Practical Protein Chemistry - A. Darbre 1989

Traditional strategy for determining protein structure
Fractionation of soluble peptides
High-voltage electrophoresis and paper chromatography

Electrophoresis and paper Chromatography serve as convenient rapid Methods for purifying medium and short Peptides (Section 10.8.2). Electrophoresis is typically performed at pH 6.5, 3.5, and 2.1. At these pH values, the side chains of Lysine and Arginine residues, as well as the N-terminal amino groups, are fully protonated. At pH 6.5, the imidazole ring of Histidine is partially protonated, while the carboxyl groups are completely ionized. At pH 3.5, histidine is fully protonated, and the carboxyl groups are partially protonated. At pH 2.1, the carboxyl groups are fully protonated, with the exception of the sulfonic acid side chain of cysteic acid. Peptide mobility during electrophoresis strictly depends on their net charge and size [46], and their localization on the paper is determined by staining a side strip of the sheet with ninhydrin (for one-dimensional maps) or by spraying with a low-concentration fluorescamine solution (for one- and two-dimensional maps). The process of electrophoretic Separation on paper is highly sensitive to the presence of salts in the sample and to carrier overloading, whereas peptide detection using these Reagents is only possible if an unblocked N-terminal amino group is present. Peptide separation via paper chromatography in the BAW system [77] (Table 10.4) occurs according to their partition coefficient between the aqueous and organic phases: hydrophobic peptides migrate faster than hydrophilic ones. Although the resolving power of chromatography in BAW is lower than that of electrophoresis, the method remains undeniably valuable for separating peptides of identical size and charge.