The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.     

Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.     

Quaternary structure of oligomeric immunoglobulin A forms from human blood serum]

A character of forces stabilyzing quaternary structure of dimer and more high molecular human immunoglobulin A oligomers is found to be different. Quaternary structure of IgA dimer is formed when joining subunits with disulfide bonds and is stabilized by non-covalent interactions between them. Disulfide bonds play a main part in the formation of trimers and tetramers. Dimer IgA reconstructs by 40% from subunits with intact interchain S--S bonds. The addition of exogenous J-chain does not significantly affect the process of dimer self-assembling from subunits with recovered and intact interchain disulfide bonds.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Free sulfhydryl in recombinant monoclonal antibodies

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other recombinant protein drugs, therapeutic mAb's need to be well characterized to ensure their structural and functional integrity. IgG mAb's are composed of two heavy and two light chains covalently linked by interchain disulfide bonds. Each domain of the heavy or light chain contains one additional disulfide bond. Native IgG mAb's, with completely formed disulfide bonds, should not bear any free sulfhydryl. This report describes detection and quantification of free sulfhydryl in recombinant mAb's produced in Chinese hamster ovary (CHO) cells using a fluorescent technique. The method utilizes the fluorescent probe N-(1-pyrenyl)maleimide (NPM). The purified mAb's appear to be homogeneous under native conditions with approximately 0.02 mol of free sulfhydryl per mole of protein. Upon denaturation, minor species related to the mAb's are observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the free sulfhydryl level is determined to be approximately 0.1 mol/mol of protein. These results suggest that a small portion of these recombinant mAb's lack in intermolecular disulfide bonds but remain noncovalently associated under native conditions. The formation of the free sulfhydryl containing mAb species is likely to occur during the culture process and/or protein folding process in the endoplasmic reticulum (ER).     

Disulfide bonding of secretory component to a single monomer subunit in human secretory IgA.

The arrangement of disulfide bonds joining secretory component (SC) to the alpha chains in secretory IgA was studied by determining the molecular size of the principal fragments resulting from CNBr digestion of secretory dimeric Fc fragments from IgA (Fc)2alpha fragments). In vitro complexes formed by incubating 125I-free SC and myeloma 131I-(Fc)2alpha fragments were isolated by gel filtration and subsequently digested with cyanogen bromide. The CNBr digests of SC-(Fc)2alpha fragments were analyzed by gel filtration in 5 M guanidine. Two principal fragments were obtained, one containing a monomeric Fc fragment from IgA (Fcalpha) associated with SC (m.w. congruent to 110,000) and a second containing the second Fcalpha monomer (m.w. congruent to 50,000) from the dimeric SC-(Fc)2alpha. Similar results were obtained when secretory (Fc)2alpha fragments isolated from native secretory IgA dimer were subjected to CNBr digestion. The data indicate that SC is disulfide bonded to a single monomer subunit in secretory IgA dimer.     

A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants

A simple method was developed for the controlled cleavage of protein disulfide bonds and the simultaneous blockage of the free sulfhydryl groups in the absence of a denaturant. The disulfide bonds of bovine serum albumin were cleaved unsymmetrically at pH 7.0 using 0.1 M sulfite in 0.1 M phosphate buffer and the free sulfhydryl groups formed were sulfonated in an oxidation-reduction cycle using molecular oxygen and 400 microM cupric sulfate as a catalyst. The reaction was affected by cupric ion concentration, sulfite concentration, reaction pH and temperature. The standardized method was successfully used to cleave the disulfide bonds of other proteins pepsin, trypsin, and chymotrypsin. The method is reliable and can be used for achieving progressive cleavage of disulfide bonds in proteins without employing a denaturant.     

Disulfide interchange reactions between cystamine and cystine peptides

A method is presented for carrying out interchange reactions on a small scale between cystamine and low molecular weight cystine peptides. The products of the reaction were separated from unchanged peptides and from excess cystamine by high-voltage electrophoresis on paper. The opening of intrachain disulfide bonds required substantially higher concentrations of cystamine than did the reaction of interchain bonds. The interchange reaction can be used to separate cysteine peptides on an analytical or a preparative scale from other components of enzymic digests of proteins. A cys-gly bond in one interchange product was hydrolyzed by trypsin, but a cys-leu bond in another interchanged peptide was insensitive to trypsin.     

2,2'-Bispyridyl disulfide rapidly induces intramolecular disulfide bonds in peptides.

A linear peptide containing two reduced cysteine residues can be rapidly converted to its oxidized cyclic form containing an intramolecular disulfide bond by adding an excess of 2,2'-bispyridyl disulfide (2,2'-dipyridyl disulfide or 2,2'-dithiodipyridine) to conventional buffer solutions. The reactants and products are easily separated by reverse-phase chromatography. This reaction will find wide application in forming intramolecular disulfide bonds because of its selectivity for free sulfhydryl groups, quickness, safety, and applicability under acidic conditions.     

Location of disulfide bonds within the sequence of human serum cholinesterase

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.     

A study of the association of human secretory component with IgA and IgM proteins.

