title: Synthesis and molecular characterization of a biotinylated analog of [Lys]bradykinin.
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.
authors.affiliation: Department of Enzymology and Receptor Biochemistry, Sterling Research Group, Sterling Drug Inc., Malvern, PA 19355.
authors.forename: D G
authors.initials: DG
authors.lastname: Sawutz
authors.forename: J
authors.initials: J
authors.lastname: Yanni
authors.forename: M
authors.initials: M
authors.lastname: Kelley
authors.forename: H
authors.initials: H
authors.lastname: Wolfe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D007082:Ileum / Q000187:drug effects / Q000502:physiology*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007609:Kallidin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D007202:Indicators and Reagents; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; C073648:biotinyl-kallidin; D007609:Kallidin; D001920:Bradykinin