Porcine odorant-binding protein: structural stability and ligand affinities measured by Fourier-transform infrared spectroscopy and fluorescence spectroscopy

Infrared spectra show that the binding of the odorants 2-isobuthyl-3-mrthox ypyrazine (PYR) and 3,7-dimethyl-1-octanol (DMO) stabilises the tertiary st ructure of porcine OBP-I against thermal denaturation. The fluorescence emi ssion spectrum of the single tryptophan shows a lambda(max) at 337 nm, indi cating 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 bin ding of the fluorescent dye 1-aminoanthracene (1-AMA), a strong ligand, doe s not modify the tryptophan fluorescence spectrum. In contrast, the lambda( max) of 1-AMA bound to OBP-I is shifted from 537 to 481 nm, with a lambda(m ax) intensity increase by a factor of 80. Bound 1-AMA is displaced by odora nt molecules in competitive binding assays and can be employed in simple an d rapid binding assay, avoiding the use of radioactive ligands. The Scatcha rd plot shows that 1-AMA binds to OBP-I with a dissociation constant of 1.3 mu M and an equimolar stoichiometry. (C) 1999 Elsevier Science B.V. All ri ghts reserved.