Intermolecular cross-links in reconstituted collagen fibrils. Evidence for the nature of the covalent bonds.

Reduction of reconstituted native type collagen fibrils with sodium borohydride produced a firmly cross-linked polymer as determined by solubility, thermal shrinkage and dissolution, and renaturation studies, and by examination of the denatured protein by ultracentrifugation, gel filtration, polyacrylamide electrophoresis, and carboxymethyl cellulose chromatography. Maximum reduction occurred within 1 min after treatment of the reconstituted fibrils; approximately 1 mole of tritium was incorporated per mole of collagen when sodium borotritide was used. About 30% of the a! chains and 30% of the 0 subunits were consumed in the reaction, yielding a heterogeneous population of higher molecular weight components. Chemical modification of the collagen aldehydes prevented cross-linking, as did limited modification of lysine or hydroxylysine c-amino groups. Lathyritic collagen also failed to cross-link, probably as a result of diminished aldehyde content. Maximum cross-linking occurred when the collagen was polymerized into native type fibrils. Collagen in solution did not cross-link, and collagen polymerized into other than native type fibrils underwent partial or minimal crosslinking. Borohydride reduction did not produce intramolecular bonds, and no relationship could be found between the degree of intramolecular cross-linking and the ability to form intermolecular cross-links. It is proposed that the effect of borohydride is due to the reduction of Schiff bases which form between the collagen molecules when they are aligned in native type fibrils.