Localization and Partial Characterization of an Aldehydic Component in Tropocollagen

Abstract

1. An alternative simplified procedure is described for the isolation of a 30-residue peptide obtained by digestion with collagenase of carp swim bladder tropocollagen (ichthyocol). The peptide contains an aldehydic component as a 2,4-dinitrophenylhydrazone. The aldehyde dinitrophenylhydrazone is present in stoichiometric proportion to the amino acids in the peptide. 2. This peptide is shown to be devoid of free terminal amino groups by lack of reactivity with ninhydrin or phenylisothiocyanate. Since it is prepared by treatment with collagenase and yet devoid of any terminal α-amino group including that of a glycine residue, the peptide most likely arises from a blocked amino-terminal position of a tropocollagen chain. 3. The aldehyde-bearing component most likely is not bound to any of the following amino acid side chains for the reasons given: (a) to side chain carboxyl groups of glutamyl or aspartyl residues in ester linkage, since treatment with aqueous hydrazine does not release the dinitrophenylhydrazone from the peptide; (b) to the hydroxyl group of a serine residue by means of a glycosidic-like attachment, since mild treatment with NaOH does not promote β elimination in the serine; (c) to side chains of either threonine, hydroxylysine, hydroxyproline, lysine, arginine, or histidine, since residues of these are not present in the peptide; (d) to the hydroxyl side chain of a tyrosine residue, since this amino acid, while present in the aldehyde-dinitrophenylhydrazone-peptide obtained following digestion with collagenase, is absent from the aldehyde-dinitrophenylhydrazone-peptide obtained by further digestion with Pronase. 4. The peptide obtained with collagenase is cleaved further into at least seven peptides which have been partially characterized. A yellow heptapeptide containing the aldehydic component as a 2,4-dinitrophenylhydrazine derivative is also without free amino groups, and this locates the aldehyde-bearing component within 7 residues of a blocked amino terminus of a tropocollagen chain. 5. Ultraviolet absorption spectra of derivatives of the aldehyde component, and its tendency to undergo acid-catalyzed α,β-dehydration to α,β-unsaturated aldehyde derivatives, suggest that its first 3 carbon atoms are involved in a β-hydroxyaldehyde structure. The aldehyde-bearing component must contain an additional functional group with which it makes covalent attachment to the protein. 6. It is suggested that the aldehyde component, by virtue of its chemical nature, reactivity, stoichiometry, and location in the protein, may be involved in the maturation and cross-linking of soluble tropocollagen to form insoluble mature tissue collagen.