Mechanism of crosslinking of proteins by glutaraldehyde II. Reaction with monomeric and polymeric collagen.

Collagen in three different states, i.e. native soluble molecules, denatured molecules and reconstituted fibers, was exposed to various concentrations of glutaraldehyde. The degree of intramolecular and intermolecular crosslink formation was evaluated by measuring collagen solubility, beta and gamma chain formation, resistance towards cleavage by CNBr or collagenase digestion. Modification of lysyl residues was measured by amino acid analysis. When dilute collagen solutions were reacted with low concentrations of glutaraldehyde, intramolecular crosslinks were observed as the predominant crosslinks. When the glutaraldehyde concentration was increased, the collagen became more insoluble, indicating the formation of intermolecular crosslinks. When reconstituted collagen fibers were reacted with low concentrations of glutaraldehyde, intermolecular crosslinks were formed, which prevented the material from being solubilized by CNBr. However, these materials could still be solubilized by collagenase. When the glutaraldehyde concentration was increased, the materials became resistant to collagenase, while there was only a small increase in the number of lysyl residues modified. This reflects an increase in the molecular length of the glutaraldehyde polymers extending from the initial glutaraldehyde and lysyl residue reaction sites rather than an increase in the actual number of crosslinking sites.