In the proposed study, various analytical and surface analysis techniques will be employed to characterize and study redox-active proteins immobilized onto metal surfaces. Particular emphasis will be placed on the electrochemical studies of metallothioneins (MTs) and the metal transfer processes accompanying with their electrochemical redox reactions. Metal/electrode surfaces will be modified in such a way that facile electron transfer (ET) reactions are facilitated without significantly altering the stable or physiologically relevant protein structures. A few useful compounds have been identified for electrode modification. Several important parameters that are related to the biological functions of MTs will be determined. These parameters include the redox potential of MT, the number of electroactive groups per MT molecule, and the heterogeneous ET rate constant. Another focus of this proposal will be on the quantification of metals released by surface-confined MT molecules upon electrochemical redox reactions. The general strategy for the proposed work is to verify an existing methodology or to establish a new procedure through the study of a model protein (e.g., cytochrome c for the redox potential measurement and for the study of heterogeneous ET and ferredoxin III for the redox- induced metal transfer). The specific plan will include the application of the following techniques: (1) Using atomic force microscopy and scanning tunneling microscopy to probe the surface coverage and orientation of immobilized MT molecules; (2) Quantifying the amount of protein attachment with quartz crystal microbalance; (3) Studying the redox properties of MT using voltammetrically based techniques (e.g., mediated thin-layer voltammetry and scanning electrochemical microscopy); (4) Examining possible structural changes associated with the MT redox reactions with electrochemical surface plasmon spectroscopy; and (5) Investigating the metal-transfer processes induced (modulated) by the redox reactions of immobilized MT molecules using electrochemistry combined on-line with atomic spectroscopic techniques. The systematic study based on our mufti-technique approach should provide new insight to the understanding of the intricate relationship between the MT redox reaction and the subsequent metal transfer in a cellular milieu. The accurate measurement of the MT redox potential will help elucidate the role of certain biological redox couples (e.g., glutathione) in the MT metal transfer process, whereas the quantification of redox-active groups and metals can allow us to determine the number of redox-active groups accessible for redox reactions and the labile metals for metal transfer in a MT molecule. Finally, we hope to probe the surface configuration and structure of the immobilized MT molecules and use the results to explain the observed redox behavior of MT (e.g., the heterogeneous ET rate and the number of redox-active groups involved).
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