scholarly journals Thiol groups of normal immunoglobulin G

1974 ◽  
Vol 137 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Barbara M. Buchwald ◽  
G. E. Connell
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin G ◽  
Specific Radioactivity ◽  
Reduced Form ◽  
Light Chains ◽  
Thiol Groups ◽  
Myeloma Proteins ◽  
Disulphide Bonds ◽  
Sh Group ◽  
Normal Human

Normal human immunoglobulin G (IgG) was treated with radioactive N-ethylmaleimide in the absence of a reducing agent but in the presence of a denaturing solvent. The sites of reaction were determined by isolation of the radioactive peptides from thermolytic digests of the heavy and light chains. Six radioactive peptides were purified from the digest of the light chain and ten from that of the heavy chain. When sequenced, all the peptides were identical with peptides that would be predicted for the half-cystine residues involved in disulphide bonds. The specific radioactivity of the peptides indicated that the proportion of the half-cystine residues in the reduced form varied from 0.57 to 2.54%. These results indicate that the disulphide bonds of IgG are not completely oxidized. The estimate of 0.2mol of SH group/mol of IgG (Cecil & Stevenson, 1965; Luks & Connell, 1968) can be accounted for if 0.8% of every half-cystine residue of the intrachain bonds was in the reduced form. Variable cysteine residues as have been described in several myeloma proteins must occur extremely infrequently among the immunoglobulins.

scholarly journals The occurrence of disulphide bonds in purified clathrin light chains

1989 ◽  
Vol 257 (3) ◽  
pp. 775-781 ◽  
Author(s):  
P Parham ◽  
F M Brodsky ◽  
K Drickamer
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Reduced Form ◽  
Light Chains ◽  
Cysteine Residues ◽  
Free Light Chains ◽  
Clathrin Heavy Chain ◽  
Disulphide Bonds ◽  
Antigenic Properties ◽  
Reduced Forms

Three forms of clathrin light chain contain two cysteine residues. These are the predominant brain-specific forms of LCa and LCb and the non-brain form of LCb. After purification in the absence of thiols they contain intramolecular disulphide bonds. The reduced and the oxidized forms show differences in electrophoretic mobility, explaining the variable and heterogeneous patterns observed on electrophoresis. Accessibility of the thiol groups in the free light chains is greater than when they are associated with the heavy chain. In contrast the cysteine residues of the clathrin heavy chain are completely inaccessible in the absence of denaturants and are not found in disulphide bonds. The antigenic properties of the oxidized and the reduced forms of the clathrin light chains are similar, as is their capacity to bind to the clathrin heavy chain. After isolation in the presence of 10 mM-iodoacetamide, the light-chain cysteine residues are fully alkylated. The results are consistent with the reduced form being the native state and the light-chain disulphide bonds an artifact of isolation.

scholarly journals Differential reduction of interchain disulphide bonds of mouse immunoglobulin G

1968 ◽  
Vol 107 (6) ◽  
pp. 823-828 ◽  
Author(s):  
Alan R. Williamson ◽  
Brigitte A. Askonas
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin G ◽  
Polyacrylamide Gel Electrophoresis ◽  
Partial Reduction ◽  
Free Light Chains ◽  
Myeloma Protein ◽  
Heavy Chains ◽  
Disulphide Bonds ◽  
Rabbit Immunoglobulin ◽  
Mouse Immunoglobulin

The relative lability of the interchain disulphide bonds of mouse G2a-myeloma protein 5563 was studied as a function of 2-mercaptoethanol concentration. Analysis of partial-reduction mixtures by polyacrylamide-gel electrophoresis and microdensitometry showed that the disulphide bonds between light and heavy chains are much more susceptible to reduction than the bonds between heavy chains. At a low concentration of 2-mercaptoethanol (10mm) the major dissociable products of mouse immunoglobulin G are heavy-chain dimers and free light chains. These findings contrast with the reported behaviour of rabbit immunoglobulin G, for which the lability of inter-heavy-chain bonds was found to exceed that of the bonds linking light and heavy chains (Hong & Nisonoff, 1965); the relative stability of rabbit immunoglobulin G interchain bonds was confirmed in the present study. Examination of human immunoglobulin G and an immunoglobulin G (γ2) of guinea pig showed that at least in the majority of molecules, as with mouse immunoglobulin G, the disulphide bonds between light and heavy chains are more susceptible to reduction than the inter-heavy-chain bonds.

