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...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 Vydae C_(18) column using 0.1% TFA in water as buffer A,and 0.08% TFA in 95% acetomtrile 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.展开更多
文摘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 Vydae C_(18) column using 0.1% TFA in water as buffer A,and 0.08% TFA in 95% acetomtrile 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.
