Longas M O, Ferguson W S, Finlay T H
J Biol Chem. 1980 Apr 25;255(8):3436-41.
Heparin accelerates the rate of reaction of antithrombin with thrombin, an effect which is abolished by mild reduction of the antithrombin with dithiothreitol. Reduced antithrombin incorporates 1.7 mol of [14C]acetamide/mol of protein, with cysteine as the only amino acid modified. Tryptic digestion of the reduced and alkylated antithrombin results in the formation of only two labeled peptides. In the absence of heparin, the second order rate constant for the reaction of thrombin with both reduced and native antithrombin is 5.9 to 9.6 x 10(5) M-1 min-1. In the presence of heparin, the rate constant for the reaction between reduced antithrombin and thrombin is 8.3 to 12.2 x 10(5) M-1 min-1, while the rate of reaction between native antithrombin and thrombin is too fast to follow under the conditions used. Reduced antithrombin elutes from a heparin-Sepharose column at 0.5 M NaCl, contrast to 10 M NaCl required for elution of the native protein. The intrinsic tryptophan fluorescence enhancement caused by heparin binding to native antithrombin is not observed with reduced antithrombin. These data indicate that cleavage of one of the three antithrombin disulfide bonds results in reduced affinity for heparin and the loss of heparin-accelerated antithrombin activity and imply that heparin and thrombin bind at different sites on the antithrombin molecule.
肝素可加速抗凝血酶与凝血酶的反应速率，用二硫苏糖醇轻度还原抗凝血酶可消除该效应。还原后的抗凝血酶每摩尔蛋白质结合1.7摩尔[14C]乙酰胺，且只有半胱氨酸这一氨基酸被修饰。对还原并烷基化的抗凝血酶进行胰蛋白酶消化，仅产生两条标记肽段。在无肝素的情况下，凝血酶与还原型及天然抗凝血酶反应的二级反应速率常数为5.9至9.6×10(5) M-1 min-1。在有肝素存在时，还原型抗凝血酶与凝血酶反应的速率常数为8.3至12.2×10(5) M-1 min-1，而在所用条件下，天然抗凝血酶与凝血酶的反应速率太快难以追踪。还原型抗凝血酶在0.5 M NaCl浓度下从肝素-琼脂糖柱上洗脱，而天然蛋白质洗脱需要10 M NaCl。肝素与天然抗凝血酶结合引起的内在色氨酸荧光增强在还原型抗凝血酶中未观察到。这些数据表明，抗凝血酶的三个二硫键之一断裂会导致对肝素的亲和力降低以及肝素加速的抗凝血酶活性丧失，这意味着肝素和凝血酶在抗凝血酶分子上的结合位点不同。
