Abstract
The heparin affinities of heat-treated type V collagen α-chains and the triple-helical molecules were evaluated in terms of the NaCl concentration required for prevention of binding to a heparin-Sepharose column. After heat treatment, α1 (V) chain required approximately two-fold higher NaCl concentration to pass through the column than the other two chains, α2 (V) and α3 (V). Thus, the heparin affinity of α1 (V) may be approximately two-fold higher than those of the other α (V)-chains. The type V collagen molecules in triple-helical conformation were separated into two fractions at 170mM NaCl in 20mM phosphate buffer, pH 7.2, containing 2M urea; bound and non-bound. The ratio of the three α-chains, α1 (V): α2 (V): α3 (V) was 2:1:0 and 1:1:1 in the bound and flow-through fractions, respectively, on analysis by SDS-PAGE. The differential affinity of the two fractions could be accounted for by the number of α1 (V) chains in the triple-helical molecule, if these fractions contained triple-helical subtypes with the chain compositions of [α1 (V)]2α2 (V) and α1 (V)α2 (V)α3 (V), respectively. From the comparison of the NaCl concentration required for prevention of the binding, [α1 (V)]2α2 (V) had about two-fold higher affinity than α1 (V)α2 (V)α3 (V), and the separated α1 (V) chain showed an intermediate affinity. A possible explanation for difference in heparin affinity among the subtypes of molecules and the separated α-chains is that the heparin affinity of type V collagen molecule is governed by the number of α1 (V) chains contained in the molecule and that steric restraint in a triple-helical conformation weakens the binding of al(V) chain to heparin.
