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A fluorimetric method for estimating cholesterolesterase activity
V. F. Pozdnev, K. S. Planutis, A. I. Tochilkin
Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR , Moscow
Abstract: A simple and highly specific method for estimating the cholesterolesterase activity is suggested. Cholesterolesterase (EC 3.1.1.13) is incubated with the emulsified substrate, cholesteryl-o-coumarate, at pH 6.6 to yield o-coumaric (trans-2-hydroxycinnamic) acid detected fluorimetrically (λexc 363 nm, λ,em 494 nm) at pH 10.4. The fluorescence associated with the unhydrolyzed substrate is negligible. Cholesteryl-o-coumarate is not hydrolyzed by pancreatic lipase, trypsin, or chymotrypsin under the above conditions. About 1 μg of pancreatic cholesterolesterase can be determined upon 15 min incubation. The substrate was synthesized by condensation of o-acetoxy-trans-cinnamic acid with cholesterol using the di-tert-butyl pyrocarbonate–pyridine–4-dimethylamino-pyridine system.
Russian Journal of Bioorganic Chemistry 1991, 17 (10):1347-1351