1. Synthesis and molecular characterization of a biotinylated analog of [Lys]bradykinin
- Author
-
Marian Kelley, John Yanni, Henry R. Wolfe, and David G. Sawutz
- Subjects
Physiology ,Stereochemistry ,Guinea Pigs ,Molecular Sequence Data ,Lysine ,Bradykinin ,Peptide ,In Vitro Techniques ,Biochemistry ,Structure-Activity Relationship ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Endocrinology ,Biotin ,Ileum ,Peptide synthesis ,Animals ,Amino Acid Sequence ,Binding site ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,Molecular Structure ,Kallidin ,Receptors, Bradykinin ,Cell Membrane ,Muscle, Smooth ,Receptors, Neurotransmitter ,Kinetics ,chemistry ,Biotinylation ,Indicators and Reagents ,Muscle Contraction - Abstract
We report the synthesis and molecular characterization of a biotinylated analog of kallidin, [Lys]bradykinin. Bradykinin was prepared by solid phase peptide synthesis. Before cleavage from the resin, a biotin moiety was coupled to the epsilon amino group of a lysine in the zeroth position of the bradykinin peptide. An omega-amino caproic acid spacer was incorporated between the biotin group and the N-terminal lysine. The biotinylated peptide was deprotected, cleaved from the resin and purified by RP-HPLC. The identity of this analog was confirmed by amino acid analysis and FAB-mass spectrometry. Biotinyl [Lys]bradykinin (BLBK, mol, wt. = 1528) inhibited [3H]-bradykinin binding to guinea pig ileum homogenates dose dependently, with an IC50 of 28.9 +/- 6 nM. The IC50 for [Lys]bradykinin was approximately 10-fold lower, 3.2 +/- 0.6 nM. BLBK induced contractility in an isolated guinea pig smooth muscle preparation with an EC50 of 129 +/- 14 nM; the corresponding value for [Lys]bradykinin was 29 +/- 8 nM. These data are consistent with the difference in binding potency observed for BLBK compared to [Lys]bradykinin. In an ELISA assay using BLBK and affinity-purified rabbit anti-bradykinin antibody, BLBK bound to anti-bradykinin antibody with an EC50 = 1.21 +/- 0.54 nM. Rank order potencies for several bradykinin peptide analogs suggest that the epitope on bradykinin recognized by the antibody is likely to be at the carboxy terminus of the peptide.
- Published
- 1991