Peculiar effect of urea on the interaction of type I collagen with heparin on chromatography.

Type I collagen in phosphate-buffered saline (PBS) bound to a heparin-Sepharose column, while heat-treated type I collagen, denatured chains of alpha 1(I) and alpha 2(I), did not. Conformation-dependent association of type I collagen with heparin was further examined in various urea concentrations. The relative amount of bound fraction decreased in proportion to the concentration of added urea; from over 90% in the absence of urea to about 30% in 3 M urea, although circular dichroism spectrum of type I collagen was not changed by the presence of 4 M urea at 25 degrees C. In 2 M urea, the relative amount of bound fraction was about 50%. Rechromatography of the flow-through fraction or bound fraction of type I collagen in 2 M urea after lyophilization showed a similar pattern to that of the initial type I collagen solution in that about a half of either sample was recovered as bound fraction. This result indicated that the association potential of type I collagen with heparin appeared to have changed reversibly. The relative amount of bound fraction was little affected by the initial protein concentration, suggesting that intermolecular interaction between type I collagen molecules or the aggregate possibly resulting from the interaction is not important in the affinity with heparin. From these results, we suggest that triple-helical type I collagen molecules undergo reversible changes of conformation in urea solution between the conformation with heparin affinity and that without the affinity. In contrast, type V collagen or alpha1 (V) chain binds to heparin under the same conditions and therefore the conformational change of type V collagen in urea solution would not be discernible in terms of heparin affinity, even if the type V collagen molecules have altered conformation in urea, as is suggested for type I collagen.