Purification, characterization and binding interactions of the Chinese-cobra (Naja naja atra) serum antitoxic protein CSAP.
The characterization of the single-chain protein in Chinese-cobra (Naja naja atra) blood serum, which yields strong specific protection against the venom of the same snake, is reported. The protein, CSAP (cobra serum antitoxic protein), was purified to electrophoretic homogeneity. Over the pH range 5-9 it formed stable complexes with the neuro- and the cardio-toxin of the snake. The molecular size of the CSAP was estimated to be 70.3 +/- 0.3 kDa. Tryptic hydrolysis of CSAP yielded several peptides that were able to bind to the toxin. The native CSAP maximally bound 8 +/- 1 toxin molecules/molecule. Six tryptic fragments, containing 5-39 residues, were sequenced. The longest of these displayed sequence similarity to rat serum albumin. The protective effect of the CSAP was demonstrated in vivo on mice and in vitro by measurement of the rate of haemolysis. Kinetic and thermodynamic parameters of the binding interactions of the neurotoxin and the CSAP were determined from the rates of displacement of 125I-labelled toxin from its complexes with the CSAP by unlabelled toxin by using a DEAE-cellulose filter assay for CSAP-toxin complexes. The toxin molecules rapidly dissociated from one type of site and slowly from a second. The binding capacity and concentration of the CSAP suffice to explain the protective effect of the latter against the toxin.
Shao
J
J
Institute of Biochemistry and Molecular Biology, Zhejiang Medical Univesity, Hangzhou, People's Republic of China.
Shen
H
H
Havsteen
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000992:Antitoxins / Q000737:chemistry / Q000302:isolation & purification*; D001665:Binding Sites; D002853:Chromatography, Liquid; D004546:Elapid Venoms / Q000037:antagonists & inhibitors* / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D000992:Antitoxins; C082394:CSAP protein, Naja atra; D004546:Elapid Venoms; D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin