Chicken apolipoprotein A-1의 완전한 일차구조를, trypsin, S. aureus V8 protease 및 cyanogen bromide를 이용하여 결정하였다. 0.1% TFA와 95% acetonitrile을 사용하여 Vydac $C_{18}$ column이 연결된 Waters HPLC를 사용하여 모든 peptide를 분리하였으며, 이 방법에 의해 분리된 대부분의 peak들은 순수하였다. Rechromatography 방법으로는 5 mM sodium phosphate와 90% acetonitrile의 두 buffer를 hypersil $C_{18}$ column에 이용하여 재분리하였다. Chicken apo A-l의 N-말단기는, 순수한 단백질의 직접적인 sequencing 방법에 의해 aspartic acid로 결정되었으며 C-말단기는 carboxypeptidase Y를 사용하여 alanine으로 동정되었다. 이러한 결과로서 mature chicken의 apo A-1은 240개의 아미노산 잔기로 이루어져 있음이 밝혀졌으며 chicken plasma의 대략적인 molar 양의 10%에 해당하는 6개의 prosegment의 sequence 역시 동정되었다.
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. All of the peptide fragments were purified on a Waters HPLC system with a Vydac $C_{18}$ 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 $C_{18}$ 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 peptide in chicken plasma.
