Nishikata M
J Biochem. 1983 Jan;93(1):73-9. doi: 10.1093/oxfordjournals.jbchem.a134180.
A Sepharose derivative coupled with a chymostatin analogue, Gly-Gly-L-Leu-L-phenylalaninal (Pheal), was prepared. A number of native and chemically modified proteases were applied on a column of the adsorbent. Bovine chymotrypsins [EC 3.4.21.1] and Streptomyces griseus protease B were adsorbed strongly at pH 8.2. The affinities of these enzymes under various conditions were measured quantitatively by frontal chromatography in terms of the dissociation constant (Kd) of the enzyme-immobilized ligand complex. The pH dependence of the Kd value of alpha-chymotrypsin was consistent with that of the inhibition constant (Ki) of the enzyme for a corresponding soluble peptide aldehyde. Anhydro-chymotrypsin, in which the active site Ser-195 is converted to dehydroalanine, was not adsorbed. Ser-195 proved to be essential for the binding. The frontal chromatography method also gave the amount of the immobilized ligand that can interact with the enzyme. It was extremely small compared with the amount of the immobilized ligand determined by amino acid analysis. This was explained on the basis of the structural features of the agarose gel.
制备了一种与抑糜酶素类似物甘氨酰 - 甘氨酰 - L - 亮氨酰 - L - 苯丙醛（Pheal）偶联的琼脂糖衍生物。将多种天然和化学修饰的蛋白酶应用于该吸附剂柱。牛胰凝乳蛋白酶[EC 3.4.21.1]和灰色链霉菌蛋白酶B在pH 8.2时被强烈吸附。通过前沿色谱法根据酶 - 固定化配体复合物的解离常数（Kd）定量测量这些酶在各种条件下的亲和力。α -胰凝乳蛋白酶的Kd值对pH的依赖性与该酶对相应可溶性肽醛的抑制常数（Ki）一致。其中活性位点Ser - 195转化为脱氢丙氨酸的脱水胰凝乳蛋白酶未被吸附。结果证明Ser - 195对于结合至关重要。前沿色谱法还给出了可与酶相互作用的固定化配体的量。与通过氨基酸分析测定的固定化配体的量相比，该量极小。这是根据琼脂糖凝胶的结构特征来解释的。
