Oliva M L, Mendes C R, Santomauro-Vaz E M, Juliano M A, Mentele R, Auerswald E A, Sampaio M U, Sampaio C A
Departamento de Bioquimica, Universidade Federal de Sao Paulo-Escola Paulista de Medicina, Rua Tres de Maio, São Paulo, Brazil.
Curr Med Chem. 2001 Jul;8(8):977-84. doi: 10.2174/0929867013372779.
A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.
用0.15M NaCl从羊蹄甲种子中提取丝氨酸蛋白酶抑制剂后，通过DEAE - 葡聚糖离子交换柱色谱、Superose 12柱凝胶过滤、Mono Q色谱法，或者通过胰蛋白酶 - 琼脂糖亲和色谱法进行纯化。通过Superose 12凝胶过滤，该抑制剂是一条分子量为20 kDa的单多肽链，但在Mono Q（FPLC系统）上进行离子交换色谱时被分离成两个峰。主要洗脱峰抑制胰蛋白酶（Ki = 0.6 nM）、血浆激肽释放酶（Ki = 0.35 nM）、纤溶酶（Ki = 33.1 nM），对胰凝乳蛋白酶的抑制作用较弱（Ki = 2700 nM），是从羊蹄甲种子中分离出的最有效的血浆激肽释放酶抑制剂。因此，它被命名为羊蹄甲激肽释放酶抑制剂（BbKI）。其活性对热不稳定，抑制胰蛋白酶的最适pH为8.0。BbKI与其他植物Kunitz抑制剂具有高度同源性，但没有二硫键，唯一的半胱氨酸残基位于C末端位置（第154位残基），这使其成为Kunitz家族的一个独特成员。通过吸附到胰蛋白酶 - 琼脂糖树脂上以及通过凝胶过滤分离胰蛋白酶 - 抑制剂复合物，证实了该抑制剂对胰蛋白酶的亲和力。合成了BbKI（GLPVRFESPLRINIIKESY）活性位点周围有变异的肽段，这些肽段含有淬灭的荧光基团。这些肽段是胰蛋白酶而非血浆激肽释放酶的底物，它们强烈抑制血浆激肽释放酶。
