title: Preparation and properties of an improved photoaffinity ligand for the N-formyl peptide receptor.
abstract: A new superior photoaffinity ligand for the N-formyl peptide receptor was prepared by derivatization of N-formyl-Met-Leu-Phe-Lys with a commercially available heterobifunctional crosslinking agent. The product, N-formyl-Met-Leu-Phe-N epsilon-(2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionyl)-Lys was readily synthesized and radiolabelled, and had increased specificity and stability as compared to previously used photoaffinity ligands. The ligand rapidly associated with the receptor with high affinity (Kd = 0.28 nM). Once bound, it was virtually non-dissociable (in the absence of photolysis) in an experimental time-frame (t1/2 (off) = 154 min). The covalent incorporation of the photoaffinity ligand into the receptor upon irradiation was complete within 5 min, minimizing cell damage, and the efficiency of covalent incorporation was approx. 40%. The derivative had enhanced biological activity, having an ED50 for superoxide anion production of 0.23 nM, 27-fold lower than its parent peptide. This derivative of the N-formyl peptide was useful for up to 3 months after radiolabelling, showing a progressive decline in specific activity during storage in the dark at 4 degrees C. The enhanced stability, reproducibility and solubility of the photoaffinity ligand is expected to aid in the purification of the N-formyl peptide receptor and will prove a useful tool for analysing receptor-mediated processes.
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism; D001386:Azides / Q000138:chemical synthesis*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D009504:Neutrophils / Q000378:metabolism; D010777:Photochemistry; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D013379:Substrate Specificity; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors
chemical_list: D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; C049721:N-formyl-methionylleucyl-phenylalanyl-N(epsilon)-(2-(4-azidosalicylamido)ethyl-1,3'-dithiopropionyl)lysine; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine