Ray A K, Guha M K, Sinha N K
Biochim Biophys Acta. 1982 May 27;716(2):126-32. doi: 10.1016/0304-4165(82)90260-4.
Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomyda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11,700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100 degrees C for 4 hr at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation constants are 7.2 x 10(-9) M for bovine trypsin and 5.5 x 10(-7) M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than that of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.
通过凝胶色谱法对三种龟鳖的蛋清进行了比较，以检测其对蛋白酶的抑制活性。三线闭壳龟（Geomyda trijuga trijuga Schariggar）的蛋清含有胰蛋白酶/枯草杆菌蛋白酶抑制剂，蠵龟（Caretta caretta Linn.）的蛋清则同时含有胰蛋白酶和糜蛋白酶抑制剂。恒河鳖（Trionyx gangeticus Cuvier）的蛋清未检测到蛋白酶抑制活性。从三线闭壳龟的蛋清中纯化出一种酸性胰蛋白酶/枯草杆菌蛋白酶抑制剂，并使其达到了均一性。它是一条由100个氨基酸残基组成的单多肽链，分子量为11,700。它含有六个二硫键，不含甲硫氨酸和碳水化合物部分。其等电点为pH 5.95，在中性pH条件下于100℃稳定4小时。该抑制剂通过形成摩尔比接近1的酶-抑制剂复合物来抑制胰蛋白酶和枯草杆菌蛋白酶。它们对牛胰蛋白酶的解离常数为7.2×10⁻⁹ M，对枯草杆菌蛋白酶的解离常数为5.5×10⁻⁷ M。用三硝基苯磺酸对氨基进行化学修饰降低了其对胰蛋白酶和枯草杆菌蛋白酶的抑制活性，但胰蛋白酶抑制活性的丧失比枯草杆菌蛋白酶抑制活性的丧失更快。它具有独立的结合位点来抑制胰蛋白酶和枯草杆菌蛋白酶。
