Use of molecular crowding for the detection of protein self-association by size-exclusion chromatography

Mrozowich, Tyler; Winzor, Donald J.; Scott, David J.; Patel, Trushar R.

Abstract

The feasibility of employing molecular crowding cosolutes to facilitate the detection of protein self-association by zonal size exclusion chromatography is investigated. Theoretical considerations have established that although the cosolute-induced displacement of a self-association equilibrium towards the oligomeric state invariably occurs in the mobile phase of the column, that displacement is only manifested as a decreased protein elution volume for cosolutes sufficiently small to partition between the mobile and stationary phases. Indeed, the use of a crowding agent sufficiently large to be confined to the mobile phase gives rise to an increased elution volume that could be misconstrued as evidence of cosolute-induced protein dissociation. Those theoretical considerations are reinforced by experimental studies of α-chymotrypsin (a reversibly dimerizing enzyme) on Superdex 200. The use of cosolutes such as sucrose and small polyethylene glycol fractions such as PEG-2000 is therefore recommended for the detection of protein self-association by molecular crowding effects in size exclusion chromatography.

Citation

Mrozowich, T., Winzor, D. J., Scott, D. J., & Patel, T. R. (2019). Use of molecular crowding for the detection of protein self-association by size-exclusion chromatography. Analytical Biochemistry, 584, Article 113392. https://doi.org/10.1016/j.ab.2019.113392

• Journal Article Type: Article
• Acceptance Date: Aug 9, 2019
• Online Publication Date: Aug 10, 2019
• Publication Date: Nov 1, 2019
• Deposit Date: Aug 13, 2019
• Publicly Available Date: Nov 20, 2019
• Journal: Analytical Biochemistry
• Print ISSN: 0003-2697
• Electronic ISSN: 1096-0309
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 584
• Article Number: 113392
• DOI: https://doi.org/10.1016/j.ab.2019.113392
• Keywords: Biophysics; Cell Biology; Biochemistry; Molecular Biology
• Public URL: https://nottingham-repository.worktribe.com/output/2421488
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0003269719305949