Studies on the primary structure of chicken apolipoprotein A-I using HPLC technique

The complete amino acid sequence of chicken plasma apolipoprotein (apo) A-I was determined by sequencing overlapping peptide fragments produced by trypsin, S. aureus V8 protease, and cyanogen bromide cleavage respectively. All of the peptide fragments were purified on a Waters or on a Beckman HPLC system with a Vydac C18 column using 0.1% TFA in water as buffer A, and 0.08% TFA in 95% acetonitrile and 5% water as buffer B. Most of the peaks separated by these systems were pure. The partially purified fractions were subjected to rechromatography with a Hypersil ODS column using 0.005M sodium phosphate, pH 6.0, as buffer A, and 90% acetonitrile and 10% water as buffer B. The N-terminus of chicken apo A-I was determined to be aspartic acid by directly sequencing the intact protein up to 30 residues, while the C-terminus was identified as alanine by carboxypeptidase Y cleavage. There are 240 amino acid residues in mature chicken apo A-I. By direct analysis of cyanogen bromide peptide, we also determined the sequence of a 6 amino acid prosegment, which is present at approximately 10% of the molar amount of the mature protein in chicken plasma.