Sective deficits in the sense of smell caused by chemical modification of olfactory epithelium

A chemically selective procedure for covalent modification of Schiff base-forming binding sites in proteins is demonstrated in vitro. In vivo studies show that the same procedure produces a selective anosmia ("odor blindness") when applied to the olfactory epithelia of experimental animals. Surgical experiments confirm that the sense of smell is specifically affected. The hypothesis that olfactory receptors are protein molecules is widely embraced (1). Here we report experimental findings to support that hypothesis. Our working model (2) is based upon the supposition that Schiff base-forming proteins in the olfactory epithelium bind carbonyl-containing odorant molecules. To assess this notion, we have developed a procedure to specifically and irreversibly modify proteins that bind simple ketones as iminium ions. We have probed its chemical selectivity and have explored the effects on the sense of smell of tiger salamanders (Ambystoma tigrinum). We have previously shown how to evaluate anosmias in this species with a behavioral assay (3). The modification procedure is derived from well-known techniques for affinity labeling of Schiff base-forming enzymes (4). We have tested our sequence of reagents, acetoacetic ester followed by sodium cyanoborohydride (NaBH3CN), by examining pure polypeptide proteins. As an example of a Schiff base-forming protein, the bacterial enzyme acetoacetate decarboxylase (AAD) (5) was chosen for model studies. AAD binds simple, uncharged ketones (5-7) at an active site lysine residue. Borohydride reducing agents convert reversible covalent complexes to irreversible covalent adducts (2). Quantitative studies with pure AAD demonstrate the selectivity of irreversible modification. Nondialyzable radioactivity is incorporated into AAD after treatment with '4C-labeled ethyl acetoacetate (EAA) (8) followed by 1092 NaBH3CN. This treatment does not radioactively label lysine-rich proteins that lack specialized Schiff base-forming NH2 + CH3COCH2COOR I-NHC(CH3)=CHCOOR Acetoacetic ester \\K /g Aminocrotonate ester