CHANGES TO BIOLOGICAL-ACTIVITY FOLLOWING ACETYLATION OF DENDROTOXIN-IFROM DENDROASPIS-POLYLEPIS (BLACK MAMBA)

The potassium channel blocker dendrotoxin I was acetylated with acetic anhydride. Mono-acetyl derivatives of all seven lysine residues (N-te rminus blocked) and a di-derivative were isolated by chromatography on the cation-exchanger Bio-Rex 70 and reversed-phase high-performance l iquid chromatography. The derivative acetyl-lys 29 and the di-derivati ve of Tyr 24 and Lys 28 had more than 1000 times lower affinity than t he native toxin, as determined by inhibition of the I-125-dendrotoxin binding to synaptosomal membranes from rat brain. Lys 29 is part of th e triplet Lys-Lys-Lys (28-30) which also occurs in the homologous a de ndrotoxin where the triplet is not in the functional site, as shown by site-directed mutagenesis, Acetylation of Lys 29 may have reduced lar ge structural perturbations that inactivated the toxin. Acetylation of Lys 28 alone had little effect, but the toxin became almost inactive when both Lys 28 and Tyr 24 were modified. Ten experiments were conduc ted under similar conditions, but a derivative of Tyr 24 was obtained only three times. In these cases the toxin apparently had a different structure, with Tyr 24 accessible to the reagent. This may depend on f reeze-drying, which call alter the structure of proteins. The third de rivative with low activity was acetyl-lys 5, with affinity decreased 2 0-fold. Lys 5 has a protruding side-chain that does not interact with any other group in the toxin molecule. Therefore, Lys 5 is probably pa rt of the functional site for dendrotoxin's binding to the voltage-dep endent K+ channels. (C) 1997 Elsevier Science Ltd.