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Voltammetric Studies of the Interactions Between Ferrocene‐Labeled Glutathione and Proteins in Solution or Immobilized onto Surface
Author(s) -
Peng Yong,
Liu YouNian,
Zhou Feimeng
Publication year - 2009
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/elan.200904601
Subject(s) - glutathione , chemistry , bovine serum albumin , binding constant , conjugate , binding site , metallothionein , ferrocene , electrochemistry , biochemistry , enzyme , electrode , gene , mathematical analysis , mathematics
Glutathione (GSH) tagged with a ferrocene (Fc) label at its C‐terminal was synthesized via coupling ferrocenyl amine to glutathione using o ‐(benzotriazol‐1‐yl)‐ N , N , N′ , N′ ‐tetramethyluronium (HBTU)/1‐hydroxybenzotrizole (HOBt). The presence of Fc yielded well defined voltammetric signals, rendering the Fc‐tagged GSH (GSH‐Fc) suitable for electrochemical studies of GSH binding to other biological species. The interaction of GSH‐Fc with bovine serum albumin (BSA) was investigated, and a binding ratio of 1.41±0.06 (GSH‐Fc/BSA) and an affinity constant K a of 6.53±2.01×10 6 M −1 were determined. These results compare well with those measured by fluorescence using untagged GSH, suggesting that the attachment of Fc to GSH does not significantly perturb the GSH structure and binding behavior. By contrasting the binding behavior to several compounds that are known to conjugate to different domains of BSA, the voltammetric study confirmed that GSH‐Fc binds at subdomain IIA of BSA with high affinity. The versatility of GSH‐Fc for studying GSH binding to surface‐confined proteins was also demonstrated with the GSH binding to electroinactive Zn‐metallothionein (Zn 7 ‐MT) through hydrogen binding at the region between the Zn 7 ‐MT α and β domains.