Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of Reduced, Denatured Lysozyme

Abstract

The oxidative refolding of reduced, denatured hen egg white lysozyme in the presence of a mixed macromolecular crowding agent containing both bovine serum albumin (BSA) and polysaccharide has been studied from a physiological point of view. When the total concentration of the mixed crowding agent is 100 g/liter, in which the weight ratio of BSA to dextran 70 is 1:9, the refolding yield of lysozyme after refolding for 4 h under this condition increases 24% compared with that in the presence of BSA and 16% compared with dextran 70. A remarkable increase in the refolding yield of lysozyme by a mixed crowding agent containing BSA and Ficoll 70 is also observed. Further folding kinetics analyses show that these two mixed crowding agents accelerate the oxidative refolding of lysozyme remarkably, compared with single crowding agents. These results suggest that the stabilization effects of mixed macromolecular crowding agents are stronger than those of single polysaccharide crowding agents such as dextran 70 and Ficoll 70, whereas the excluded volume effects of mixed macromolecular crowding agents are weaker than those of single protein crowding agents such as BSA. Both the refolding yield and the rate of the oxidative refolding of lysozyme in these two mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating that mixed macromolecular crowding agents are more favorable to lysozyme folding and can be used to simulate the intracellular environments more accurately than single crowding agents do.