Photometric assay of antithrombin III with a chromogenic substrate

A simple assay procedure for antithrombin III is described. The synthetic product benzoyl-Phe-Val-Arg-p-nitroanilide (S-2160) is used as substrate. By thrombin p-nitroaniline is split from the tripeptide molecule. The yellowish color of this split product can be measured in a photometer. Inactivation of thrombin by antithrombin III results in inhibition of this reaction. The percentage of thrombin inhibited is in direct relation to the activity of antithrombin III. Various modifications were tested to optimate the method. Comparable results were obtained between the coagulation method of HENSEN and LOELIGER and the photometric method.