Static and time-resolved fluorescence studies of fluorescent phosphatidylcholine bound to the phosphatidylcholine-transfer protein of bovine liver

Abstract

Phosphatidylcholine analogs containing a cis-parinaroyl chain at the sn-1, sn-2 or both sn-1 and sn-2 positions (1-PnA-PC, 2-PnA-PC and diPnA-PC, respectively), were used to investigate the lipid binding site of the phosphatidylcholine transfer protein (PC-TP) from bovine liver by fluorometric techniques. Binding of these fluorescent lipids to the protein was registered by measuring the enhancement of parinaroyl fluorescence and the quenching of the tryptophanyl fluorescence. The fluorescence intensity of 1-PnA-, 2-PnA- and diPnA-PC bound to PC-TP was proportional to the chromophore content. The energy-transfer efficiency between the tryptophan residues and the bound chromophores was .apprx. 40% for 1-PnA- and 2-PnA-PC and 60% for diPnA-PC. Quenching of the tryptophanyl fluorescence was, in part, accounted for by a decrease of the fluorescence lifetimes. The orientation of the 1 and 2 fatty acyl chains of the PnA-PC analogs on the transfer protein was analyzed by time-resolved fluorescence anisotropy measurements. The fluorescence anisotropy decayed according to a single exponential function yielding a rotational correlation time of 26 ns for 1-PnA-PC, 11 ns for 2-PnA-PC and 15 ns for diPnA-PC. These correlation times indicate that both fatty acyl chains are immobilized at different positions on the protein. The difference in correlation time suggests that the shape of the phosphatidylcholine transfer protein is ellipsoidal (axial ratio .simeq. 2.5) with the 1 fatty acyl chain oriented parallel to the long symmetry axis and having an angle of 60-90.degree. with the 2 fatty acyl chain.