Dithiothreitol (DTT)-mercury and DTT-solid amalgam electrodes are proposed for protein microanalysis by means of constant current chronopotentiometric stripping (CPS). At the DTT-modified hanging mercury drop electrode (DTT-HMDE), proteins at nanomolar concentrations produce the CPS peak H, which is due to the protein catalyzed hydrogen evolution. Self-assembled monolayers (SAMs) of DTT at the electrode surface protected surface-attached proteins from the electric field-driven denaturation, but did not interfere with the electrocatalysis. Using CPS peak H, native and denatured forms of bovine serum albumin (BSA) and of other proteins were easily distinguished. On the other hand, in usual slow scan voltammetry (scan rates between 50 mV/s and 1 V/s), the adsorbed BSA behaved as fully or partially denatured. BSA-modified DTT-HMDE was exposed to different potentials, E(B) for 60 s, followed by CPS measurement. Three E(B) regions were observed, in which either BSA remained native (A, -0.1 to -0.3 V), was denatured (B, -0.35 to -1.4 V), or underwent desorption (C, at potentials more negative than -1.4 V). At potentials more positive than the reduction potential of the DTT Hg-S bond (approximately -0.65 V against Ag|AgCl|3 M KCl), the densely packed DTT SAM was impermeable to [Ru(NH(3))(6)](3+). At more negative potentials, the DTT SAM was disturbed, but under conditions of CPS (with very fast potential changes), this SAM still protected the protein from surface-induced denaturation. Thiol-modified Hg electrodes in combination with CPS represent a new tool for protein analysis in biomedicine and proteomics.

We developed an innovative electrochemical method for monitoring conformational transitions in proteins using constant current chronopotentiometric stripping (CPS) with dithiothreitol-modified mercury electrodes. The method was applied to study the effect of oncogenic mutations on the DNA-binding domain of the tumor suppressor p53. The CPS responses of wild-type and mutant p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins. Further, we were able to monitor the loss of an essential zinc ion resulting from mutation (R175H) or metal chelation. We envisage that our CPS method can be applied to the analysis of virtually any protein as a sensor for conformational transitions or ligand binding to complement conventional techniques, but with the added benefit that only relatively small amounts of protein are needed and instant results are obtained. This work may lay the foundation for the wide application of electrochemistry in protein science, including proteomics and biomedicine.

Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.