19F NMR studies of tryptophan/serum albumin binding.
19F NMR provides direct measures of the Trp binding avidity of 'fatty acid free' bovine serum albumin when D- and L-6-fluorotryptophan are used as the probes. Both a high and low affinity binding site are present. The addition of octanoate either displaces the ligand from both sites or greatly decreases the affinity such that little binding occurs at 2 mM levels. In the case of L-6-fluorotryptophan separate signals are observed for the high and low affinity binding sites and titrations with competing ligands can be used to establish the relative affinities of ligands at the high affinity site. Binding at this site appears to be hydrophobic and shape specific with L-Phe being a very poor ligand (K(D)[L-Phe]/K(D)[L-Trp]=800) while both GHKalphaNal and GHKW displace L-6-fluorotryptophan from this site. In tripeptides of the general formula GHK[ epsilon NH(CH(2))(n)(CO)W], affinity increases with tether length and binding at the low affinity site is restored. This NMR assay appears well-suited for the discovery of selective binding agents in this and other biorecognition phenomena.
Cao
Bolong
B
Department of Chemistry, University of Washington, Seattle 98195, USA.
Endsley
Stephanie
S
Andersen
Niels H
NH
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D005462:Fluorine Radioisotopes; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D009281:Naphthalenes / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
D005462:Fluorine Radioisotopes; D008024:Ligands; D009281:Naphthalenes; D009842:Oligopeptides; C031721:naphthalene; D012710:Serum Albumin, Bovine; C001244:6-fluorotryptophan; D014364:Tryptophan