Protein crosslinking improves the thermal resistance of plastocyanin immobilized on a modified gold electrode.
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.
['Olloqui-Sariego|José Luis|JL|', 'Díaz-Quintana|Antonio|A|', 'De la Rosa|Miguel Ángel|MÁ|', 'Calvente|Juan José|JJ|', 'Márquez|Inmaculada|I|', 'Díaz-Moreno|Irene|I|', 'Andreu|Rafael|R|']
D002942:Circular Dichroism D003300:Copper D003432:Cross-Linking Reagents D000458:Cyanobacteria D004566:Electrodes D006046:Gold D006358:Hot Temperature D010084:Oxidation-Reduction D010970:Plastocyanin D055550:Protein Stability D011506:Proteins D013056:Spectrophotometry, Ultraviolet D013816:Thermodynamics
Covalent crosslinking Multilayered protein film Protein thermostability Temperature variable voltammetry Thermophilic plastocyanin