scholarly journals The disulphide bonds of the heavy chain of rabbit immunoglobulin G

1970 ◽  
Vol 116 (2) ◽  
pp. 261-268 ◽  
Author(s):  
I. J. O'Donnell ◽  
B. Frangione ◽  
R. R. Porter
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin G ◽  
Good Yield ◽  
Sequence Variants ◽  
Lower Yield ◽  
Disulphide Bonds ◽  
Rabbit Immunoglobulin ◽  
Minor Sequence

Six peptides containing eight half-cystine residues were isolated in good yield, after either oxidation or reduction and carboxymethylation of fragment C-1, which contains the N-terminal half of the heavy chain of rabbit immunoglobulin G. The sequences of five of these peptides had been reported previously (Cebra, Steiner & Porter, 1968b; Wilkinson, 1969) and that of the sixth was established. Other peptides containing half-cystine residues were isolated in much lower yield and are presumed to be derived from minor sequence variants. The cystine-containing peptides from enzymic digests of whole immunoglobulin G and Fc fraction were studied by several techniques and the results obtained enable us to put forward a scheme of the arrangement of the inter- and intra-chain disulphide bonds.

scholarly journals Intrachain disulphide bridges in immunoglobulin G heavy chains. The Fc fragment

1968 ◽  
Vol 106 (1) ◽  
pp. 15-21 ◽  
Author(s):  
B. Frangione ◽  
C. Milstein ◽  
Edward C. Franklin
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin G ◽  
The Other ◽  
Light Chains ◽  
Fc Fragment ◽  
Heavy Chains ◽  
Human Immunoglobulins ◽  
Other Hand ◽  
Terminal Loop ◽  
Heavy Chain Disease

The disulphide bridges of the Fc fragment (C-terminal half of the heavy chain) have been studied in several human immunoglobulins, containing heavy chains of different antigenic types (γ1, γ2, γ3 and γ4), and in heavy-chain-disease proteins. Two intrachain disulphide bridges were found to be present. The sequences appear to be identical in the Fc fragments of two types of chain studied (γ1 and γ3), and very similar to corresponding sequences of the Fc fragment in rabbit. These results suggest that the C-terminal half of the heavy chains is covalently folded (in a similar fashion to the light chains) with a C-terminal loop and an N-terminal loop. The similarity is emphasized by comparison of the sequence and location of the disulphide-bridged peptides of the C-terminal loop of heavy and light chains. The N-terminal loop, on the other hand, appears to be very different in Fc fragments and light chains. The C-terminal loop is the only one present in the F′c fragment.

scholarly journals The recombination of dimers of immunoglobulin peptide chains

1970 ◽  
Vol 118 (5) ◽  
pp. 703-712 ◽  
Author(s):  
G. T. Stevenson ◽  
K. J. Dorrington
Keyword(s):  
Immunoglobulin G ◽  
Sodium Acetate ◽  
Optical Rotatory Dispersion ◽  
Acetate Buffer ◽  
Light Chains ◽  
Sodium Acetate Buffer ◽  
Covalent Bonds ◽  
Myeloma Protein ◽  
Disulphide Bonds ◽  
Antigenic Properties

