Phenylalanyl-2-Sulfanilylglycine as Substrate for Leucine Aminopeptidase Assay

A chromogenic mimic of phenlyalanyl-dipeptide, L-phenylalanyl-L-2-sulfanilylglycine (PSG), was synthesized and examined for its usability in leucine aminopeptidase (LAP) assay. The enzyme activity was easily determined by measuring the amount of diazotized adduct of sulfanilic acid released upon hydrolysis of PSG ($\varepsilon^{420}$=18,000/M/cm). Under the experimental conditions employed, PSG showed a Km of 0.063 mM and a Kcat of 1683/min, assessable less than 0.1 $\mu$ g of LAP per milliliter. And the presence of aminopeptidase M (APM) was suggested to be negligible in LAP assay. This novel assay can circumvent the occasional yellow background in biological systems, i.e., serums, etc..