Mizuno K, Hayashi T
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo.
J Biochem. 1996 Nov;120(5):934-9. doi: 10.1093/oxfordjournals.jbchem.a021509.
The heparin affinities of heat-treated type V collagen alpha-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, alpha 1(V) chain required approximately two-fold higher NaCl concentration to pass through the column than the other two chains, alpha 2(V) and alpha 3(V). Thus, the heparin affinity of alpha 1(V) may be approximately two-fold higher than those of the other alpha (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 alpha-chains, alpha 1(V): alpha 2(V): alpha 3(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 alpha 1(V) chains in the triple-helical molecule, if these fractions contained triple-helical subtypes with the chain compositions of [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), respectively. From the comparison of the NaCl concentration required for prevention of the binding, [alpha 1(V)]2, alpha 2(V) had about two-fold higher affinity than alpha 1(V) alpha 2(V) alpha 3(V), and the separated alpha 1(V) chain showed an intermediate affinity. A possible explanation for difference in heparin affinity among the subtypes of molecules and the separated alpha-chains is that the heparin affinity of type V collagen molecule is governed by the number of alpha 1(V) chains contained in the molecule and that steric restraint in a triple-helical conformation weakens the binding of alpha 1(V) chain to heparin.
通过防止与肝素 - 琼脂糖柱结合所需的NaCl浓度，评估了热处理的V型胶原α链和三螺旋分子的肝素亲和力。热处理后，α1（V）链通过柱子所需的NaCl浓度比其他两条链α2（V）和α3（V）高约两倍。因此，α1（V）的肝素亲和力可能比其他α（V）链高约两倍。在含有2M尿素的pH 7.2的20mM磷酸盐缓冲液中，三螺旋构象的V型胶原分子在170mM NaCl下被分离成两个部分：结合部分和未结合部分。通过SDS - PAGE分析，结合部分和流过部分中三条α链α1（V）：α2（V）：α3（V）的比例分别为2：1：0和1：1：1。如果这些部分分别包含链组成为[α1（V）]2α2（V）和α1（V）α2（V）α3（V）的三螺旋亚型，则两个部分的差异亲和力可以由三螺旋分子中α1（V）链的数量来解释。通过比较防止结合所需的NaCl浓度，[α1（V）]2α2（V）的亲和力比α1（V）α2（V）α3（V）高约两倍，分离出的α1（V）链表现出中等亲和力。分子亚型和分离出的α链之间肝素亲和力差异的一个可能解释是，V型胶原分子的肝素亲和力由分子中所含α1（V）链的数量决定，并且三螺旋构象中的空间限制削弱了α1（V）链与肝素的结合。
