title: Porcine odorant-binding protein: structural stability and ligand affinities measured by fourier-transform infrared spectroscopy and fluorescence spectroscopy.
abstract: Infrared spectra show that the binding of the odorants 2-isobuthyl-3-methoxypyrazine (PYR) and 3,7-dimethyl-1-octanol (DMO) stabilises the tertiary structure of porcine OBP-I against thermal denaturation. The fluorescence emission spectrum of the single tryptophan shows a lambdamax at 337 nm, indicating that the residue is not directly exposed to the solvent. Tryptophan does not appear to be involved in the odorant binding process and it is not accessible to the fluorescence quenchers NaI, CsCl and acrylamide. The binding of the fluorescent dye 1-aminoanthracene (1-AMA), a strong ligand, does not modify the tryptophan fluorescence spectrum. In contrast, the lambdamax of 1-AMA bound to OBP-I is shifted from 537 to 481 nm, with a lambdamax intensity increase by a factor of 80. Bound 1-AMA is displaced by odorant molecules in competitive binding assays and can be employed in simple and rapid binding assay, avoiding the use of radioactive ligands. The Scatchard plot shows that 1-AMA binds to OBP-I with a dissociation constant of 1.3 microM and an equimolar stoichiometry.
authors: Dipartimento di Biologia Cellulare e Molecolare, Università di Perugia, Via del Giochetto 6, 06126, Perugia, Italy.
authors: S
authors: S
authors: Paolini
authors: F
authors: F
authors: Tanfani
authors: C
authors: C
authors: Fini
authors: E
authors: E
authors: Bertoli
authors: Paolo Pelosi
mesh_terms: D000818:Animals; D000873:Anthracenes; D001667:Binding, Competitive; D008024:Ligands; D015394:Molecular Structure; D009297:Nasal Mucosa / Q000737:chemistry*; D017433:Protein Structure, Secondary; D018035:Receptors, Odorant / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013552:Swine; D013696:Temperature
chemical_list: D000873:Anthracenes; D008024:Ligands; D018035:Receptors, Odorant; C045648:odorant-binding protein; C049062:1-aminoanthracene