Heithier H, Ward L D, Cantrill R C, Klein H W, Im M J, Pollak G, Freeman B, Schiltz E, Peters R, Helmreich E J
Department of Physiological Chemistry, University of Würzburg, F.R.G.
Biochim Biophys Acta. 1988 Oct 7;971(3):298-306. doi: 10.1016/0167-4889(88)90145-0.
The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 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-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.
本文报道了胰高血糖素的单荧光素、单罗丹明和单 -4 - 硝基苯 -2 - 恶唑 -1,3 - 二唑（NBD）衍生物的合成。通过使用硫醇特异性荧光试剂将色氨酸 -25 转化为 2 - 硫醇色氨酸来引入荧光基团。与胰高血糖素相比，所有衍生物都保留了激活腺苷酸环化酶的能力，因此被认为是完全激动剂。在大鼠肝细胞膜中，NBD -、荧光素 -、罗丹明 -Trp25 - 胰高血糖素和天然胰高血糖素的 IC50 值分别测定为 6.8×10⁻⁹、1.7×10⁻⁸、1.8×10⁻⁸ 和 5.4×10⁻⁹ M。根据 IC50 值分别计算出 NBD -、荧光素 -、罗丹明 -Trp25 - 胰高血糖素和天然胰高血糖素结合的 Kd 值为 2.16×10⁻⁹、4×10⁻⁹、2×10⁻⁹ 和 1.72×10⁻⁹ M。在磷酸盐缓冲盐水（pH 7.4）中，荧光素 -Trp25 - 胰高血糖素的单体衍生物获得了最高的量子产率（0.18）。通过使组氨酸 -1 和赖氨酸 -12 的氨基与异硫氰酸荧光素反应制备了双荧光素 - 胰高血糖素，并使用荧光素标记的 2 - 硫醇Trp25 - 胰高血糖素制备了二聚体衍生物。双荧光素 - 胰高血糖素与胰高血糖素受体的结合较弱，并表现出拮抗特性。由两个双荧光素 -2 - 硫醇Trp25 - 胰高血糖素分子形成的二聚体衍生物具有相似的弱结合性质，而由双荧光素 -2 - 硫醇Trp25 - 胰高血糖素和 2 - 硫醇Trp25 - 胰高血糖素形成的二聚体在低浓度下表现出与单荧光素 - 胰高血糖素相似的性质。两种二聚体衍生物在水性介质中的溶解度都很小。使用流式细胞术跟踪荧光素 -Trp25 - 胰高血糖素和双荧光素 - 胰高血糖素与大鼠肝细胞的特异性结合。
