Two-dimensional electrophoresis of the fatty acid binding protein from human heart: evidence for a thiol group which can form an intermolecular disulfide bond.
A 100,000 g supernatant from human heart muscle, containing cytosolic proteins with some contaminating plasma proteins, was analyzed for fatty acid binding protein (FABP) by two-dimensional electrophoresis (2-DE) using isoelectric focusing under nondenaturing conditions in the first dimension. FABP purified from human heart muscle was found to comigrate with a major spot in 2-DE gels of the supernatant. This spot was comparable with those of the myoglobins in staining intensity. When purified FABP was charged with [3H]palmitate and subjected to nondenaturing 2-DE, radioactivity always comigrated with this protein. Under denaturing and reducing conditions in the second dimension, FABP was found to have a pI of 5.3 and an apparent molecular weight of 15,000. Isoforms of FABP, reported here for the first time to occur in human heart muscle, were observed as minor spots focusing at pH 5.1 and 5.7. When electrophoresis in the second dimension was carried out under denaturing but nonreducing conditions, an additional protein appeared at pH 5.3 with an apparent molecular weight of about 30,000. This protein was identified as a dimer of FABP and evidence for the involvement of an intermolecular disulfide bond in this dimerization is presented.
Nielsen
S U
SU
Institut für Biochemie, Universität Münster, Germany.
Vorum
H
H
Spener
F
F
Brodersen
R
R
D002352:Carrier Proteins / Q000032:analysis*; D003432:Cross-Linking Reagents / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D006801:Humans; D007536:Isomerism; D009206:Myocardium / Q000737:chemistry*; D009363:Neoplasm Proteins; D010168:Palmitates / Q000032:analysis; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014316:Tritium; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D010168:Palmitates; D013438:Sulfhydryl Compounds; D025521:Tumor Suppressor Proteins; D014316:Tritium