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It is now well appreciated that both crowding and confinement influence enzyme structure and function due to excluded volume effects; however, the relative efficacies of these environments on protein fates remain unclear due to a lack of direct comparison studies. In this study, we explore the use of the biopolymer alginate to develop an in vitro platform to investigate the effects of both crowding and confinement on the behavior of two model enzymes - horseradish peroxidase and β-galactosidase. Alginate, in its solution phase, can be used as a crowding agent and, in its gel phase by crosslinking using divalent cations, to encapsulate and confine proteins, thereby allowing us to use the same system to directly compare the effects of crowding and confinement. Different degrees of crowding and confinement were achieved by varying the alginate concentration, and these studies demonstrated a clear dependence of enzyme activity on the degree of crowding and confinement. Moreover, our data also suggested that protein confinement in crosslinked alginate gels led to higher enhancements in enzyme activity under denaturing conditions relative to non-crosslinked crowded environments. Results from the kinetic analyses were corroborated using structural measurements of protein denaturation using the 8-anilinonaphthalene-1-sulfonic acid fluorescence assay.

Influence of Macromolecular Crowding and Confinement on Enzyme Activity and Structure under Native and Denaturing Conditions