Increasing the thermal stability of immobilized proteins is a motivating goal for improving the performance of electrochemical biodevices. In this work, we propose the immobilization of crosslinked plastocyanin from the thermophilic cyanobacterium Phormidium laminosum by simultaneous incubation of a mixture of plastocyanin and the coupling reagents. The thermal stability of the so built covalently immobilized protein films has been assessed by cyclic voltammetry in the 0–90 °C temperature range and has been compared to that of physisorbed films. It is shown that the protein loss along a thermal cycle is significantly reduced in the case of the crosslinked films, whose redox properties remain unaltered along a cyclic heating-cooling thermal scan, and can withstand the contact with 70 °C solutions for four hours. Comparison of thermal unfolding curves obtained by circular dichroism spectroscopy of both free and crosslinked protein confirms the improved thermic resistance of the crosslinked plastocyanin. Notably, the electron transfer thermodynamics of physisorbed and crosslinked plastocyanin films are quite similar, suggesting that the formation of intra- and inter-protein amide bonds do not affect the integrity and functionality of the copper redox centers. UV–Vis absorption and circular dichroism measurements corroborate that protein crosslinking does not alter the coordination geometry of the metal center.

提高固定化蛋白质的热稳定性是改善电化学生物装置性能的一个激励目标。在这项工作中，我们建议从嗜热的蓝细菌Phrmidium laminosum固定交联的质体蓝素通过同时孵育质体蓝蛋白和偶联剂的混合物。如此构建的共价固定蛋白膜的热稳定性已通过循环伏安法在0–90°C的温度范围内进行了评估，并已与物理吸附的膜进行了比较。结果表明，在交联膜的情况下，沿着热循环的蛋白质损失显着减少，该交联膜的氧化还原特性在循环加热-冷却热扫描中保持不变，并且可以承受与70°C的溶液接触4小时。通过游离和交联蛋白的圆二色性光谱获得的热展开曲线的比较证实了交联的质体蓝蛋白的改进的耐热性。尤其，物理吸附和交联的质体蓝蛋白膜的电子转移热力学非常相似，这表明蛋白内和蛋白间酰胺键的形成不会影响铜氧化还原中心的完整性和功能性。UV-Vis吸收和圆二色性测量结果证实了蛋白质交联不会改变金属中心的配位几何形状。