METHOD FOR MEASURING THE ACTIVITIES OF CHOLESTERYL ESTER TRANSFER PROTEIN (LIPID TRANSFER PROTEIN)

A continuous recording fluorescence assay was developed for cholestery l ester transfer protein (CETP). The assay measures the increase in fl uorescence accompanying the relocation of fluorescent lipids, choleste ryl esters and triglycerides, from a donor emulsion to an acceptor emu lsion. In the absence of CETP, the quantum yields of the fluorescent l ipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the flu orescent lipids from the donor to the acceptor emulsions and fluoresce nce increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly incre ased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within <30 min and the ki netics of this process are consistent with a simple, first-order react ion pathway. The redistribution kinetics support a mechanism of adsorp tion double right arrow exchange double right arrow desorption double right arrow diffusion.