Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition | Zendy

Stacy Uchendu | Zendy; Angelika Rafalowski | Zendy; Erin Frances Cohn | Zendy; Luke W Davoren | Zendy; Erika A. Taylor | Zendy

Open Access

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Author(s) -

Stacy Uchendu,

Angelika Rafalowski,

Erin Frances Cohn,

Luke W Davoren,

Erika A. Taylor

Publication year - 2018

Publication title -

journal of visualized experiments

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.596

H-Index - 91

ISSN - 1940-087X

DOI - 10.3791/58307

Subject(s) - chemistry , chromatography , dioxygenase , clark electrode , anaerobic exercise , oxygen , substrate (aquarium) , reagent , enzyme , biochemistry , electrode , biology , organic chemistry , electrolyte , physiology , ecology

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

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