A fluorescently labeled intestinal fatty acid binding protein. Interactions with fatty acids and its use in monitoring free fatty acids.

摘要: The fatty acid-binding protein from rat intestine (I-FABP) has been covalently modified with the fluorescent compound Acrylodan. Acrylodan was found to label Lys27, one of few amino acid residues by x-ray diffraction studies change orientation upon (FA) binding I-FABP. Binding FA this Acrylodan-modified I-FABP (ADIFAB) induces a large shift in fluorescence emission wavelength 432 505 nm. As consequence, ratio intensities provides direct measure concentration bound protein. is well described single site equilibrium for concentrations below critical micelle concentration. ADIFAB dissociation constants (Kd) determined at 37 degrees C and oleate, palmitate, linoleate, arachidonate, linolenate were, respectively, 0.28, 0.33, 0.97, 1.6, 2.5 microM. variation these Kd values molecular species highly correlated solubility water, suggesting that all bind similar conformation site. response together measured allows determination long chain free (FFA) range, depending species, between 1 nM > 20 an example its use as probe FFA levels, used here monitor time course release IgE receptor- ionomycin-activated basophilic leukemia (RBL) cells.