Cholesterol aggregation and interaction with cholesterol oxidase in supercritical carbon dioxide

High-pressure EPR spectroscopy indicates that cholesterol forms aggregates in supercritical carbon dioxide. In pure carbon dioxide, changes in cholesterol aggregate size or packing structure are observed with changing pressure. Near the critical point of carbon dioxide, cholesterol solubility is too low to permit significant aggregation, and monomeric cholesterol is observed. Addition of small amounts of dopants to supercritical carbon dioxide strongly affects cholesterol aggregation. Branched butanols (2-methyl-1-propanol and 2-methyl-2-propanol) and ethanol (to a lesser degree) promote cholesterol aggregation, while methanol, acetone, and 1-butanol do not. Cosolvents that promote aggregation also increase the rate at which cholesterol oxidase from Gloeocysticum chrysocreas catalyzes the oxidation of cholesterol. In supercritical carbon dioxide solutions, the EPR spectroscopy reveals little or no conformational change in cholesterol oxidase as 2-methyl-2-propanol or methanol is added. Damp cholesterol oxidase binds multiple cholesterol molecules; dry enzyme loses the ability to bind cholesterol. When molecular oxygen is the oxidizing agent, the rate of enzymatic cholesterol oxidation is greatly reduced in bone-dry carbon dioxide compared to that in water-saturated carbon dioxide.