title: Interfacial behavior and activity of laccase and bilirubin oxidase on bare gold surfaces.
abstract: Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL⁻¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm⁻², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O₂) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s⁻¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s⁻¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes.
authors.affiliation: Biomedical Sciences, Health & Society, Malmö University , 205 06 Malmö, Sweden.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Pankratov
authors.forename: Javier
authors.initials: J
authors.lastname: Sotres
authors.forename: Alejandro
authors.initials: A
authors.lastname: Barrantes
authors.forename: Thomas
authors.initials: T
authors.lastname: Arnebrant
authors.forename: Sergey
authors.initials: S
authors.lastname: Shleev
mesh_terms: D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D042845:Laccase / Q000737:chemistry / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D006046:Gold; D042845:Laccase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase