Avidin binding of biotinylated analogues of substance P

We report the bioactivities of three biotinylated analogues of Substance P, [alpha-biotinyl-Lys3]Substance P-(3-11), [alpha-biotinyl-Arg1]Substance P, [epsilon-biotinyl-Lys3]Substance P, as well as the bioactivities of their complexes with avidin on guinea pig ileum. The rate of dissociation of an [alpha-biotinyl-Arg1]Substance P-avidin complex has been determined in the presence of 2-(4'-hydroxyazobenzene) benzoic acid and [3H]biotin. The biphasic dissociation of a 4:4 complex between [alpha-biotinyl-Arg1]Substance P and avidin led us to postulate the existence of two sets of binding sites, with the following rates of dissociation, at 4 degrees C, kappa-1 = 6 x 10(-4) x s-1 and kappa-1 = 1.2 x 10(-5) x s-1. We have confirmed this non-equivalence of the four binding sites of avidin by nuclear magnetic resonance using a spin-echo technique. The [alpha-biotinyl-Arg1]Substance P-avidin may be used for the affinity chromatography of Substance P receptors and the decreased affinity of this complex may be taken as an advantage since it can be displaced under mild conditions, i.e. by biotin-containing buffer.