Whereas to date binding studies have been restricted to the use of radiolabeled compounds or specific fluorescent probes for competitive binding experiments, we set up a general method (volatile-odorant binding assay, VOBA) for airborne odorant binding studies under physiological conditions without specific labeling. Here we report the purification and the cloning of a novel rat OBP-1 variant called OBP-1F. The presence of four OBP subtypes in mouse suggests that more than two OBP could be isolated from the rat mucus. Whereas OBP-1 preferentially binds heterocyclic compouds, such as pyrazines, OBP-2 seems to be more specific for long-chain aliphatic aldehydes and carboxylic acids, providing the first evidence that different OBP subtypes are specially tuned towards a distinct class of odorants. Recently, their binding properties have been investigated demonstrating that the two rat OBP have distinct ligand specificity ]. The latter was specifically found in the vomeronasal organ and not in the olfactory epithelium ]. In rat, two OBP (called OBP-1 and OBP-2) have been cloned with quite different sequences ]. Different OBP subtypes have been reported to occur simultaneously in the same animal species, two in pig ], four in mouse ], three in rabbit ] and at least eight in porcupine ]. OBP have been identified in a variety of species, including pig, rabbit, mouse and rat, ] since the discovery of the first vertebrate OBP isolated from the bovine nasal mucus ]. X-ray analysis has revealed that bovine and porcine OBP are folded in the typical β-barrel structure of lipocalins ]. Most OBP are homodimers, but monomers and heterodimers have also been reported ]. Although their functions are still unclear, OBP are also suspected to participate in the deactivation of odorants ]. These proteins reversibly bind odorants with dissociation constants in the micromolar range. The odorant-binding proteins (OBP), which are abundant low-molecular mass soluble proteins (≈ 20 kDa) secreted by the olfactory epithelium in the nasal mucus of vertebrates, have been thought to play such a role ]. In order to reach their membrane receptors embedded in the membrane of the olfactory neurons, airborne odorants, which are commonly hydrophobic molecules, have to be conveyed through the aqueous nasal mucus by carriers. Uptake of airborne odorants in nearly physiological conditions strengthens the role of OBP as volatile hydrophobic odorant carriers in the mucus of the olfactory epithelium through the aqueous barrier towards the chemo-sensory cells. The assay permitted observations on the binding of airborne odorants of different chemical structures and odors (2-isobutyl-3-methoxypyrazine, linalool, isoamyl acetate, 1-octanal, 1-octanol, dimethyl disulfide and methyl thiobutyrate). Owing to the large OBP-1F amounts expressed, we set up a novel biomimetic assay (volatile-odorant binding assay) to study the uptake of airborne odorants without radiolabelling and attempted to understand the odorant capture by OBP in the nasal mucus under natural conditions. Fluorescence experiments revealed that 1-AMA was displaced efficiently by molecules including usual solvents such as EtOH and dimethylsulfoxide. OBP-1F interacts with fluorescent probe 1-aminoanthracene (1-AMA) with a dissociation constant of 0.6 ± 0.3 µ m. We observed that, in contrast with porcine OBP-1, purified recombinant OBP-1F is a homodimer exhibiting two disulfide bonds (C44–C48 and C63–C155), a pairing close to that of hamster aphrodisin. L −1 in a form identical to the natural protein as shown by MS, N-terminal sequencing and CD.Recombinant OBP-1F, the sequence of which is close to that of previously reported rat OBP-1, has been secreted by the yeast Pichia pastoris at a concentration of 80 mg After characterization of a novel odorant-binding protein (OBP) variant isolated from the rat nasal mucus, the corresponding cDNA was cloned by RT-PCR.
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