Spectrophotometric Determination of Affinities of α‐Factors for Their G Protein‐Coupled Receptors in Saccharomyces cerevisiae

A new and relatively simple spectrophotometric technique has been developed for the accurate determination of α-factor pheromone affinities for Ste2p whole cell receptor in yeast a-cells. We designed and tested nine detector peptides containing mono- (e412 = 14 500) or tri-cysteine residues (e412 = 43 660). The free unbound detector was detected using Ellman's reagent at 412 nm. Saturation binding studies using Saccharomyces cerevisiae Y 7925 ( MATa ) at a concentration of 2.5 × 1011 cells/mL with the highest affinity detector 1, [Orn6]α-factor-[Cys]3, resulted in a dissociation constant ( K D ) of 1.67 × 10−7 and total binding sites per cell (B cell = 29 500 sites/cell) comparable with those obtained using radiolabeled binding assays. Competitive binding assay using five nonchromogenic α-factor analogs allowed for the determination of each K D value. [Orn6,d-Ala9]α-factor showed the highest receptor affinity ( K D  = 1.03 × 10−7 M), which was threefold higher than that of native α-factor. This assay provides rapid and convenient results for determining the relative affinities of nonchromogenic α-factors and eliminates the need for radioactive waste disposal.