Commercially available synthetic substrates for the fibrinolytic enzymes plasmin and urokinase yield 4-nitroaniline during hydrolysis. A method is described for intensifying the color of this chromogen produced during enzyme catalyzed amidolysis of these synthetic chromogenic substrates. The liberated chromogen is diazotized and coupled with N-(1-Naphthyl)-ethylenediamine dihydrochloride to yield a colored complex with absorption maximum at 545 nm and a molar absorbancy of 4.54 × 104. This contrasts with an absorption maximum of 380 nm and a molar absorbancy at 405 nm of only 9850 for the 4-nitroaniline. When combined with a modification of the assay procedure a substantial increase in sensitivity can be obtained. This method is applicable to all synthetic substrates which use 4-nitroaniline as chromogen when amplification of the color yield is desired.
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