Isolation and identification of the cyanogen bromide peptides of D-glyceraldehyde-3-phosphate dehydrogenase from human muscle.

D-Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from human muscle was S-carboxymethylated, cleaved with cyanogen bromide and citraconylated. The enzymatic protein contains 344 amino acid residues, nine of which are methionines. The respective ten cyanogen bromide fragments have been isolated and characterized. Each peptide was purified to homogeneity by Bio-Gel chromatography, high voltage electrophoresis and descending paper chromatography, and characterized by electrochromatography, N-terminal sequence and amino acid composition.

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

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.

Application of Pluronic copolymer liquid crystals for the capillary electrophoretic separation of collagen type I cyanogen bromide fragments.

A capillary electrophoretic method exploiting the properties of Pluronic copolymer liquid crystals (F127) was developed for the separation of collagen cyanogen bromide (CNBr) fragments. The separations obtained were at least comparable (if not better) to those obtained by other methods applicable to this category of compounds. In the optimized version a bare silica capillary [47 cm (40 cm to the detector) x 75 microm I.D.] was used with 10 mM Tris and 75 mM phosphate buffer (pH 2.5) containing 7.5% Pluronic F127 copolymer. The separation mechanism which involves both the molecular sieving and surfactant properties of the Pluronic F127 gel phase is discussed.