Maturation Analysis in Connective Tissue Proteins by [14C]Cyanide Incorporation

The use of a reaction involving [14C]cyanide and ammonia to stabilize and identify aldehydic cross-links in elastin and collagen is described. The products resulting from acid hydrolysis are in the form of 14C-labeled amino acids. The reaction was first studied with periodate-treated collagens, used as a model of collagen with aldehyde side chains. In these collagens the α-aminoglutaryl γ-semialdehyde residues react with [14C]cyanide and ammonia almost quantitatively and the expected product, after acid hydrolysis, is predominantly [14C]α, δ-diaminoadipic acid. This product indeed was isolated and identified. In elastin three amino acids derived from cross-links were isolated and their structures established. They are α,e-diaminopimelic acid, 1,9-diamino-1,9-dicarboxy-4-glycino-non-4-ene (carboxy amino aldol), and 2-amino-6-lysinopimelic acid (carboxylysinonorleucine). These compounds arise, respectively, from modification of residues of α-amino adipic acid δ-semialdehyde,aldol condensate of α-aminoadlpic acid δ-semialdehyde, and dehydrolysinonorleucine, and are in full agreement with structures of such compounds previously deduced using established sodium borohydride reduction procedures. Aldimines are modified with cyanide directly, not requiring the presence of ammonia. Cross-links already in the reduced form in the native protein, and the pyridinium forms of the desmosines do not incorporate the label and can be directly observed and quantitated by established procedures. The contents of modified cross-links can be estimated when the specific activity of Na14CN is known.