Plasmin determination based on enzymatic digestion of a β-casein layer at the air/water interface

Abstract

The possibility of enzyme activity determination based on natural substrate digestion at the air/water interface has been investigated through the interaction of a β-casein layer with the protease plasmin. The protein layers were spread from aqueous solutions of β-casein in a 0.1–0.4 g/L concentration range to the air/water interface. 200 μL plasmin was introduced into the water subphase (15 mL) in various initial concentrations (25–1000 nM), corresponding to 0.33–13.3 nM final concentration. The decrease of the surface pressure due to the enzymatic degradation of the β-casein monolayers was followed in time, and calibration curves were obtained by plotting either the total surface pressure change, or the maximum rate of the surface pressure change against the plasmin concentration. The method is suitable for label-free determination of plasmin, with assay linearity down to 0.7 nM as the final concentration of plasmin in the system, corresponding to 50 nM for the initial, undiluted plasmin sample in the given experimental setup.