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

Autor: Demant EJ; Department of Medical Biochemistry and Genetics, University of Copenhagen, Copenhagen, DK-2200, Denmark. erland@imbg.ku.dk, Nystrøm BT
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2001 Aug 01; Vol. 295 (1), pp. 38-44.
DOI: 10.1006/abio.2001.5181
Abstrakt: 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.
(Copyright 2001 Academic Press.)
Databáze: MEDLINE
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