Upon venom fractionation on Sephadex G50 seven fractions were obtained (Fig. 1A). F3–F6 were found toxic for mice and were further screened using toxicity in mice by i.c.v. injection, off-line MALDI/TOF-MS and ELISA tests with the specific serum raised against native KTX. The smallest molecular weight
Pifithrin (ranging between 3000 and 4500 Da) were detected in F5, which was analyzed using reverse-phase HPLC (Fig. 1B). A single component at the HPLC Retention Time (RT) 10 min was identified as positive to the ELISA test. It was submitted to a second HPLC step, which led to a perfect homogenous peptide. It was named Aam-KTX (Fig. 1C). Its molecular mass of 4184.08 Da (Fig. 1D) differed slightly from that of KTX (4150 Da). Aam-KTX constitutes 0.76% in weight of the whole crude venom. With a LD50 (i.c.v. injection) of 100 ng/mouse, Aam-KTX was found four times less toxic than KTX. Aam-KTX was tested for its ability to compete with the 125I-KTX bound to its specific antibodies in liquid-phase RIA. Unlabelled Aam-KTX fully inhibited the binding of 125I-KTX, but with a four times weaker affinity: EC50 of 5.6 nM for Aam-KTX instead of 1.5 nM for KTX (data not shown). This result was in favor of high affinity recognition of Aam-KTX by the KTX antibodies. Consequently no major modifications in the conformational epitopes were expected, as well as no large amino
acid sequence variations, since it was previously demonstrated that scorpion toxins epitopes are mainly conformational [20].
