Function, structure, and stability of enzymes confined in agarose gels.
Research over the past few decades has 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 average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.
Department of Bioengineering, Santa Clara University, Santa Clara, California, United States of America.
Jeffrey
J
Kunkel
Department of Bioengineering, Santa Clara University, Santa Clara, California, United States of America.
Prashanth
P
Asuri
D004795:Enzyme Stability; D020100:Hydrogels / Q000737:chemistry*; D007700:Kinetics; D016062:Porosity; D012685:Sepharose / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry
D020100:Hydrogels; D012996:Solutions; D012685:Sepharose