Dityrosine-based substrates for the selective and sensitive assay of thermolysin

Thermolysin family proteases are related to various diseases such as bacterial infections, cholera, gastritis and peptic ulcers, and gastric carcinoma. Hence, assay materials capable of the sensitive and specific assessment of thermolysin activity need to be developed for the detection of those diseases. In this study, simple peptides containing an unusual fluorescent amino acid, dityrosine, were tested for the assay of thermolysin. Various types of DBDY-(amino acid-INH)2 were synthesized from the conjugation of N,N′-diBoc-dityrosine (DBDY) with two molecules of amino acid and isoniazid (INH). Among these, DBDY-(Phe-INH)2 and DBDY-(Ile-INH)2 were found to be the most promising substrates for thermolysin assays. The hydrolysis reaction occurring between DBDY and Phe (or Ile) resulted in the release of Phe-INH and Ile-INH, and the fluorescence of DBDY was recovered. The reaction was found to follow Michaelis-Menten kinetics, and the KM and kcat/KM values were in the ranges of 1.91–3.95 μM and 2.43 × 104–6.03 × 104 M−1 s−1, respectively, which were comparable to other protease assay materials. Considering the simple preparations of DBDY-(Phe-INH)2 and DBDY-(Ile-INH)2, they are believed to be useful for the selective and sensitive assay of thermolysin.