Analysis of a conformation-independent epitope and a conformational epitope in a protein: a study on cobrotoxin from Taiwan cobra venom.
The antibodies against cobrotoxin were separated into two antibody preparations by successive affinity chromatographies on reduced and S-carboxymethylated (RCM)-cobrotoxin-Sepharose, and cobrotoxin-Sepharose columns. The antibodies (abbreviated as Abcf-i) that bound with the RCM-cobrotoxin-Sepharose were verified to specifically recognize the continuous epitopes of cobrotoxin, which were insensitive to conformational changes. Whilst the antibodies (abbreviated as Abcf-d) that did not bind with the RCM-cobrotoxin-Sepharose column recognized the conformational epitopes in cobrotoxin. The two antibody preparations were employed to screen the antigenic peptides derived from the proteolytic hydrolysate of cobrotoxin and RCM-cobrotoxin. Five antigenic peptides (AP-4, AP-5, AP-10, AP-11, and AP-12) were obtained from the acid protease A-digested hydrolysate of cobrotoxin, and two antigenic peptides (V8-2 and V8-4) were found in the hydrolysate of RCM-cobrotoxin after hydrolysis with Saccharomyces aureus V8 protease. The segments at positions 1-21 and 22-38 encompassed the peptide fractions, AP-4, AP-5, V8-2, and V8-4, that reacted with Abcf-i, indicating that the two segments bore the continuous epitopes of cobrotoxin. Alternatively, AP-10, AP-11, and AP-12 reacted with both Abcf-i and Abcf-d. The structures of the three peptides had a common segment at positions 43-62, suggesting that this region comprised the conformation-independent epitopes as well as conformational epitopes in cobrotoxin. These results reflected that the conformation-independent and conformational epitopes in a protein can be separately identified.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
Kuo
K W
KW
Lin
J
J
Lin
S R
SR
Chang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002846:Chromatography, Affinity; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000276:immunology*; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D003039:Cobra Neurotoxin Proteins; D004546:Elapid Venoms; D000939:Epitopes; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D016282:Aspartic Acid Endopeptidases