Ando Y, Imamura S, Yamagata Y, Kikuchi T, Murachi T, Kannagi R
Department of Dermatology, Kyoto University Faculty of Medicine.
J Biochem. 1987 Jun;101(6):1331-7. doi: 10.1093/oxfordjournals.jbchem.a122000.
A high-performance liquid chromatographic method was developed for the assay of transglutaminase [EC 2.3.2.13] activity. Casein and dansylcadaverine were used as substrates and the reaction was stopped by adding an excess amount of EGTA. Casein-bound dansylcadaverine was separated from free dansylcadaverine by high-performance liquid chromatography on a TSK SW gel column on the basis of the differences in the molecular weight and hydrophobicity. The sensitivity was approximately 0.04 nmol of casein-bound dansylcadaverine in the assay mixture. With this assay method, human erythrocyte transglutaminase and platelet factor XIII were purified by successive chromatographies on DEAE-cellulose and Sephacryl S-300, which were common for both enzymes, followed by Blue Sepharose CL-6B and DEAE Bio-Gel A for erythrocyte transglutaminase or Phenyl-Sepharose CL-4B for platelet factor XIII. The purification factors and activity yields were 15,300-fold and 22% for erythrocyte transglutaminase and 43.8-fold and 33% for platelet factor XIII.
建立了一种高效液相色谱法用于测定转谷氨酰胺酶[EC 2.3.2.13]的活性。以酪蛋白和丹磺酰尸胺作为底物，通过加入过量的乙二醇双（2-氨基乙基醚）四乙酸（EGTA）终止反应。基于分子量和疏水性的差异，采用高效液相色谱法在TSK SW凝胶柱上分离结合在酪蛋白上的丹磺酰尸胺和游离的丹磺酰尸胺。该测定方法的灵敏度约为测定混合物中0.04 nmol结合在酪蛋白上的丹磺酰尸胺。使用该测定方法，通过在DEAE-纤维素和Sephacryl S-300上连续进行色谱分离对人红细胞转谷氨酰胺酶和血小板因子XIII进行纯化，这两种酶都适用这两种色谱柱，随后对红细胞转谷氨酰胺酶使用Blue Sepharose CL-6B和DEAE Bio-Gel A进行纯化，对血小板因子XIII使用苯基琼脂糖CL-4B进行纯化。红细胞转谷氨酰胺酶的纯化倍数和活性回收率分别为15300倍和22%，血小板因子XIII的纯化倍数和活性回收率分别为43.8倍和33%。
