Dodt J, Machleidt W, Seemüller U, Maschler R, Fritz H
Biol Chem Hoppe Seyler. 1986 Aug;367(8):803-11. doi: 10.1515/bchm3.1986.367.2.803.
A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 IU/mg and dissociation constants Ki of the thrombin-inhibitor complexes close to 3 X 10(-11) mol/l. The inhibitor with N-terminal isoleucine was designated hirudin PA. This inhibitor contains 66 amino-acid residues and has a molecular mass of 7,087 Da. The complete amino-acid sequence of hirudin PA was established by automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments. On the basis of the primary structures two types of thrombin inhibitors from the leech can be distinguished, designated hirudin and hirudin PA. The degree of structural homology of both isoinhibitors is approximately 82%; both have a tyrosine-O-sulfate residue near the C-terminus.
已开发出一种五步分离程序，用于从整条水蛭中纯化水蛭素同工型（异水蛭素）。通过反相高效液相色谱法对两种凝血酶抑制制剂进行最终纯化，得到了几种N端为缬氨酸或异亮氨酸但抑制特性相同的异水蛭素，即特异性凝血酶抑制活性为680 - 720 IU/mg，凝血酶 - 抑制剂复合物的解离常数Ki接近3×10⁻¹¹ mol/l。N端为异亮氨酸的抑制剂被命名为水蛭素PA。该抑制剂含有66个氨基酸残基，分子量为7087 Da。通过对天然、氧化的抑制剂及其两个胰蛋白酶片段进行自动固相埃德曼降解，确定了水蛭素PA的完整氨基酸序列。根据一级结构，可区分出水蛭中的两种凝血酶抑制剂，分别命名为水蛭素和水蛭素PA。两种异抑制剂的结构同源性约为82%；两者在C端附近都有一个酪氨酸 - O - 硫酸酯残基。
