The Introduction of Intramolecular Covalent Cross-linkages into Ichthyocol Tropocollagen with Monofunctional Aldehydes

The presence of covalent cross-linkages between the peptide chains of collagen has been postulated to account for many of the properties of collagen (l), particularly the insolubility of mature fibrous mammalian collagen in water or dilute acids at low temperature. There is as yet no direct chemical evidence as to the nature and number of the cross-linkages, although ester bonds (2-4) and aldehyde bridges (5) have been implicated. One of the strongest bits of evidence for the covalent interchain cross-linkage is that gelatins may be prepared, either from mature fibrous collagen or from mammalian tropocollagen, that can be reversibly renatured to the fibrous or segment-long-spacing (SLS) forms (6-9). Gelatins of the renaturable type (7) represent a distinct molecular species and presumably consist of three peptide chains held together by cross-linkages that were imposed on the tropocollagen framework after the tropocollagen was incorporated into the fibrous collagen structure (10). The crosslinkages are thought to hold the chains in the proper register to provide nucleation sites for the reformation of the trihelical structure. It occurred to us that it might be possible to introduce intramolecular cross-linkages into a native tropocollagen molecule that would not ordinarily yield the renaturable y-component upon denaturation, and thus synthesize a y-component that would exhibit the proper renaturation properties. Such a synthesis would constitute a formal proof that covalent crosslinkages could account for the observed behavior of natural collagens and would provide a suitable model for investigation of the renaturation phenomena. In this paper we report the successful synthesis of a y-gelatin from an ichthyocol and an analysis of its stability and renaturability. Several factors influenced our choice of starting material. The collagen had to have a low content of naturally cross-linked components, it had to be already characterized or be quite similar to the collagens described in the literature, and, hopefully, it had to be available in reasonably large amount. The ichthyocol collagens as a class fulfilled the first two requirements. The buffalo fish (Ostariophysi ictiobus), a member of the sucker family and closely related to the carp, was available locally. Buffalo fish swim bladder collagen was therefore used.