Infrared spectra show that the binding of the odorants 2-isobuthyl-3-mrthoxypyrazine (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 lambda(max) 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 lambda(max) of 1-AMA bound to OBP-I is shifted from 537 to 481 nm, with a lambda(max) 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 mu M and an equimolar stoichiometry. (C) 1999 Elsevier Science B.V. All rights reserved. RI Tanfani, Fabio/F-1441-2010
Porcine odorant-binding protein: structural stability and ligand affinities measured by Fourier-transform infrared spectroscopy and fluorescence spectroscopy
PELOSI, PAOLO
1999-01-01
Abstract
Infrared spectra show that the binding of the odorants 2-isobuthyl-3-mrthoxypyrazine (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 lambda(max) 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 lambda(max) of 1-AMA bound to OBP-I is shifted from 537 to 481 nm, with a lambda(max) 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 mu M and an equimolar stoichiometry. (C) 1999 Elsevier Science B.V. All rights reserved. RI Tanfani, Fabio/F-1441-2010I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
