunit of either bovine thyrotropin or luteinizing hormone with dithioerythritol results primarily in the opening of a single disulfide bridge. The partially reduced subunits were alkylated with [1-¹⁴C] iodoacetic acid, followed by complete reduction and alkylation with non-radioactive iodoacetic acid. Isolation and degradation of the radioactive tryptic peptides shows that the bond primarily reduced in each β subunit links analogous half-cystine residues in the two sequences (88–95 in TSH-β and 93–100 in LH-β). These results are the first direct evidence of similar disulfide structures in hormone specific subunits of glycoprotein hormones.     

Effect of partial reduction of disulfide bonds on the enzymatic activity of thrombin

It was demonstrated that partial reduction of disulfide bonds in thrombin by dithiothreitol in the absence of denaturating agents leads to a decrease of enzymatic activity with respect to fibrinogen coagulation and tosylarginine methyl ester hydrolysis. Polyacrylamide gel electrophoresis and determination of the number of SH-groups liberated in the course of reduction suggest that the observed inactivation is primarily due to the disruption of the S-S-bridge between the A- and B-chains of thrombin.     

Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry

A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.