Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum al bumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluore scence emission. As determined by spectrofluorometric titration, binding af finities for palmitoyl-, stearoyl-, and oleoyl-CoA (K-d = 0.2-0.4 muM) are 5-10 times lower than those for the corresponding nonesterified fatty acids . In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) abov e the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescen t probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl- CoA synthetase activities are measured down to 0.05 muM/min with a data sam pling rate of 10 points per second. This new method provides a very promisi ng spectrofluorometric approach to the study of acyl-CoA synthetase reactio n kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays. (C) 2001 Academic Press.