Procedure:
1. Preparation of Substrate Dilutions: Prepare at least 7-9 individual BAEE substrate concentrations in Tris-HCl buffer (pH 8.0). Ensure concentrations span below, around, and above the expected K_m. Keep solutions at 25°C.
2. Spectrophotometer Setup:
* Zero the spectrophotometer with Tris-HCl buffer (pH 8.0).
* Set the wavelength to 253 nm, as BAEE hydrolysis product (Nα-benzoyl-L-arginine) absorbs strongly at this wavelength, while BAEE does not. The change in absorbance over time (ΔA/Δt) is directly proportional to the reaction velocity.
* Set the temperature of the cuvette holder to 25.0 ± 0.1°C using a circulating water bath.
3. Reaction Initiation and Data Acquisition:
* For each substrate concentration:
* Add a fixed volume (e.g., 950 μL) of the specific BAEE substrate solution into a cuvette.
* Place the cuvette in the spectrophotometer and allow it to equilibrate to 25°C (approx. 5 minutes).
* Initiate the reaction by rapidly adding a small, precise volume (e.g., 50 μL) of trypsin solution, mixing gently but thoroughly. The final trypsin concentration should be in the nM range, ensuring [E]₀ << [S].
* Immediately record the increase in absorbance at 253 nm over time for 2-5 minutes. Ensure the initial velocity (v₀) is measured within the linear range of the reaction (i.e., less than 10-15% of substrate conversion).
4. Blank Reactions: Run control reactions for each substrate concentration with buffer instead of enzyme to correct for any non-enzymatic hydrolysis or instrumental drift. Record absorbance change.
5. Data Analysis:
* Calculate Initial Velocity (v₀): For each substrate concentration, determine v₀ from the slope of the initial linear portion of the absorbance vs. time plot (ΔA/Δt).
* Convert ΔA/Δt to [Product]/time: Use Beer-Lambert Law: A = εbc. The molar extinction coefficient (ε) for Nα-benzoyl-DL-arginine at 253 nm is 1150 M⁻¹cm⁻¹. v₀ (μM·s⁻¹) = (ΔA/Δt) / (ε × b), where b is path length (1 cm). This gives product formation rate.
* Plot Michaelis-Menten Curve: Plot v₀ against [S].
* Linearized Plots (for parameter estimation):
* Lineweaver-Burk Plot: Plot 1/v₀ vs. 1/[S]. Y-intercept = 1/V_max, X-intercept = -1/K_m.
* Equation: 1/v₀ = (K_m/V_max)(1/[S]) + 1/V_max
* Hanes-Woolf Plot: Plot [S]/v₀ vs. [S]. Y-intercept = K_m/V_max, Slope = 1/V_max.
* Equation: [S]/v₀ = (1/V_max)[S] + K_m/V_max
* Eadie-Hofstee Plot: Plot v₀ vs. v₀/[S]. Slope = -K_m, Y-intercept = V_max.
* Equation: v₀ = -K_m(v₀/[S]) + V_max
* Non-linear Regression: Use software (e.g., OriginLab, GraphPad Prism) to fit the Michaelis-Menten equation directly to the v₀ vs. [S] data for more accurate K_m and V_max determination.