Structure-function relations of biotin derivatives of apamin

Apamin was biotinylated at various residues so as to produce an apamin derivative that was suitable for labelling apamin sensitive K+ channels. We labelled the sole histidine residue (His-18) of apamin with diazobenzoyl biocytin (DBB) and Lys-4 with NHS-biotin and NHS-XX-biotin. We found that at least two labelled species were produced by DBB. Proton NMR spectroscopy revealed that in addition to labelling the His-18, DBB labelled the Gln-16 and Gln-17 of apamin. Both NHS-biotin and NHS-XX-biotin appeared to specifically label Lys-4. To test the potency of these apamin derivatives, we developed an assay using apamin reversal of the adrenaline induced relaxation of the mouse ascending colon. The biological activity of the His-18 derivative was 46-fold less than that of native apamin. Biotinylation of Lys-4 with NHS-biotin reduced the activity by only 6-fold. The inclusion of a 14-carbon spacer between the Lys-4 and the biotin resulted in a derivative with only a 4-fold reduction in potency.