(446a) Role of Protein Confinement On Enzyme Function and Stability

Research over the past few decades have attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior as the cell environment is very crowded with the average macromolecular space much smaller than the macromolecules themselves. In our study, we attempt to address this question using a model enzyme – horseradish peroxidase (HRP) encapsulated in agarose hydrogels of different porosities. Our studies reveal that the activity of the agarose encapsulated HRP is lower than that of the solution phase enzyme. However, the encapsulated enzyme retains a higher percentage of its activity in the presence of denaturants. Moreover, we observe that the degree of confinement (achieved using the different percentages of agarose – 0.5%, 2%) has an effect on the stabilization effect; HRP encapsulated in 2% agarose is significantly more stable than HRP encapsulated in 0.5% agarose. Furthermore we observed similar results through structural measurements of HRP denaturation using 8-anilinonaphthalene-1-sulfonic acid assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly crowded environments; furthermore, these observations are in accord with previous studies of macromolecular crowding on protein structure and function.