Cleavage at acyl-proline bonds with sodium in liquid ammonia: application with nanomolar amounts of peptides and separation of products by high-performance liquid chromatography for structural analysis.
Cleavage of X-Pro bonds with metallic sodium in liquid ammonia is a little-used method due to difficulties with handling of reagents, variable cleavage yields, and separation of peptides from salt byproducts. Construction of a small distillation/reaction apparatus permitted peptide incubations at the nanomole scale. Reverse-phase high-performance liquid chromatography (HPLC) of the residue after removal of NH3 allows separation of the salts and fractionation of the cleaved peptides which may be taken directly for sequence analysis. HPLC also allows rapid assessment of the degree of cleavage before structural analysis. Cleavage of some peptides proceeded in high yield while others were cleaved poorly or not at all, modifying earlier generalizations on factors influencing cleavage.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases; D000444:Aldehyde Dehydrogenase; D000595:Amino Acid Sequence; D000641:Ammonia; D000818:Animals; D002766:Cholecystokinin; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D008832:Microchemistry; D010446:Peptide Fragments; D010455:Peptides; D012964:Sodium; D013552:Swine
D010446:Peptide Fragments; D010455:Peptides; D000641:Ammonia; D002766:Cholecystokinin; D012964:Sodium; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D000444:Aldehyde Dehydrogenase