| Abstract
| - Photoreactive analogues of substance P (biotin sulfone−spacer (amino pentanoic or Gly3)-Arg-Pro-Lys-Pro-(pBzl)Phe-Gln-Phe-Phe-Gly-Leu-Met(O2)NH2) with or withoutisotope (deuterium) labeling have been synthesized. Deuteriums were present on (d)-biotin or epibiotin sulfone(D3), on the Gly3 spacer linker (D6), or on the Gly inposition 9 of SP (D2). Therefore, peptide analogues couldbe either unlabeled or tri-, penta-, or hexadeuterated.Results obtained with the use of these peptide analoguesshow that (d)-biotin sulfone and epibiotin sulfone are notrecognized with the same affinity by streptavidin, with (d)-biotin sulfone displaying better affinity for the protein.Photolabeling of the human NK-1 receptor with a 1:1molar ratio of nondeuterated and deuterated photoreactive substance P (SP) analogues in position 5, followedby combined digestions, purification, and MALDI-TOFmass spectrometry analysis, made the identification of thedomain of the receptor covalently linked by the photoreactive SP analogue easier. Indeed, doublets in massspectra were specific for the covalent complex whereassingle peaks could be attributed to contaminating species.This method is particularly suitable when minute amountsof complex have to be analyzed, as in the case of highlyhydrophobic G-protein coupled receptors.
|
