Performing Anaerobic Stopped-flow Spectrophotometry Inside of an Anaerobic Chamber

Abstract

The catalytic cycle of most flavin-dependent enzymes can be divided into oxidative and reductive half-reactions. Although some enzymes are oxidized by electron carrier proteins or organic compounds, many use oxygen as the final electron acceptor. In order to properly study the reductive half-reaction of flavin-dependent enzyme that react with oxygen, as in the case of oxidases and monooxygenases, it is necessary to establish anaerobic conditions that will only allow the reduction process to be monitored. The reduced flavoenzyme can be further studied by exposing it to oxygen to monitor the oxidative half-reaction. Anaerobic chambers provide an ideal environment for performing these experiments as they reliably maintain an anaerobic atmosphere inside a large workspace. A common tool used to study flavin-dependent enzymes is the stopped-flow spectrophotometry. This chapter describes methods for performing stopped-flow experiments in an anaerobic chamber. We include information about the chamber components, setting up a stopped-flow spectrophotometer inside of a chamber, preparing anaerobic solutions, and performing experiments to measure the reductive and oxidative half-reactions of flavin-dependent monooxygenases.

Department(s)

Chemistry

Comments

National Science Foundation, Grant CHE-1506206

Keywords and Phrases

Anaerobic chambers; Anaerobiosis; Enzyme assays; Enzyme kinetics; Flavoenzymes; Oxidation; Reduction; Schlenk line; Stopped-flow spectrophotometry

International Standard Book Number (ISBN)

978-012816829-5

International Standard Serial Number (ISSN)

1557-7988; 0076-6879

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 Elsevier, All rights reserved.

Publication Date

01 Jan 2019

PubMed ID

31072501

Link to Full Text

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