Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels.
Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels.
复制标题
肝素与抗凝血酶 III 的结合：丹磺酰和罗丹明标记的使用。
DOI：
10.1016/0003-9861(80)90113-7
复制
发表时间：
1980
影响因子：
3.9
通讯作者：
Schmer,G
中科院分区：
文献类型：
--
作者：
Piepkorn,MW;Lagunoff,D;Schmer,G
Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limitedN-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (Ka) of 4.9 × 105,m−1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent Ka= 7.9 × 105,m−1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.
