PREPARATION AND CHARACTERIZATION OF BIOTINYLATED ANALOGS OF THE CALCIUM-CHANNEL BLOCKER OMEGA-CONOTOXIN

We have prepared a series of biotinylated analogs of ω-conotoxin (ωCgTx) as potent, selective markers for N-type calcium channels. At pH 9.5, reaction of ωCgTx with amidocaproylbiotin succinimidyl ester gives three biotinylated conjugates, labeled at lysines 2 or 24, or at both positions. Kinetic competition assays of 125I-ωCgTx binding to rat brain synaptic membranes show that each conjugate has a similar rate constant for association (1-1.3 × 106 M-1 s-1) but not dissociation (1-4 × 10-4 s-1). Comparison with rate constants obtained for the association (1.2 × 107 M-1 s-1) and dissociation (5 × 10-5 s-1) of native ωCgTx indicates that while biotinylation reduces ωCgTx potency (K(d(kin)) = k-2/k2 = 4 pM for ωCgTx), binding of these labels to membranes is nevertheless of very high affinity (K(d(kin)) 0.1-0.3 nM). © 1993 Academic Press, Inc.