Human secretory component (SC) was isolated from colostral whey, and the binding of 125I-SC to purified IgA and IgM monoclonal proteins was studied using two methods to separate free from immunoglobulin-bound 125I-SC: a) gel filtration on Sephadex G-200, and b) precipitation of 125I-SC-Ig complexes with anti-Ig antibody. Both IgA dimeric proteins and IgM pentamers bound 125I-SC with approximately one SC-binding site per mole of polymer and similar affinity. Assuming a reversible equilibrium, an apparent association constant congruent to 10-8 M-1 was calculated to govern the binding of 125I-SC to immunoglobulin polymers. The assignment of a single association constant may be an oversimplication, particularly for the case of IgA polymers, since evidence was obtained that disulfide bonds were formed in the 125I-SC-IgA complex. Despite the complexity of the reaction, binding of 125I-SC to both IgA and IgM polymers could be analyzed by standard methods of saturation analysis, and both were shown to have a similar affinity for 125I-SC. No differences were noted in the affinity of 125I-SC binding to the IgA1 and IgA2 subclasses. Binding of monomeric IgA and IgM proteins could not be measured and was at least 100-fold lower than that found for IgA and IgM polymers. Complexes of 125I-SC with IgA dimers were presumed to involve covalent bond formation, since these complexes did not dissociate in guanidine-HCl. One IgA2 trimer did not form a covalent bond since it was completely dissociated in guanidine. In contrast, 125I-SC-IgM complexes were dissociated in denaturing solvent, indicating that such complexes were held together primarily by non-covalent bonds. Experiments with (Fc)5 mu isolated by high temperature tryptic digestion of IgM showed that binding of 125I-SC was to the Fc region of IgM proteins. It was suggested that the binding of SC with similar affinity to both IgA and IgM polymers may be important in the biologic function of both these immunoglobulin classes.     

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.     

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.     

Effect of the intermolecular disulfide bond on the conformation and stability of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of proteins. It exists as a covalent dimer in solution, with the 15 kDa monomers linked by an interchain disulfide bond through the Cys101 residues. Sedimentation equilibrium and velocity experiments demonstrated that, after removal of the interchain disulfide bond, GDNF remains as a non-covalent dimer and is stable at pH 7.0. To investigate the effect of the intermolecular disulfide on the structure and stability of GDNF, we compared the solution structures of the wild-type protein and a cysteine-101 to alanine (C101A) mutant using Fourier transform infrared (FTIR), FT-Raman and circular dichroism (CD) spectroscopy and sedimentation analysis. The elimination of the intermolecular disulfide bond causes only minor changes (approximately 4%) in the secondary structures of GDNF. The far- and near-UV CD spectra demonstrated that the secondary and tertiary structures were similar for both wild-type and C101A GDNF. Heparin binding and sedimentation velocity experiments also indicated that the folded structure of the wild-type and C101A GDNF are indistinguishable. The thermal stability of GDNF does not appear to be affected by the absence of the interchain disulfide bond and the biological activity of the C101A mutant is identical with that of the wild-type protein. However, small but significant changes in side chain conformations of tyrosine and aliphatic residues were observed by FT-Raman spectroscopy upon removal of the intermolecular disulfide bond, which may reflect structural changes in the area of dimeric contact. By comparing the Raman spectrum of wild-type GDNF with that of the C101A analog, we identified the conformation of the intermolecular disulfide as trans-gauche-trans geometry. These results indicate that GDNF is an active, properly folded molecule in the absence of the interchain disulfide bond.     

On the Role of Disulfide Bonds in Polymerization of Soybean 7S Globulin during Storage

Modification of soybean 7S globulin with N-ethylmaleimide (NEM) was effective for preventing humidity-induced insolubilization during storage. The sulfhydryl-disulfide interchange reaction and/or oxidation of the sulfhydryl groups to form disulfide bonds closely related to the polymerization of the 7S globulin. A dimer, consisting of α′ and α subunits linked with disulfide bonds, was observed in the polymerized 7S globulin fractions, but this dimer was not readily apparent in the NEM-7S globulin. The formation of the dimer certainly initiates the insolubilization of the 7S globulin during storage. Although the β subunit had sulfhydryl groups, it did not take a part in the formation of the dimer.     

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.     

Antiparallel alignment of the two protomers of the regulatory subunit dimer of cAMP-dependent protein kinase I

The purified type I regulatory subunit of cAMP-dependent protein kinase is a dimeric protein, and the two protomers of the dimer are linked by two interchain disulfide bonds. The disulfide linkages that join these two polypeptide chains have been identified in order to provide a structural basis for the orientation of the two chains in the asymmetric dimer. Disulfide bonds were found to exist exclusively between Cys-16 and Cys-37, and this assignment, thus, establishes a general antiparallel alignment of the two chains. Two other homologous proteins, the type II regulatory subunit and the cGMP-dependent protein kinase also are dimeric proteins. In all three proteins, a relatively small, nonhomologous, amino-terminal segment of the polypeptide chain is essential for maintaining the dimeric aggregation state.     

Interaction of S-sulfonated human IgG with human complement and its components.

S-sulfonated human IgG (S-sIgG) was prepared by treating IgG with sodium sulfite and sodium tetrathionate. The treatment resulted in the selective cleavage of interchain disulfide bonds of the IgG to give S-sulfonate groups. Complement fixing activities of aggregated S-sIgG and the immune complex formed with the S-sIgG antibody were very weak. S-sIgG at a high dose reduced the activity of the first complement component (C1) in normal human serum without any reduction of other complement components activites, but S-alkylated IgG at the same dose did not. Loss of C1 activity was not caused by either S-sulfonated myeloma proteins (IgA and IgE) or urea-treated S-sIgG, in which both inter- and intra-chain disulfide bonds were cleaved. These results suggest that the selective reduction of C1 by S-sIgG is due to a conformational change of the immunoglobulin.     

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.