Submitted on March 8, 2005
Revised on April 12, 2005
Accepted on April 12, 2005
A simple and rapid method to measure cholesterol binding to P450s and other proteins
Natalia Mast and Irina A. Pikuleva
Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031
Corresponding Author: irpikule{at}utmb.edu
Cholesterol plays an important role in cellular function and membrane compartmentalization and is involved in the interaction with more than a dozen of different proteins. By using three cholesterol-metabolizing cytochromes P450 (P450s 7A1, 46A1, and 11A1), we have developed a rapid and simple assay for measurements of nanomolar-to-micromolar cholesterol affinities. In this assay, the P450 is incubated with a fixed amount of radiolabeled cholesterol and varying concentrations of cold cholesterol followed by separation of free and protein-bound cholesterol via filtration through a membrane. Free cholesterol is found in the flow-through fraction while P450 binds to the membrane. Radioactivity of the membranes is then measured and a saturation curve is generated after correction for non-specific binding of cholesterol to the filter. The validity of the filter assay was confirmed by spectral assay, a traditional method to evaluate interaction of the P450 enzymes with their substrates. Two types of membranes, one binding positively charged proteins and another negatively charged proteins, were identified. These membranes were also found to hold proteins through hydrophobic interactions. Thus, cholesterol-binding properties of a wide variety of proteins could be characterized by using this filter assay.
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