University of Regensburg Trenner Faculty of Chemistry Trenner Analytical Chemistry
Single molecule analysis in femtoliter arrays

Mechanistic aspects of horseradish peroxidase elucidated through single-molecule studies

In contrast to β-galactosidase, the product formation rate of single horseradish peroxidase (HRP) molecules in the fiber bundle array is on average 10 times lower than in bulk solution. The redox-reaction mechanism of HRP involves two separate steps of product formation. HRP (purple) first oxidizes Amplex Red in a one-electron transfer step (red) to non-fluorescent radical intermediates, which subsequently undergo an enzyme independent dismutation reaction to form fluorescent resorufine (yellow). If HRP is confined in a femtoliter container the dismutation reaction decreases such that less product is formed. This two-step oxidation mechanism of the widely used Amplex Red and other fluorogenic substrates is often overlooked. It is important for single-molecule studies with HRP as well as for bulk reactions at low substrate turnover rates.

(JACS 2009)

© 2014, Thomas Hirsch, Gisela Emmert.