1. Both the γ and light peptide chains of human pooled and myeloma immunoglobulin G can be prepared as non-aggregating dimers at pH5.4 in 4mm-sodium acetate buffer. The dimeric state is maintained by non-covalent bonds, since the formation of interchain disulphide bonds was prevented by alkylation of the thiol groups. In the case of the light chains there is some evidence that the dimers are in equilibrium with a small amount of monomer. 2. When such dimers of the γ and light chains are mixed at pH5.4 in 4mm-sodium acetate buffer they combine rapidly, yielding a product that resembles the original immunoglobulin G in its physicochemical and antigenic properties. However, the original optical rotatory dispersion spectrum was regained only with the homogeneous myeloma protein. The recombined pooled immunoglobulin G had a spectrum slightly different from the original, suggesting that at least some of the recombinant molecules had not regained native conformations. 3. Dimers of γ chains stabilized by interchain disulphide bonds were able to recombine with light chains. However, light chains stabilized in the dimeric state by interchain disulphide bonds would not combine with γ chains. 4. The chains of rabbit immunoglobulin G behave similarly to the human chains in this system, apart from the alkylated light chains showing clearer evidence of monomeric components.

scholarly journals Loss of an individual idiotype on chemical modification. A strategy for assigning idiotypic determinants.

1981 ◽  
Vol 153 (5) ◽  
pp. 1275-1285 ◽  
Author(s):  
J Dickerman ◽  
B Clevinger ◽  
B Friedenson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chemical Modification ◽  
Heavy Chain ◽  
Light Chains ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Low Levels ◽  
The Individual ◽  
Complementary Strategy

Two dextran-binding myeloma proteins, J558 and Hdex 24, which possess the same individual idiotype (IdI) were diazotized to low levels (1-3.3 groups per subunit) with 1-[14C]-p-aminobenzoate. Both proteins lost the IdI idiotype under these conditions with most of the label incorporated on the heavy chains of each protein. When the diazotization ws carried out in the presence of the hapten 1-O-methyl-alpha-D-glucopyranoside the loss of idiotypic reactivity could be prevented for J558 but not for Hdex 24. Under these conditions most of the label was incorporated on the light chains of J558, but on the heavy chains of Hdex 24. For J558, these results show that a major determinant of the individual idiotype is within the hypervariable positions of the heavy chain. For Hdex 24 the determinant being modified is on the heavy chain but not involved in hapten binding. These results are consistent with previous work showing that J558 and Hdex 24 differ in amino acid sequence in the D and the J segments of the heavy chain and offer an alternative and complementary strategy for assigning idiotypic determinants.

scholarly journals Oxidative refolding of recombinant prochymosin

1999 ◽  
Vol 340 (1) ◽  
pp. 345-351 ◽  
Author(s):  
Chongjuan WEI ◽  
Bin TANG ◽  
Yuying ZHANG ◽  
Kaiyu YANG
Keyword(s):  
Inclusion Bodies ◽  
Tertiary Structure ◽  
Reduced Form ◽  
Thiol Groups ◽  
Terminal Domain ◽  
Free Thiol ◽  
Rate Limiting Step ◽  
Oxidative Refolding ◽  
Disulphide Bonds ◽  
Two Stages

The disulphide-coupled refolding of recombinant prochymosin from Escherichia coli inclusion bodies was investigated. Prochymosin solubilized from inclusion bodies is endowed with free thiol groups and disulphide bonds. This partially reduced form undergoes renaturation more efficiently than the fully reduced form, suggesting that some native structural elements existing in inclusion bodies and remaining after denaturation function as nuclei to initiate correct refolding. This assumption is supported by the finding that in the solubilized prochymosin molecule the cysteine residues located in the N-terminal domain of the protein are not incorrectly paired with the other cysteines in the C-terminal domain. Addition of GSH/GSSG into the refolding system facilitates disulphide rearrangement and thus enhances renaturation, especially for the fully reduced prochymosin. Based on the results described in this and previous papers [Tang, Zhang and Yang (1994) Biochem. J. 301, 17-20], a model to depict the refolding process of prochymosin is proposed. Briefly, the refolding process of prochymosin consists of two stages: the formation and rearrangement of disulphide bonds occurs at the first stage in a pH 11 buffer, whereas the formation and adjustment of tertiary structure leading to the native conformation takes place at the second stage at pH 8. The pH 11 conditions help polypeptides to refold in such a way as to favour the formation of native disulphide bonds. Disulphide rearrangement, the rate-limiting step during refolding, can be achieved by thiol/disulphide exchange initiated by free thiol groups present in the prochymosin polypeptide, GSH/GSSG or protein disulphide isomerase.

