SELECTED RECENT ABSTRACTS

Liping Zhou, Jing Yang, Carmelita Estavillo, James D. Stuart, John B. Schenkman, and James F. Rusling, "Toxicity Screening by Electrochemical Detection of DNA Damage by Metabolites Generated In-situ in Ultrathin DNA-Enzyme Films", J. Am. Chem. Soc. 2003, 125, 1431-1436.

Rapid detection of DNA damage could serve as a basis for in-vitro genotoxicity screening for new organic compounds. Ultrathin films (20-40 nm) containing myoglobin or cytochrome P450_cam and DNA grown layer-by-layer on electrodes were activated by hydrogen peroxide, and the enzyme in the film generates metabolite styrene oxide from styrene. This styrene oxide reacted with double stranded (ds)-DNA in the same film, mimicking metabolism and DNA damage in human liver. DNA damage was detected by square wave voltammetry (SWV) by using catalytic oxidation with Ru(bpy)₃²⁺ (bpy = 2,2'-bipyridine) and by monitoring the binding of Co(bpy)₃³⁺. Damaged DNA reacts more rapidly than intact ds-DNA with Ru(bpy)₃³⁺, giving SWV peaks at ~1 V vs. SCE that grow larger with reaction time. Co(bpy)₃³⁺ binds more strongly to intact ds-DNA, and its SWV peaks at 0.04 V decreased as DNA was damaged. Little change in SWV signals was found for incubations of DNA/enzyme films with unreactive organic controls or hydrogen peroxide. Capillary electrophoresis and HPLC-MS suggested formation of styrene oxide adducts of DNA bases under similar reaction conditions in thin films and in solution. The catalytic SWV method was more sensitive than the Co(bpy)₃³⁺ binding assay, providing multiple measurements over a 5 min. reaction time.

(Supported by NIEHS, NIH)

Bernard Munge, Carmelita Estavillo, John B. Schenkman and James F. Rusling, "Optimizing Electrochemical and Peroxide-Driven Oxidation of Styrene with Ultrathin Polyion Films containing Cytochrome P450_cam and Myoglobin", ChemBiochem, 2003, 4, 82-89.

Ultrathin films of myoglobin (Mb) and cytochrome P450_cam grown layer-by-layer with polyions were optimized for electrochemical and hydrogen-peroxide driven epoxidation of styrene. Very thin films (ca. 12-25 nm) using nmol amounts of protein gave the best rates for the catalytic epoxidation. Classical bell-shaped activity-pH profiles and turnover rates similar to those in solution were obtained. (Supported by NIEHS, NIH)

Venkateswarlu Panchagnula, Challa V. Kumar, and James F. Rusling, "Ultrathin Layered Myoglobin-Polyion Films Functional and Stable at Acidic pH Values", J. Am. Chem. Soc., 2002, 124, 12515-12525.

Cross-linking of myoglobin promoted by 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide within films of polystyrene sulfonate after layer-by-layer self-assembly provided remarkable stabilization. Crosslinking greatly improved adhesion of the films to fused silica slides and allowed extensive optical studies over a wide pH range. Circular dichroism and visible absorbance spectra showed that Mb retained its native conformation when films were placed in solutions of pH as low as 2 and up to pH 11. Linear dichroism revealed an average orientation of the Mb iron heme cofactors of 58^O to the film normal. High concentrations of urea did denature the protein in the films, however. At pH 1, Mb in solution is fully unfolded, but retained considerable a-helical content in the crosslinked films. Both the polyion film environment and crosslinking seem to play roles in stabilizing protein secondary structure and function at low pH. Crosslinked Mb-polyion films on pyrolytic graphite electrodes were used in strongly acidic solutions for the electrochemical catalytic reduction of trichloracetic acid, hydrogen peroxide, and oxygen. The pH-dependent catalytic reduction of trichloracetic acid was faster in 0.1 M HCl than in the medium pH range. The crosslinked films are also stable in microemulsions. (Supported by NSF and NIEHS, NIH).

Abhay Vaze and James F. Rusling, ^"Optimizing Turnover of a Cobalt Corrin-Polyion Scaffold on Electrodes in Microemulsions with a Flow Reactor", J. Electrochemical Soc., 2002, 149, D193.

Electrochemical reactors employing catalytic films of the cobalt corrin vitamin B₁₂ hexacarboxylic acid [B₁₂(COOH)₆] attached to poly(L-lysine) (PLL) covalently bound to carbon cloth electrodes were optimized for the reduction of 1,2-dibromocyclohexane (DBCH) to cyclohexene in a microemulsion. An improved initial electrode oxidation method enabled attachment of films with higher surface concentrations of catalyst than previously. A parallel plate flow reactor containing a catalyst-coated carbon cloth cathode gave 6-fold larger turnover rates compared to a simple stirred batch reactor, especially for 20 and 90 nm thick films of B₁₂(COOH)₆-PLL. The 90 nm thick films showed high turnover rates in the flow reactor while maintaining 100% current efficiency, whereas in the batch reactor current efficiency dropped well below 100% at the higher applied currents. Significantly thicker films of B₁₂(COOH)₆-PLL on carbon cloth than reported in previous work were shown to operate efficiently in microemulsions for catalyzing DBCH conversion to cyclohexene. The key factor was eliminating mass transport limitations in the microemulsion by using an efficient flow reactor, resulting in turnover rates up to 3000 min^-1 at 100% current efficiency. (Supported by NSF)