Application of high-resolution ultrasonic spectroscopy for real-time monitoring of trypsin activity in β-casein...

High-resolution ultrasonic spectroscopy (HR-US) was applied for real-time monitoring of β-casein hydrolysis by trypsin at various conditions for the first time. The technique is based on the precision measurement of hydration changes proportional to the number of peptide bond hydrolyzed. As HR-US exhibits ultrasonic transparency for most solution, the analysis did not require optical transparency like for 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay. Appropriate enzymatic models were fitted with degree of hydrolysis (d_h) profiles to provide kinetic and mechanistic description of proteolysis in terms of initial hydrolysis rate, r⁰, and rate constant of hydrolysis, k_h, and enzyme inactivation, k_d. Maximal r⁰ and d_h were obtained at 45 °C and pH 8. The exponential dependence of kinetic parameters allowed determination of the activation (E_A = 50.3 ± 7 kJ/mol) and deactivation (E_D = 62.23 ± 3 kJ/mol) energies of hydrolysis. The ultrasonic assay provided rapid detection of trypsin activity even at sub-nanomolar concentration.