MACROMOLECULAR CROWDING IN BIOLOGICAL SYSTEMS: DYNAMIC LIGHT SCATTERING (DLS) TO QUANTIFY THE EXCLUDED VOLUME EFFECT (EVE)

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Abstract
Macromolecules crowd defined spaces, thereby excluding other like-sized molecules from the volume they occupy. These excluded-volume effect(s) (EVE) are well characterized for intracellular and partially for extracellular compartments such as blood plasma. We showed that EVE in fibroblast culture leads to faster enzymatic procollagen conversion and matrix deposition. Apparently, EVE can be applied to emulate in vivo conditions in an in vitro setting. Thus, we attempted to quantitatively capture the crowding potential of various macromolecules using dynamic light scattering under physiological conditions. We found that charged macromolecules like dextran sulfate (negative, 500 kDa) have a hydrodynamic radii of 46.4 ± 0.3 nm i.e. ~4 fold larger than that of neutral macromolecules like Ficoll (neutral, 400 kDa) and thus show greater EVE potential. At biologically effective concentrations viscosity was not increased. Unexpectedly, we observed a dramatic drop of hydrodynamic radii of all macromolecules tested above a threshold concentration. This suggested a hyper-crowding state in which the crowders compacted themselves mutually. We will use this hyper-crowding threshold to determine retrogradely rules that allow to predict the conditions for optimum crowding effects (such as the half-hyper-crowding concentration) in biological systems. We propose Dynamic Light Scattering (DLS) as a potential tool to estimate EVE in biotechnical applications.

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