Practical Protein Chemistry - A. Darbre 1989

Traditional Strategy for Protein Structure Determination
Fractionation of Soluble Peptides
Dowex-50 Column Chromatography

Elution of Peptides from a Column packed with Dowex-50 resin (sulfopolystyrene) is carried out using an increasing gradient of pH and pyridine concentration [58, -60] (details are given in Table 10.5). At pH 2, the carboxyl groups of C-terminal residues and the side chains of aspartic and glutamic acids are protonated, as are the N-terminal amino groups and the side chains of Arginine, Lysine, and Histidine. Under these conditions, the peptides are adsorbed onto the sulfo groups of the resin. As the pH increases, the carboxyl groups become ionized and the peptides are released from the resin. Thus, peptide fractionation occurs according to their net charge and the $ ext{p}K$ of their carboxyl groups. Acidic peptides elute first, followed by neutral and basic ones. Neutral peptides typically emerge from the column in two groups: first, "true" neutral peptides lacking internal basic or acidic residues, and subsequently peptides whose internal charges are compensated. This Separation is due to the higher $ ext{p}K$ values of the side-chain carboxyl groups compared to those of the C-terminal residues. Large, hydrophobic, and highly basic peptides (bearing positive charges from multiple groups) are generally eluted in low yield. Because pyridine is "opaque" in the ultraviolet region at 230 and 280 nm, Peptide elution profiles are constructed by analyzing small aliquots of the collected fractions using high-voltage paper Electrophoresis at pH 6.5; based on the analysis results, the fractions are pooled and concentrated on a rotary evaporator.