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 (Naj a naja atra) blood serum, which yields strong specific protection agai nst 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 est imated to be 70.3+/-0.3 kDa. Tryptic hydrolysis of CSAP yielded severa l peptides that were able to bind to the toxin. The native CSAP maxima lly bound 8+/-l toxin molecules/molecule. Six tryptic fragments, conta ining 5-39 residues, were sequenced. The longest of these displayed se quence similarity to rat serum albumin. The protective effect of the C SAP was demonstrated in vivo on mice and in vitro by measurement of th e rate of haemolysis. Kinetic and thermodynamic parameters of the bind ing interactions of the neurotoxin and the CSAP were determined from t he rates of displacement of I-125-labelled toxin from its complexes wi th the CSAP by unlabelled toxin by using a DEAE-cellulose filter assay for CSAP-toxin complexes. The toxin molecules rapidly dissociated fro m 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.