Separation of fatty acid binding protein by high-performance mixed-mode chromatography.
Fatty acid binding protein (FABP) (14 kDa), can regulate the levels of tissue free fatty acids by binding them with high affinity. Since free fatty acids are known to accumulate in the ischemic myocardium, it is likely that FABP has a significant role in regulating their concentration in ischemic heart. FABP has recently been purified from other proteins, but the method requires several hours and special techniques. In this report, we describe a rapid high-performance liquid chromatographic method for separating and isolating the FABP from myocardial tissue biopsies. About 25-50 micrograms of rat heart cytosol was incubated with 2 nmol of the potassium salt of [9,10-3H]oleate (25,000 cpm) for 10 min at 37 degrees C. This was then injected onto a Bio-Rad (Richmond, CA, U.S.A.) TSK-125 column. The sample was run using a low-salt isocratic mobile phase containing 10 mM potassium phosphate buffer (pH 7) and 1 mM dithiothreitol, and at a flow-rate of 0.8 ml/min. The heart cytosol, when incubated with isotopic oleate, was resolved into two radioactive peaks, one eluting in the area of serum albumin (retention time 9.6 min) and the other corresponding to a retention time of 12.9 min. The sodium dodecyl sulfate polyacrylamide gel electrophoretic profile of the later peak revealed a major protein band of ca. 14 kDa. Rat heart FABP purified by gel filtration and ion-exchange chromatography coeluted with the second radioactive peak.(ABSTRACT TRUNCATED AT 250 WORDS)
Samanta
A
A
Department of Surgery, University of Connecticut School of Medicine, Farmington 06032.
Cordis
G A
GA
Prasad
M R
MR
Das
D K
DK
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005230:Fatty Acids, Nonesterified / Q000032:analysis; D009206:Myocardium / Q000032:analysis; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D051381:Rats
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005230:Fatty Acids, Nonesterified; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins