An enzymic cycling method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA hydrolase.
A method for measuring free fatty acids by enzymic cycling is described. Free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase, then the acyl-CoAs are hydrolyzed back to the free fatty acids by acyl-CoA hydrolase in a cyclic fashion. The amounts of AMP produced during this cyclic reaction are determined from the absorbance at 340 nm in the presence of AMP deaminase and glutamate dehydrogenase. This method is sensitive to as low as 0.1 nmol of free fatty acids, and the standard curve is linear up to 1.0 nmol. This method shows a broad specificity for long-chain fatty acids (C12--C20) and the recoveries of fatty acids added to bacterial cell-free extracts are more than 90%.
D000659:AMP Deaminase; D001419:Bacteria; D055598:Chemical Phenomena; D002621:Chemistry; D003066:Coenzyme A Ligases; D004926:Escherichia coli / Q000201:enzymology; D005230:Fatty Acids, Nonesterified / Q000302:isolation & purification*; D005969:Glutamate Dehydrogenase; D006863:Hydrogen-Ion Concentration; D010170:Palmitoyl-CoA Hydrolase; D013869:Thiolester Hydrolases
D005230:Fatty Acids, Nonesterified; D005969:Glutamate Dehydrogenase; D013869:Thiolester Hydrolases; D010170:Palmitoyl-CoA Hydrolase; D000659:AMP Deaminase; D003066:Coenzyme A Ligases