Studies on the formation, separation, and characterization of cyanogen bromide fragments of human AI apolipoprotein

Analytical Biochemistry
J R MorrisonB Grego

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 ami...Continue Reading

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Citations

Jun 22, 1999·Molecular and Biochemical Parasitology·P J Bradley, J C Boothroyd
Feb 8, 2003·Biochimica Et Biophysica Acta·Anh HoangDmitri Sviridov
Jul 30, 2010·Journal of Lipid Research·Rachel J BraceSally P A McCormick
Apr 27, 2004·Proceedings of the National Academy of Sciences of the United States of America·Brian G DrewBronwyn A Kingwell
Jan 20, 2004·The Journal of Biological Chemistry·Ying FuDmitri Sviridov
Jul 12, 2008·Arteriosclerosis, Thrombosis, and Vascular Biology·Andrew J MurphyJaye Chin-Dusting
Sep 17, 2002·Arteriosclerosis, Thrombosis, and Vascular Biology·Dmitri SviridovPaul Nestel
Dec 27, 1996·The Journal of Biological Chemistry·D SviridovN Fidge
Jan 18, 2003·The Journal of Biological Chemistry·Genevieve EscherDmitri Sviridov

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