title: Studies on the formation, separation, and characterization of cyanogen bromide fragments of human AI apolipoprotein.
abstract: We have sought to obtain conditions for cyanogen bromide (CNBr) cleavage of apolipoprotein AI which would preserve, as far as possible, the biological activity of the resulting fragments. We found that the choice of solvent is an important consideration since modification of amino acids in different proteins varies with cleavage conditions. Initially, an analytical technique employing reversed-phase (RP)-HPLC which separates the four CNBr fragments in a single chromatographic step was established to monitor the products and extent of cleavage. In developing this technique, spectral data indicated damage to tyrosine and tryptophan residues during CNBr digestion. This problem was resolved by using 70% trifluoroacetic acid instead of 70% formic acid as the solvent, which had the added benefit of increasing the extent of cleavage of the Met86-Ser87 bond by 50%. We applied the information derived from the analytical RP-HPLC method to achieve the preparative isolation of CNBr fragments. This procedure included a gel permeation chromatography step using a citrate/urea buffer before RP-HPLC to isolate pure fragments in volatile buffers. Finally, we discuss aspects of structural integrity with an emphasis on modification of aromatic amino acids and deamidation of asparagine and glutamine residues.
authors.affiliation: Protein Chemistry and Molecular Biology Unit, Baker Medical Research Institute, Prahran, Victoria, Australia.
authors.forename: J R
authors.initials: JR
authors.lastname: Morrison
authors.forename: N H
authors.initials: NH
authors.lastname: Fidge
authors.forename: B
authors.initials: B
authors.lastname: Grego
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000302:isolation & purification*; D003720:Densitometry; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data
chemical_list: D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D003488:Cyanogen Bromide