A homogeneous assay was developed to evaluate ligands that target the membrane progesterone receptor alpha (mPRα) of goldfish. This was achieved by employing graphene quantum dots (GQDs), a type of semiconductor nanoparticle conjugated to the goldfish mPRα. When progesterone-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was combined with the other agents, fluorescence was observed through Förster resonance energy transfer (FRET). However, this fluorescence was quenched by binding between the ligand and receptor. This established method demonstrated the ligand selectivity of the mPRα protein. Then, the methylotrophic yeast Pichia pastoris was used to express the goldfish mPRα (GmPRα) protein. The recombinant purified GmPRα protein was coupled with graphene quantum dots (GQDs) to generate GQD-conjugated goldfish mPRα (GQD-GmPRα). Fluorescence at a wavelength of 520 nm was observed through FRET upon the combination of P4-BSA-FITC and subsequent activation by ultraviolet (UV) light. Adding free P4 to the reaction mixture resulted in a decrease in fluorescence intensity at a wavelength of 520 nm. The fluorescence was reduced by the administration of GmPRα ligands but not by steroids that do not interact with GmPRα. The findings indicated that the interaction between the ligand and receptor led to the formation of a complex involving GQD-GmPRα and P4-BSA-FITC. The interaction between the compounds and GQD-GmPRα was additionally validated by a binding experiment that employed the radiolabeled natural ligand [³H]-17α,20β-dihydroxy-4-pregnen-3-one. We established a ligand-binding assay for the fish membrane progesterone receptor that is applicable for screening compounds.

开发了一种均质测定法来评估针对金鱼膜孕酮受体 α (mPRα) 的配体。这是通过采用石墨烯量子点（GQD）实现的，石墨烯量子点是一种与金鱼 mPRα 结合的半导体纳米颗粒。当黄体酮-BSA-异硫氰酸荧光素（P4-BSA-FITC）与其他试剂组合时，通过福斯特共振能量转移（FRET）观察到荧光。然而，这种荧光因配体和受体之间的结合而被猝灭。该建立的方法证明了 mPRα 蛋白的配体选择性。然后，利用甲基营养酵母毕赤酵母表达金鱼mPRα（GmPRα）蛋白。将重组纯化的 GmPRα 蛋白与石墨烯量子点 (GQD) 偶联，生成 GQD 缀合的金鱼 mPRα (GQD-GmPRα)。 P4-BSA-FITC 组合并随后被紫外 (UV) 光激活后，通过 FRET 观察到波长为 520 nm 的荧光。向反应混合物中添加游离 P4 会导致 520 nm 波长处的荧光强度降低。施用 GmPRα 配体可减少荧光，但不与 GmPRα 相互作用的类固醇不会减少荧光。研究结果表明，配体和受体之间的相互作用导致形成包含 GQD-GmPRα 和 P4-BSA-FITC 的复合物。化合物与 GQD-GmPRα 之间的相互作用还通过采用放射性标记的天然配体 [ ³ H]-17α,20β-diHydroxy-4-pregnen-3-one 的结合实验进行了验证。我们建立了鱼膜黄体酮受体的配体结合测定法，适用于筛选化合物。