Redox modification of proteins using sequential-parallel electrochemistry in microtiter plates.
Redox modification of proteins has frequently been used to improve the electron-transfer properties in amperometric biosensors. One approach is the coordinative labelling of histidine residues with metal complexes like [Ru(bpy)2Cl2] and [Ru(bpy)2CO3]. Although the reaction depends on a variety of parameters no detailed optimisation of these modification procedures has been done, most probably due to the complexity of the parameter matrix and the expected differences for any individual protein. A multi electrode sequential analyser (MESA) system has been developed which allows one to follow in a sequential-parallel scheme a number of modification reactions by performing electrochemical measurements such as cyclic voltammetry or differential pulse voltammetry in individual wells of a conventional microtiter plate. Using this system, the ligand exchange reaction leading to the binding of the Ru-complex to histidine residues could be investigated with imidazole as a model compound. Furthermore, the selective labelling of soluble PQQ (pyrrolochinolinquinone)-dependent glucose dehydrogenase (sGDH) and glucose oxidase (GOx) with Ru complexes could be optimised and the electrochemical and biological properties of the obtained 'electroenzymes' were examined.