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

8.4K Views

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08:31 min

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October 3rd, 2018

DOI :

10.3791/58307-v

October 3rd, 2018

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8,355 Views

Stacy N. Uchendu¹, Angelika Rafalowski¹, Erin F. Cohn¹, Luke W. Davoren¹, Erika A. Taylor¹

¹Department of Chemistry, Wesleyan University

Chapters

0:04

Title

0:33

Anaerobic DesB Purification

2:29

Anaerobic Protein Concentration, Buffer Exchange, and DesB Desalting

4:10

Oxygen Electrode Preparation

5:41

Oxygen Electrode Kinetic Assay

6:48

Results: Representative DesB Dioxygenase Induction, Activity, and Inhibition

7:50

Conclusion

Transcript

This method is designed to answer key questions in the field inorganic biochemistry, about how to maintain enzyme activity in proteins that contain oxygen-sensitive metals. The main advantage of this technique is that it helps you visualize ways i

Summary

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

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