Dinitrophenol derivatization of proteolytic products and its application in the assay of protease(s) activity

A spectrophotometric method based on dinitrophenol (DNP) derivatization of proteolytic products was developed for monitoring the increase in NH(2)-groups as a function of protease activity. DNP derivatization of amino acids and proteolytic products was carried out at an alkaline pH of 8.8, in presence of 2,4-dinitrofluorobenzene (DNFB), followed by the stabilization of products by adjusting the pH to approximately 2.5. Using casein as substrate, under the defined assay conditions for proteases, trichloroacetic acid soluble proteolytic products were derivatized with DNFB reagent. Though alkaline pH favored the DNP derivatization of primary amino compounds, the products formed were found to be unstable. However, upon adjusting the pH to 2.5+/-0.1, DNP derivatives of amino acids and proteolytic products were found to be stable with identical lambda(max) of 395 nm. The utility of the method was evaluated by assaying the proteolytic activities of trypsin and calcium activated neutral protease (CANP). Proteolytic activity was quantified by employing the molar extinction coefficient of DNP derivatives of an equimolar concentration of glutamate and glycine. By employing this method, CANP activity in different regions of rat brain was determined. The proposed method to monitor the increase in NH(2)-end groups as a function of proteolytic activity could be employed to assay the activity of proteases.