Homogeneous noncompetitive assay of protein via Förster-resonance-energy-transfer with tryptophan residue(s) as intrinsic donor(s) and fluorescent ligand as acceptor.
Homogeneous noncompetitive assay of a protein in biological samples based on Förster-resonance-energy-transfer (FRET) was proposed by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor that was defined as an analytical FRET probe. Conjugate of a suitable fluorophore, which should have an excitation peak around 340 nm but an excitation valley around 280 nm, with a moiety binding to a protein of interest gave an analytical FRET probe to the protein. To test this method, N-biotinyl-N'-(1-naphthyl)-ethylenediamine (BNEDA) was used as an analytical FRET probe for homogeneous noncompetitive assay of streptavidin (SAV). The occurrence of FRET between the bound BNEDA and tryptophan residues was supported by the modeled geometry of the complex. By excitation at 280 nm, free BNEDA produced negligible fluorescence at 430 nm, but the bound BNEDA produced much higher stable fluorescence at 430 nm after 2 min of binding reaction. The competitive binding between BNEDA and biotin gave the dissociation constant of (16+/-3) fM for BNEDA (n=3). By excitation at 280 nm, fluorescence at 430 nm of reaction mixtures containing 32.0 nM BNEDA responded linearly to SAV subunit concentrations ranging from 0.40 to 30.0 nM with the desirable resistance to common interferences in biological samples. Therefore, by using tryptophan residue(s) in a protein of interest as intrinsic donor(s) and its fluorescent ligand as the corresponding FRET acceptor, this homogeneous noncompetitive assay of the protein in biological samples was effective and advantageous.
Liao
Fei
F
Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Chongqing Medical University, Chongqing 400016, China. liaofeish@yahoo.com
Xie
Yanling
Y
Yang
Xiaolan
X
Deng
Ping
P
Chen
Yiwen
Y
Xie
Guoming
G
Zhu
Sha
S
Liu
Beizhong
B
Yuan
Huidong
H
Liao
Juan
J
Zhao
Yunsheng
Y
Yu
Ming'an
M
D001710:Biotin / Q000032:analysis / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D021122:Protein Subunits; D019809:Streptavidin / Q000032:analysis / Q000378:metabolism*; D014364:Tryptophan / Q000032:analysis / Q000378:metabolism*
D005456:Fluorescent Dyes; D008024:Ligands; D021122:Protein Subunits; D001710:Biotin; D014364:Tryptophan; D019809:Streptavidin