Giuliano Migliorini — University of Padua #
Diffusion and enzymatic reactions in crowded solutions #
Biochemical reactions in living systems are affected by macromolecular crowding, a phenomenon
attributed to excluded volume effects and nonspecific interactions, yet these reactions are usually
studied in dilute solutions. Here, we investigate crowding effects on the activity of some key
enzymes found in the extracellular matrix (ECM), such as elastase and collagenase, using
controlled solutions of the branched polymer dextran.
Dextran solutions are characterized via rheology and diffusion measurements. Polymer transport
properties exhibit scaling-invariant behavior governed by degree of polymerization and branching,
whereas water diffusivity and solution density do not depend on the degree of polymerization.
These results enable the construction of volume fractions tailored to the physical observable of
interest.
The enzymatic activity of ECM proteases is quantified through spectrophotometric assays. We
introduce a theoretical framework for fluorescence detection in non-ideal mixtures and apply it to
the full progress curve assays of key ECM enzymes. For elastase, the degradation of a peptide is
enhanced by crowding and reveals an equilibrium constant exhibiting the same scaling behavior as
dextran transport properties, suggesting polymer size and topology as tunable parameters in
crowding experiments.