Anisotropic orientation of horseradish peroxidase by reconstitution on a thiol-modified gold electrode.
Horseradish peroxidase (HRP) was reconstituted on the surface of a gold electrode that was modified first with a hemin-carbon-chain-thiol derivative followed by addition of the apo protein to the contacting solution. To facilitate the reconstitution of the holo enzyme, the hemin needs to be immobilised on a carbon-chain spacer arm. To achieve this, an immobilisation protocol was developed that is based on the initial formation of a mixed self-assembled monolayer on the gold surface consisting of 3-carboxypropyl disulphide and an activated disulphide (3,3'-dithiodipropionic acid di-(N-succinimidyl ester)) followed by binding of a diaminoalkane to the activated disulphide. The hemin was then coupled to the second amino group of the diaminoalkane by means of a carbodiimide coupling reagent. Finally, the enzyme was reconstituted on the hemin-modified surface by immersion of the electrode in a solution containing apo-HRP. The advantage of this method is that the length of the spacer arm can be changed easily, because diaminoalkanes of different chain lengths are available. The electrochemistry of the hemin and the reconstituted HRP electrodes was studied by means of cyclic voltammetry and differential-pulse voltammetry. The catalytic ability for reduction of hydrogen peroxide was investigated for both direct and mediated electrochemistry with a soluble electron donor (ortho-phenylenediamine).
Zimmermann
H
H
Analytische Chemie, Elektroanalytik und Sensorik, Ruhr-Universität Bochum, Germany. heiko@anachem.ruhr-uni-bochum.de
Lindgren
A
A
Schuhmann
W
W
Gorton
L
L
D004563:Electrochemistry; D004566:Electrodes; D006046:Gold; D006427:Hemin / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013499:Surface Properties
D013438:Sulfhydryl Compounds; D006427:Hemin; D006046:Gold; D006735:Horseradish Peroxidase