Diffusion and enzymatic reactions in crowded solutions: statistical physics approaches

Properties of biochemical reactions, happening in the extracellular matrix (ECM), are affected by macromolecular crowding in hard to predict ways. Nevertheless, these reactions are usually studied in dilute solutions. In this thesis, which is part of the ANR-funded X-CROWD project, how macromolecular crowding affects enzymatic activity is investigated using controlled solutions of the branched polymer dextran. Preliminary characterization of polymer solutions is fundamental and has been conducted through rheological and diffusion measurements. Dextran transport properties are found to scale with polymer size, revealing the signature of branching. The rich phenomenology found is discussed and modelled, opening interesting perspectives on the role of dextran as crowding agent. The enzymatic activity of ECM proteases is tracked through spectrophotometric assays. However, under crowded conditions, undesired absorption must be accounted for. A theoretical framework for fluorescence detection in non-ideal mixtures is introduced and applied to the full progress curve assays of two key ECM enzymes. In the case of elastase, the degradation of a peptide is enhanced by crowding and reveals an equilibrium constant satisfying the same scaling features as dextran transport properties, suggesting polymer size and topology as tunable parameters in crowding experiments. However, the same result is not found in the case of a matrix metalloprotease under the same crowding conditions, evidencing that also enzyme and biochemical specificities determine the response to macromolecular crowding.