Separation of the Subtypes of Type V Collagen Molecules, [ l(V)]2 2(V) and 1(V) 2(V) 3(V), by Chain Composition-Dependent Affinity for Heparin: Single 1(V) Chain Shows Intermediate Heparin Affinity between Those of the Type V Collagen Subtypes Composed of [ 1(V)]2 2(V) and of 1(V) 2(V) 3(V)
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 170 mM NaCl in 20 mM phosphate buffer, pH 7.2, containing 2 M urea; bound and non-bound. The ratio of the three a-chains, αl(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 αl(V) chain to heparin.