Assay of the binding of fatty acids by proteins: evaluation of the Lipidex 1000 procedure.
Fatty acid (FA) binding by fatty acid-binding protein (FABP) is frequently monitored with the so-called Lipidex 1000 assay, in which protein associated and non-protein bound FA are separated by selectively binding the latter to Lipidex 1000. Careful evaluation of this assay showed that the use of aqueous FA solutions resulted in a marked decrease (60 to 70%) of FA concentration due to their aspecific binding to the surface of the test-tube used. In addition, solutions of rat heart FABP in the micromolar range also showed a concentration decrease up to 80% due to protein binding to the surface of the test-tube. Introduction of detergents, Triton X-100 or Tween 20, limited the FA loss to less than 20% and totally eliminated FABP adsorption. Kinetic parameters for the binding of [1-14C]oleic acid by purified rat heart FABP, assayed in the presence of Triton X-100, were found to be similar to those assayed in the absence of detergent, when adequate corrections were made for losses of FA and FABP due to surface adsorption. Use of Tween 20 resulted in a substantial increase of the dissociation constant. The addition of 100 microM Triton X-100 to the assay medium considerably facilitates the determination of kinetic parameters of fatty acid-binding by proteins.
Department of Physiology, University of Limburg, Maastricht, The Netherlands.
M M
MM
Vork
J F
JF
Glatz
D A
DA
Surtel
G J
GJ
van der Vusse
D000327:Adsorption; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D003911:Dextrans; D005069:Evaluation Studies as Topic; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009206:Myocardium / Q000737:chemistry; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D017830:Octoxynol; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism*; D011092:Polyethylene Glycols; D011136:Polysorbates; D051381:Rats; D013499:Surface Properties
D002352:Carrier Proteins; D003911:Dextrans; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; D011136:Polysorbates; C010599:lipidex; D019301:Oleic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol