A dityrosine-based substrate for a protease assay: Application for the selective assessment of papain and chymopapain activity

[Display omitted] ► N,N′-diBoc-dityrosine (DBDY) was conjugated with two isoniazid (INH) molecules. ► Due to the quenching effect of INH, DBDY–(INH)2 lost the fluorescence of DBDY. ► Only papain and chymopapain catalyzed the hydrolysis of DBDY–(INH)2. ► DBDY–(INH)2 can be used as a selective and sensitive assay of papain and chymopapain. N,N′-diBoc-dityrosine (DBDY), which was synthesized by the oxidative C–C coupling of 2 N-Boc-l-tyrosine molecules, was conjugated with two isoniazid (INH) molecules. Due to the quenching effect of INH, DBDY–(INH)2 lacks the fluorescence of DBDY. As such, it was tested for use in the detection of proteases by measuring fluorescence recovery. In this study, serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), aspartic proteases (pepsin and aspergillopepsin) and cysteine proteases (papain and chymopapain) were chosen. Reported optimum assay conditions were chosen for each enzyme. Only papain and chymopapain catalyzed the hydrolysis of DBDY–(INH)2 and led to fluorescence recovery, possibly due to their extensive binding sites and the INH-mediated inhibition of metalloproteases and aspartic proteases.