title: Peculiar effect of urea on the interaction of type I collagen with heparin on chromatography.
abstract: 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.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo.
authors.forename: K
authors.initials: K
authors.lastname: Mizuno
authors.forename: T
authors.initials: T
authors.lastname: Hayashi
mesh_terms: D002845:Chromatography / Q000379:methods; D003094:Collagen / Q000737:chemistry* / Q000302:isolation & purification; D004347:Drug Interactions; D006493:Heparin / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010944:Plants / Q000737:chemistry; D011489:Protein Denaturation; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; C037405:heparin-sepharose; D014508:Urea; D006493:Heparin; D003094:Collagen; D012685:Sepharose