The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media.

Detailed knowledge of the rates, equilibria, and mechanism of biochemical reactions has traditionally been acquired through experiments conducted on solutions containing low concentrations (less than about 1 mg/ml) of total protein, nucleic acid, and/or polysaccharide together with buffer salts, low molecular weight substrates, and cofactors as required. In contrast, biochemical reactions in living systems take place in media containing substantially greater total concentrations (50–400 mg/ml) of macromolecules that may be present in solution and/or in indefinitely large arrays (e.g. cytoskeletal fibers) (1, 2). Because no single macromolecular species may be present at high concentration, but all species taken together occupy a significant fraction of the volume of the medium, such media are referred to as “crowded” (3) and/or “confining” (4) rather than “concentrated,” depending upon whether the macrosolutes are soluble and/or structured. Fig. 1 provides a schematic illustration of crowding and confinement in eukaryotic cytoplasm. In such media, nonspecific interactions between macrosolutes contribute significantly to the total free energy of the medium. High concentrations of “background” macromolecules that do not participate directly in a particular test reaction have been observed to induce order-of-magnitude or greater changes in the rates and equilibria of numerous test reactions (see below). To properly assess the physiological role of a particular reaction or set of reactions characterized in vitro, it is important to consider the possible influence of crowding and/or confinement upon the reaction in its physiological milieu.