A diagonal paper electrophoretic method for the selective isolation of histidyl peptides.

Abstract

Thiolysis of an imidazolyl-dinitrophenyl-histidyl peptide at either pH 3.5 or 6.5 results in an increase in the net positive charge on the peptide. It is shown that this property can be used to form the basis of a diagonal paper electrophoretic purification of histidyl peptides from proteins. The amino groups of the protein are first reacted with citraconic anhydride and then the citraconyl protein is reacted with 1-fluoro-2,4-dinitrobenzene. The dinitrophenyl-citraconyl protein is digested with pepsin in 10% formic acid and, if necessary, with other proteolytic enzymes. The enzymatic digest is subjected to high-voltage paper electrophoresis at either pH 3.5 or 6.5. A guide strip is removed, thiolyzed with 2-mercaptoethanol, and subjected to electrophoresis at the same pH at right angles to the original direction of electrophoresis. The histidyl peptides are displaced off the diagonal toward the cathode. The off-diagonal peptides are isolated from the original electrophoretogram by thiolysis and electrophoresis using the diagonal electrophoretogram to locate the positions of the dinitrophenyl-histidyl peptides.