Preparation and Characterization of Biotinylated Analogs of the Calcium Channel Blocker ω-Conotoxin

• Published in: Analytical biochemistry 1993-10, Vol.214 (1), p.227-232
• Main Authors: Jones, O.T., So, A.P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Animals; Binding, Competitive; Biological and medical sciences; Biotin; Brain - metabolism; Calcium Channel Blockers - chemical synthesis; Calcium Channel Blockers - metabolism; Chromatography, High Pressure Liquid - methods; Fundamental and applied biological sciences. Psychology; Iodine Radioisotopes; Kinetics; Mathematics; Models, Theoretical; Molecular Sequence Data; omega-Conotoxins; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptides - chemical synthesis; Peptides - metabolism; Peptides, Cyclic - chemical synthesis; Peptides, Cyclic - metabolism; Proteins; Radioligand Assay - methods; Rats; Synaptic Membranes - metabolism; Thermodynamics; Trypsin
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1993.1481

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 (Kdkin = k−2/k2 = 4 pM for ωCgTx), binding of these labels to membranes is nevertheless of very high affinity (Kdkin 0.1-0.3 nM).