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Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(- 1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.
An amperometric biosensor for the determination of glycated hemoglobin in human whole blood is proposed. The principle is based on the electrochemical measurement of ferroceneboronic acid (FcBA) that has been specifically bound to the glycated N-terminus. Hemoglobin is immobilized on a zirconium dioxide nanoparticle modified pyrolytic graphite electrode (PGE) in the presence of didodecyldimethylammonium bromide (DDAB). The incubation of this sensor in FcBA solution leads to the formation of an FcBA-modified surface due to the affinity interaction between boronate and the glycated sites of the hemoglobin. The binding of FcBA results in well-defined redox peaks with an E-0' of 0.299 V versus Ag/AgCl (1 M KCl). The square wave voltammetric response of the bound FcBA reflects the amount of glycated hemoglobin at the surface. This signal increases linearily with the degree of glycated hemoglobin from 6.8 to 14.0% of total immobilized hemoglobin. The scheme was applied to the determination of the fraction of glycated hemoglobin in whole blood samples.