Primary structure of insoluble tendon collagen: I. N- and C-terminal positional analysis of collagenolytic breakdown products of native collagen

Abstract

Purified insoluble native Achilles tendon collagen was digested with Clostridium histolyticum collagenase. The N-terminal sequence of the mixed peptides to position 3 was determined by a modification of the Edman degradation procedure, C-terminal amino acids by hydrazinolysis. Experimental losses and non-specific cleavage of the peptides were estimated from model compounds and appropriate corrections made. At the N-terminus 182 residues out of 200 were glycine, 10 were alanine, while isoleucine, phenylalanine, and valine accounted for the remainder. In the second position 77 residues out of 200 were proline, but significant amounts of other amino acids were present showing that the specificity requirement of the enzyme is less rigorous than originally suspected. Ten residues of hydroxyproline in this position were unexpected. In the third N-terminal position the distribution was even more heterogenous, but hydroxyproline and alanine, mostly in tripeptides, predominated. C-terminally, 58 residues were hydroxyproline, 42 alanine, 29 arginine, with other amino acids in considerably lower amounts. No glycine was detected at the C-terminus. Attempts were made to deduce the distribution of third position residues between tripeptides and longer chain peptides and comparisons made with results obtained by others with different collagens.