Ludek Havran

Determination of glutathione-S-transferase traces in preparations of p53 C-terminal domain (aa320-393).

Tumor suppressor protein p53 is often expressed as a fusion protein with glutathione-S-transferase (GST). The sensitive determination of GST in p53 samples is thus necessary. We propose a method for the determination of traces of GST in the p53 C-terminus based on the constant current chronopotentiometric stripping analysis (CPSA) with hanging mercury drop electrode (HMDE). GST produces a catalytic signal in cobalt-containing solutions due to cysteine residues. A large excess of the C-terminus does interfere with the determination because of the lack of cysteines in the molecule. This method is simple and very sensitive and is capable of detecting <1% GST in the p53 sample.

Determination of the level of DNA modification with cisplatin by catalytic hydrogen evolution at mercury-based electrodes.

Electrochemical methods proved useful as simple and inexpensive tools for the analysis of natural as well as chemically modified nucleic acids. In particular, covalently attached metal-containing groups usually render the DNA well-pronounced electrochemical activity related to redox processes of the metal moieties, which can in some cases be coupled to catalytic hydrogen evolution at mercury or some types of amalgam electrodes. In this paper we used voltammetry at the mercury-based electrodes for the monitoring of DNA modification with cis-diamminedichloroplatinum (cisplatin), a representative of metallodrugs used in the treatment of various types of cancer or being developed for such purpose. In cyclic voltammetry at the mercury electrode, the cisplatin-modified DNA yielded catalytic currents the intensity of which reflected DNA modification extent. In square-wave voltammetry, during anodic polarization after prereduction of the cisplatinated DNA, a well-developed, symmetrical signal (peak P) was obtained. Intensity of the peak P linearly responded to the extent of DNA modification at levels relevant for biochemical studies (rb = 0.01-0.10, where rb is the number of platinum atoms bound per DNA nucleotide). We demonstrate a correlation between the peak P intensity and a loss of sequence-specific DNA binding by tumor suppressor protein p53, as well as blockage of DNA digestion by a restriction endonuclease Msp I (both caused by the DNA cisplatination). Application of the electrochemical technique in studies of DNA reactivity with various anticancer platinum compounds, as well as for an easy determination of the extent of DNA platination in studies of its biochemical effects, is discussed.

Biophysical and electrochemical studies of protein–nucleic acid interactions

This review is devoted to biophysical and electrochemical methods used for studying protein–nucleic acid (NA) interactions. The importance of NA structure and protein–NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein–DNA and protein–RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein–NA interactions. Recent applications of electrochemical methods in studies of protein–NA interactions are discussed in detail.Graphical abstract