Tcc 240a

The enzymatic activity of rt-PA and t-ELIP were measured using a spectrophotometric method [7 (link), 23 (link)]. Thawed rt-PA or reconstituted t-ELIP were diluted into a solution of 0.5 % BSA and 1 % Triton-X (Sigma-Aldrich, St. Louis, MO, USA) to achieve concentrations of rt-PA between 0.3 and 3 µg/mL in disposable cuvettes used for spectrophotometric measurement. The amount of Triton-X employed in the solution exceeded the critical micelle concentration (0.015 %) (Triton-X Product Information Sheet, Sigma- Aldrich, St. Louis, MO, USA) to ensure rupture of the lipid shell surrounding t-ELIP and release of the associated rt- PA. The solution was aspirated to remove echogenic microbubbles [24 (link)], and diluted into a pre-warmed solution (37 °C) of 0.5 % phosphate buffer solution and a chromogenic substrate (S-2288, Chromogenix, DiaPharma Group, Inc., Westchester, OH, USA). The chromogenic substrate is hydrolyzed by rt-PA and allows spectrophotometrical measurement of the change of absorbance at 405 nm over time, which is proportional to the enzymatic activity of rt-PA. A spectrophotometer (UV-1700, Shimadzu, Japan) with temperature controller (TCC-240A, Shimadzu, Japan) was used to record the absorbance of the solution over the course of 5 min at 37 °C. The rt-PA activity was reported in terms of the change in absorbance over time (ΔAbs/min).

The enzymatic activity of WT MbNHase and each variant was determined using acrylonitrile as the substrate (acrylamide; Δε₂₂₅ = 2.9 mM⁻¹ cm⁻¹). The rate of nitrile hydration was determined by continuously monitoring the formation of acrylamide at 225 nm using a Shimadzu UV-2450 spectrophotometer equipped with a TCC-240A temperature controlled cell holder [19 (link)]. A typical 1 mL reaction consisted of 50 mM Tris-HCl buffer pH 7.0 at 25 °C and various concentrations of acrylonitrile (0 to 450 mM). One unit (U) of MbNHase activity is defined as the formation of 1 μmol of acrylamide per minute. To obtain the kinetic parameters, V_max and K_m, the initial velocities from at least three independent measurements were fitted to the Michaelis-Menten equation using OriginPro 9.0 (OriginLab, Northampton, MA). Kinetic data for each MbNHase variant was determined more than three times for multiple purifications, all of which provided consistent results.