Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives (original) (raw)

1988, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

The synthesis of monofluerescein, monorhodamine, and mono-4-nitrobenz-2-oxa-l,3-diazule (NBD) derivafives of glucagon is reported. The fluorescent groups were introduced by conve~ng tryptophan-25 to 2.thloltryptophan using thiol-speeific fluorescent reagents. All derivatives retained the ability to activate adenylate cydase when compared to glucagon and thus were considered full agonists. ICs0 values of 6.8.10 -9, 1.7.10 -s, 1.8.10 -s and 5.4" 10 `-9 M were measured in rat liver membranes for NBD-, fluerescein-, rhedamine-TrpZS.glueagon and native glueagon, respeefivaly. From the ICs0 values Kd values of 2.16.10 -9, 4.10 -9, 2-10 -9 and 1.72.10 -9 M were calculated for the binding of NBD-, fluorescein-, rhodamlue-Ts~-glueagon and native glueagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-TrpZS-glucagon in phosphate-buffered saline (pH 7.4). Difluerescein-glueagon was also prepared by reacting the amino groups of histidine-I and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiulTrp 2sglueagon. Difluorescein.glueagon bound only weakly to glucagon receptors and displayed antagonist properties, The dimer derivative formed from two difluorescoin.2-thlolTq~.glucagon molecules had similar poor binding qualities, whereas the dimer formed from dlfluorescein-2-thioiTrp~-glueagon and 2-thiolTrl~glueagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp~-glueagon and difluores~in-glueagon to rat hepatueytes was followed using flow cytometry.