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.
D000818:Animals; D001360:Avidin / Q000378:metabolism*; D001681:Biological Assay; D001710:Biotin / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D007082:Ileum / Q000187:drug effects; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009119:Muscle Contraction / Q000187:drug effects; D010047:Ovalbumin / Q000031:analogs & derivatives*; D010446:Peptide Fragments; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology
D010446:Peptide Fragments; D001360:Avidin; D013373:Substance P; D001710:Biotin; C039226:substance P (3-11), alpha-biotinyl-Lys(3)-; C039225:substance P, alpha-biotinyl-Arg(1) -; C039224:substance P, epsilon-biotinyl-Lys(3)-; D010047:Ovalbumin