Further Studies on the Chromogenic Substrate Assay Method for Bacterial Endotoxins Using Horseshoe Crab (Tachypleus tridentatus) Hemocyte Lysate1

摘要

Basic studies on the chromogenic substrate method for the assay of bacterial endotoxins were performed usingTachypleushemocyte lysate. From the substrate specificity studies,Tachypleusclotting enzyme was found to prefer tripeptidyl-p-nitroanilide (pNA) having glycine and leucine (valine) in theP2andP3positions, and the apparentKmvalues for these peptidyl-pNA's were in the order of 10−5M. Coagulogen, a natural substrate of the clotting enzyme, competitively inhibited the amidase-catalyzed hydrolysis of Tos-Ile-Glu-Gly-Arg-pNA with aK1of 3.8×10−6M, indicating that the amidase activity is due to catalytic action of the clotting enzyme.Using the chromogenic substrate, the optimal conditions for the endotoxin-induced activation of the latent amidase inTachypleushemocyte lysate was examined. As a result, it was shown that the following points should be taken into consideration for the assaying of endotoxins; (1) the endotoxin-induced amidase activity in the lysate occurs optimally at pH 8.0 and at 40°C, (2) the induction requires Mg2+, (3) the endotoxin-induced amidase activity is unstable on prolonged incubation, (4) the sensitivity of the lysate to endotoxin is highly dependent upon its protein concentration, and (5) the latent amidase activity in the lysate is induced by a variety of endotoxin preparations with considerable differences in reactivity. In conclusion, the chromogenic substrate method using Tachypleus hemocyte lysate is valuable for the detection and quantitation of bacterial endotoxins, and under certain conditions the method is fifty-times more sensitive than theLimulusgelation