Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments.
Over the past years, bottom-up bionanotechnology has been developed as a promising tool for future technological applications. Many of these biomolecule-based assemblies are characterized using various single-molecule techniques that require strict anaerobic conditions. The most common oxygen scavengers for single-molecule experiments are glucose oxidase and catalase (GOC) or protocatechuate dioxygenase (PCD). One of the pitfalls of these systems, however, is the production of carboxylic acids. These acids can result in a significant pH drop over the course of experiments and must thus be compensated by an increased buffer strength. Here, we present pyranose oxidase and catalase (POC) as a novel enzymatic system to perform single-molecule experiments in pH-stable conditions at arbitrary buffer strength. We show that POC keeps the pH stable over hours, while GOC and PCD cause an increasing acidity of the buffer system. We further verify in single-molecule fluorescence experiments that POC performs as good as the common oxygen-scavenging systems, but offers long-term pH stability and more freedom in buffer conditions. This enhanced stability allows the observation of bionanotechnological assemblies in aqueous environments under well-defined conditions for an extended time.
B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307 Dresden, Germany.
Marko
M
Swoboda
Jörg
J
Henig
Hsin-Mei
HM
Cheng
Dagmar
D
Brugger
Dietmar
D
Haltrich
Nicolas
N
Plumeré
Michael
M
Schlierf
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry; D002374:Catalase / Q000737:chemistry; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D036103:Nanotechnology; D010100:Oxygen / Q000737:chemistry* / Q000302:isolation & purification; D038761:Photobleaching; D055668:Photochemical Processes; D011520:Protocatechuate-3,4-Dioxygenase / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D002374:Catalase; D011520:Protocatechuate-3,4-Dioxygenase; D010100:Oxygen