Effect of oxazolidine E on collagen fibril formation and stabilization of the collagen matrix.

Oxazolidine E, an aldehydic cross-linking agent, is used to impart hydrothermal stability to collagen. The purpose of this study was to investigate the exact nature of oxazolidine E induced cross-links with collagen by using synthetic peptides having sequence homology with collagen type I. Tandem mass spectrometry revealed the formation of methylol and Schiff-base adducts upon reaction of oxazolidine E with the peptides. This was confirmed by allowing the reaction to proceed under reducing conditions using cyanoborohydride. Mass spectrometry (MS)-MS analysis clearly showed interaction of tryptophan and lysine residues with oxazolidine E and demonstrated that arginine could be cross-linked with glycine in the presence of oxazolidine E through the formation of a methylene bridge. Collagen fibrils regenerated from monomers in the presence and absence of oxazolidine E were studied using atomic force microscopy to investigate morphological alterations. Regenerated fibrils showing the typical 65 nm D-banding pattern were obtained from those formed both in the presence and absence of oxazolidine E, and there was no evidence of a change in the D-periodicity of these fibrils. This indicated that oxazolidine E does not hinder collagen molecules from correctly aligning to form the quarter-stagger structure.

Unravelling the mechanism of the interactions of oxazolidine A and E with collagens in ovine skin.

Cross-linking agents play an important part in the physical properties of collagen based biomaterials. Oxazolidines are novel aldehydic tanning agents that are widely used to stabilise collagens in the leather industry. The exact mechanism through which they cross-link collagens is, however, not well understood. When they are combined with vegetable tannins, it is thought that oxazolidines form carbocationic intermediates through ring opening, which are then able to interact with the amino acid side chains of collagens and flavonoid ring systems of vegetable tannins. In this study, the interactions of oxazolidines, with collagens, have been investigated using a number of analytical techniques. High pressure liquid chromatography (HPLC) analysis of oxazolidine tanned collagen samples showed that there is an irreversible reaction with tyrosine side chains. Mass spectrometry (MS) revealed the formation of a Schiff's base adduct with lysine residues, which was reversible in nature. MS analysis of reaction of oxazolidines with a model peptide Suc-Ala-Phe-Lys-AMC in presence of NaCNBH(3), indicated the formation of a product with an increase in molecular weight of 28 kD characteristic of the addition of two methyl groups to lysine. Differential scanning calorimetry showed a synergistic effect for combination tannage, with best results being obtained when vegetable tan was added prior to the aldehydic tanning agents. Circular dichroism (CD) studies of collagen in presence of the more reactive oxazolidine A showed that there was a loss in ellipticity simply because of aggregation of collagen molecules rather than a change in the secondary structure. Based on the results obtained, a scheme has been proposed to explain the possible mechanism of action of oxazolidines with the collagen amino acid side chains.