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 albumin 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 fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent 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 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction 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.
Demant
E J
EJ
Department of Medical Biochemistry and Genetics, University of Copenhagen, Copenhagen, DK-2200, Denmark. erland@imbg.ku.dk
Nystrøm
B T
BT
D000085:Acetates / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002793:Cholic Acids / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D017830:Octoxynol / Q000378:metabolism; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D017186:Titrimetry
D000085:Acetates; D002793:Cholic Acids; D003374:Coumarins; D003902:Detergents; D005227:Fatty Acids; D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine; C102522:7-hydroxycoumarin-4-acetic acid; D017830:Octoxynol; D003066:Coenzyme A Ligases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate