Synthesis of peptide labelled with p-iodophenylalanine which corresponds to the first amphipathic region of apolipoprotein C-I.
The amino terminal fragment (1-15) of apolipoprotein C-1, H-Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe14-Gly was prepared by the solid phase method. However, the phenylalanine residue at position 14 was replaced with p-iodophenylalanine in the chemical synthesis. The preparation of t-butyloxy-carbonyl-p-iodophenylalanine is described. After completion of the synthesis, the product was deprotected and cleaved from the resin with liquid HF. The peptide was purified by gel filtration on Sephadex G-25 and preparative high performance liquid chromatography. The purified material was shown to be homogeneous by amino acid analytical data and by chromatography in three different analytical reversed phase HPLC systems. The peptide was then labelled by the chloroamine-T procedure and good incorporation of 125I was obtained. After purification of the product by gel filtration on Biogel P-2, the labelled pentadecapeptide was tested for ability to bind to Very Low Density Lipoproteins (VLDL) in the following manner: VLDL was isolated from normolipemic serum by ultracentrifugal flotation and 100-microliter samples were incubated with labelled material dissolved in 200 microliter of 0.5 M NaCl, 0.02 M sodium phosphate buffer, pH 7.4 at 37 degrees for 30 min. The VLDL fraction was again isolated by ultracentrifugal flotation and the incorporation of radioactivity into the lipoprotein was measured. Under these conditions, a sample of [3H]-native apolipoprotein C-I was incorporated to an extent of 83% of the total sample, while the [125I]-pentadecapeptide exhibited an incorporation of 8.7%.
D000595:Amino Acid Sequence; D053302:Apolipoprotein C-I; D001056:Apolipoproteins C / Q000138:chemical synthesis*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007202:Indicators and Reagents; D010455:Peptides / Q000138:chemical synthesis*; D010649:Phenylalanine / Q000031:analogs & derivatives*; D011485:Protein Binding
D053302:Apolipoprotein C-I; D001056:Apolipoproteins C; D007202:Indicators and Reagents; D010455:Peptides; D010649:Phenylalanine; C049181:4-iodophenylalanine