High-performance liquid chromatographic assay of transglutaminase and its application to the purification of human erythrocyte transglutaminase and platelet factor XIII

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.