Characterization of the cleavage products of human serum immunoglobulin disulphide bonds. I. S-Sulpho derivatives

1967 ◽  
Vol 20 (6) ◽  
pp. 1243 ◽  
Author(s):  
CJ Brackenridge
Keyword(s):  
Human Serum ◽  
Serum Immunoglobulin ◽  
Amino Terminal ◽  
Disulphide Bonds ◽  
Exclusion Chromatography ◽  
Polypeptide Chains ◽  
Antibody Interactions ◽  
Dispersing Agents ◽  
Time Of Reaction

An examination of variables, including amount of catalyst, pH, sulphite concentration, specific buffer salts, and time of reaction led to conditions for the quantitative sulphitolysis of human serum immunoglobulin disulphide bonds. The cleavage was carried out in the absence of dispersing agents, using air as oxidant and cupric ions as catalyst. The treated protein was characterized by solubility, exclusion chromatography, ultracentrifugation, and immunoelectrophoresis. Separation into three fractions of different molecular weight was achieved by passage through Sephadex G-200 gel. Efforts to eliminate the proportion of aggregated material, which showed no tendency to establish an equilibrium with the remaining two unaggregated fractions, were largely unsuccessful. It was concluded that non-covalent forces play a significant role in antibody interactions. The isolated fractions were individually characterized by sedimentation, heat precipitation, immunoelectrophoresis, and quantitative analysis of amino-terminal residues. This led to the demonstration of at least two polypeptide chains present in each fraction.

Characterization of the cleavage products of human serum immunoglobulin disulphide bonds. II. S-Cyano derivatives

1967 ◽  
Vol 20 (6) ◽  
pp. 1265 ◽  
Author(s):  
CJ Brackenridge
Keyword(s):  
Amino Acid ◽  
Human Serum ◽  
Native Protein ◽  
Terminal Amino Acid ◽  
Disulphide Bonds ◽  
Exclusion Chromatography ◽  
Terminal Amino ◽  
Oxidative Sulphitolysis ◽  
Polypeptide Chains

The displacement by cyanide ions of S-sulpho substituents from sulphitolysed human serum immunoglobulins yielded S-cyano derivatives, the solubility and sedimentation characteristics of which were examined with respect to pH. Exclusion chromatography gave rise to an aggregated and an unaggregated fraction in proportions similar to those found after oxidative sulphitolysis. Evidence obtained from immunoelectrophoresis, N-terminal amino acid assay, and hexose and amino acid analysis showed that both S-cyano fractions contained polypeptide chains in the same ratios as in the native protein.

scholarly journals Localization and Characterization of the Cleavage-Associated Neoantigen in the Edomain of Fibrinogen

1977 ◽  
Author(s):  
E. F. Plow ◽  
T. S. Edgington
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Conformational Changes ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Terminal ◽  
Exclusion Chromatography

Plasmic cleavage of fibrinogen to generate fragment X partially exposes a specific cryptic molecular site, fg-Eneo. This site in the E domain of the molecule is further exposed during subsequent cleavage. We now report on localization of this site which provides an incisive marker for the structural and conformational changes associated with plasmic cleavage of fibrinogen. Fg-Eneo was stable to reduction and alkylation and the chains of the E fragment were separated by ion exchange chromatography on DEAE-cellulose. An active component was obtained and subjected to molecular exclusion chromatography on Sephadex G-50 to insure removal of intact fg-E. A fg-Eneo positive chain was recovered and identified as Eγ with respect to amino-terminal tyrosine, amino acid composition, and immunochemical analysis. The fg-Eneo site was stable to tryptic degradation, and tryptic peptides were prepared and separated by multiple molecular exclusion chromatographic steps. Final separation of two peptides of similar size was achieved on the basis of carbohydrate content by affinity chromatography on Concanavalin A. Only the active peptide was bound by the lectin. Purity and identification of the active tryptic peptide as γ36–53 was established by amino acid composition and sequence. These results establish that this region of the γ chain of fibrinogen is not present at the hydrated surface of the native molecule but that, in association with plasmic cleavage and conformational changes, this site is progressively exposed and provides a dynamic marker of the cleavage sequence.

Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

Biochemistry ◽  
1990 ◽  
Vol 29 (6) ◽  
pp. 1654-1660 ◽  
Author(s):  
Walter D. Funk ◽  
Ross T. A. MacGillivray ◽  
Anne B. Mason ◽  
Stephen A. Brown ◽  
Robert C. Woodworth
Keyword(s):  
Recombinant Protein ◽  
Human Serum ◽  
Cultured Cells ◽  
Serum Transferrin ◽  
Human Serum Transferrin ◽  
Amino Terminal

scholarly journals Characterization of Human Serum Immunoglobulin G Modified with Singlet Oxygen

2013 ◽  
Vol 29 (1) ◽  
pp. 63-68 ◽  
Author(s):  
Aniket Gupta ◽  
Aadil Wani ◽  
Anamika Joshi ◽  
Haseeb Ahsan ◽  
Rizwan Ahmad
Keyword(s):  
Singlet Oxygen ◽  
Human Serum ◽  
Immunoglobulin G ◽  
Serum Immunoglobulin

scholarly journals Isolation and partial characterization of a D-galactose-binding lectin from the latex of Synadenium carinatum

2005 ◽  
Vol 48 (5) ◽  
pp. 705-716 ◽  
Author(s):  
Maria Aparecida Souza ◽  
Francielle Amâncio-Pereira ◽  
Cristina Ribeiro Barros Cardoso ◽  
Adriano Gomes da Silva ◽  
Edmar Gomes Silva ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Terminal Sequence ◽  
Native Page ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Polypeptide Chains ◽  
Binding Lectin

A lectin from the latex of Synadenium carinatum was purified by affinity chromatography on immobilized-D-galactose-agarose and shown to be a potent agglutinin of human erythrocytes. The haemagglutination of human red cells was inhibited by 3.0 mM N-acetyl-D-galactopyranoside, 6.3 mM methyl-beta-D-galactopyranoside, 50 mM methyl-alpha-D-galactopyranoside and 50 mM D-fucose but not by L-fucose, demonstrating an anomeric and a conformational specificity. According to SDS-PAGE analysis, the lectin appeared to be a glycoprotein composed of two polypeptide chains of ca. 28 and 30 kDa, but size exclusion chromatography (Sephadex G-100) and native PAGE revealed a protein of apparent molecular weight 120 - 130 kDa made up of 28 and 30 kDa subunits. The lectin was stable in the range pH 6 - 9, and 4 - 56ºC. The N-terminal sequence of the 30 kDa subunit contained the conserved consensus sequence GPN observed in other D-galactose-binding lectins found in latex of members of the Euphorbiaceae.

scholarly journals Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 582
Author(s):  
Cristian Franco-Servín ◽  
Edgar Neri-Castro ◽  
Melisa Bénard-Valle ◽  
Alejandro Alagón ◽  
Ramsés Alejandro Rosales-García ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Biochemical Characterization ◽  
Two Dimensions ◽  
Size Exclusion ◽  
Muscle Twitch ◽  
Amino Terminal ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Main Components

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30–35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve–hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

scholarly journals Characterization of tyrosyl kinase activity in human serum.

1985 ◽  
Vol 260 (3) ◽  
pp. 1582-1587 ◽  
Author(s):  
M F Lin ◽  
P L Lee ◽  
G M Clinton
Keyword(s):  
Human Serum ◽  
Kinase Activity
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