scholarly journals Goat immunoglobin G. Peptide chains and terminal residues

1969 ◽  
Vol 115 (3) ◽  
pp. 371-375 ◽  
Author(s):  
D. Givol ◽  
E. Hurwitz
Keyword(s):  
Carboxylic Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Immunoglobulin G ◽  
Light Chains ◽  
Terminal Sequence ◽  
Terminal Residue ◽  
Heavy Chains ◽  
Molecular Weights ◽  
Immunoglobin G

Goat immunoglobulin G (IgG) was isolated and characterized. The molecular weights of the IgG and its heavy chains and light chains were found to be 144000, 53600 and 23000 respectively. The light chain corresponds to human L type as was shown by the absence of C-terminal S-carboxymethylcysteine and its high content of N-terminal pyrrolid-2-one-5-carboxylic acid (PCA). The major C-terminal residue of the light chain was serine and the major N-terminal dipeptide was PCA-Ala (0·6mole/mole). The major C-terminal residue of the heavy chain was glycine and the N-terminal sequence of the heavy chain is PCA-Val-Gln. This tripeptide was obtained in a 70% yield.

Studies on the amino acid sequence of heavy chains from rabbit immunoglobulin G

1966 ◽  
Vol 166 (1003) ◽  
pp. 159-175 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heavy Chain ◽  
Immunoglobulin G ◽  
Light Chains ◽  
Antibody Specificity ◽  
Sequence Heterogeneity ◽  
Specific Antibodies ◽  
Heavy Chains ◽  
Insight Into

It is now generally agreed that the four-chain subunit structure of Immunoglobulins which was first proposed by Porter (1962), accurately represents the gross structure of immunoglobulin G (IgG) and specific antibodies (Fleischman, Porter & Press 1963; Edelman & Gally 1964; Marler, Nelson & Tanford 1964; Nelson et al . 1965). However, an understanding of the structural basis of antibody specificity requires greater insight into the amino acid sequence of the polypeptide chain components of specific antibodies. Isolated light chains from specific antibodies and inert IgG, show a considerable degree of electrophoretic heterogeneity (Edelman & Gally 1964; Cohen & Porter 1964; Poulik 1964). Tryptic peptide maps of light chains (Nelson et al . 1965) have suggested that this heterogeneity may be accounted for by differences in amino acid sequence. This view has received considerable support from the observation that Bence-Jones proteins, which may be regarded as light chains, vary significantly in amino acid sequence (Hilschman & Craig 1965; Milstein 1966; Titani, Whitley & Putman 1966). A similar but less well-defined sequence heterogeneity has been suggested to exist in the heavy chains of specific antibodies (Feinstein 1964). However, the Fc fragment of the heavy chains has been thought to possess a regular amino acid sequence which may be similar, if not identical, among all specific antibodies (Porter 1959; Nelson et al . 1965). This paper summarizes the results of studies on the amino acid sequence of heavy chains and that portion of heavy chain, Fc fragment, which is obtained on treatment of rabbit IgG with papain (Porter 1959). These studies were designed to determine how much of the amino acid sequence of heavy chain could be accounted for by a unique, regular amino acid sequence which was common to most, if not all, IgG antibodies. In addition, attempts were made to locate regions of heavy chains which varied in amino acid sequence. Although structural variants appear to occur among the heavy chains found in non-specific IgG, it would be desirable to know what portion of the heavy chain sequence is invariant among all antibodies. If antibody specificity results from sequence heterogeneity in light and heavy chains, then knowledge of the variant and invariant portions of these chains may provide insight into the nature of specific binding sites in anti-­bodies.

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