An alternative substrate of arginase was synthesized in which the bridging guanindium nitrogen was replaced with a sulfur. This compd. is a good substrate for arginase, resulting in urea and 2-amino-5-mercaptovaleric acid, with a kcat that was similar to and a Km that was less than half that of arginine. The thiol product reacted with 5,5'-dithiobis(2-nitrobenzoate)to yield a disulfide adduct and release 2-nitro-5-thiobenzoate as a chromophoric product. The new continuous spectrophotometric assay of arginase, which was designed and optimized based on the reaction between thiol product and DTNB, was considerably more sensitive than existing continuous assays, and was much less time consuming than the fixed-point assays of arginase that are presently being used. The significantly higher kcat with thioarginine for arginase, coupled with a 10-fold greater extinction coeff. for 2-nitro-5-benzoate as compared with m-nitroanaline, led to greater than a million-fold enhanced sensitivity for the improved spectrophotometric arginase assay. ©2001 Academic Press.