Enzymatic Assay of Cathepsin B

Introduction

This procedure applies to all products that have a specification for Cathepsin B activity, such as product numbers C0150 and C8571, determined by the liberation of 7-amino-4-methylcoumarin from Z-Arg-Arg 7-amido-4-methylcoumarin.

Cathepsin B is a lysosomal cysteine proteinase that will hydrolyze proteins with a broad specificity for peptide bonds, but will preferentially cleave at the carboxyl side of Arg-Arg bonds in small molecule substrates. Lysosomal Cathepsin B has also been shown to degrade soluble monomeric collagen and insoluble polymeric collagen in vitro.

            Nα–CBZ–Arg–Arg–7–amido–4–methylcoumarin + H2O    Cathepsin B   > Arg–Arg + 7–AMC

The substrate Nα–CBZ–Arg–Arg–7–amido–4–methylcoumarin is used for the fluorometric detection of Cathepsin B activity. The Km value for this substrate is 0.39 mM, with an optimum pH of 6.0. The fluorescence of the free aminomethylcoumarin released.

Definitions

• Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC
• CBZ – carbobenzoxy.
• Arg-Arg – arginylarginine
• 7-AMC – 7-amino-4-methylcoumarin.
• Unit definition – one unit will liberate 1 nanomole of 7-amino-4-methylcoumarin from Z-Arg-Arg 7-amido-4-methylcoumarin per min at pH 6.0 at 40 ºC.

Procedure

CONDITIONS: T = 40 °C, pH = 6.0, Excitation = 348 nm, Emission = A_440nm, Light path = 1 cm

METHOD: Fluorometric Rate Determination

REAGENTS:

• 352 mM potassium phosphate buffer, 48 mM sodium phosphate, and 4.0 mM ethylenediaminetetraacetic Acid; pH 6.0 at 40 °C (Buffer). Prepare a solution in purified water using 47.9 mg/mL of potassium phosphate monobasic (Product No. P5379); 6.8 mg/mL of sodium phosphate dibasic, such as product number S0876; 1.7 mg/mL of Eethylenediaminetetraacetic acid (Product No. ED4SS). Adjust the pH to 6.0 at 40 °C using 1 N HCl or 1 N KOH.
• 8.0 mM L-Cysteine HCL Solution, pH 6.0 at 40 °C (L-Cys). Prepare a fresh solution in Reagent 7.3.1 (Buffer) using 1.4 mg/ml of L-Cysteine hydrochloride, such as product number C7880. Adjust to pH 6.0 at 40 °C with 1 N NaOH.
• 0.1% (v/v) Brij 35 Solution (Brij 35). Prepare a 0.1% (v/v) solution in purified water using Brij 35 Solution, 30% (w/v) solution (Product No. B4184).
• 0.02 mM Nα–CBZ–Arg–Arg–7–amido–4–methylcoumarin (Arg-Arg-7-AMC).Prepare a fresh solution in Dimethyl Sulfoxide (Product No. D5879)using 7.1 mg/mL of Nα–CBZ–Arg–Arg–7–amido–4–methylcoumarin (Product No. C5429.) Dilute to a final concentration of 0.02 mM with Reagent 7.3.3 (Brij 35) and use within 3 hours of preparation. Protect this solution from light.
• 5.0 μM 7–amino–4–methylcoumarin (Standard). Prepare a solution in dimethyl sulfoxide (Product No. D5879) using 1 mg/mL of 7–amino–4–methylcoumarin (Product No. A9891). Dilute to a final concentration of 5.0 μM with Brij 35 reagent. Protect this solution from light.
• Cathepsin B Enzyme Solution (Enzyme). Immediately before use, prepare a solution containing 5-10 units/mL of Cathepsin B in cold 0.1% (v/v) Brij 35 Solution.

PROCEDURE:

For measuring enzymatic activity, pipette (in milliliters) the following reagents into fluorometric cuvettes:

Mix by inversion and equilibrate to 40 °C. Monitor the intensity of fluorescence at the excitation wavelength of 348 nm and the emission wavelength of 440 nm until constant using a suitably thermostatted fluorometer.

Then pipette (in milliliters) the following reagents into fluorometric cuvettes:

Immediately mix by inversion and record the increase in intensity of fluorescence at the excitation wavelength of 348 nm and the emission wavelength of 440 nm for 5 minutes. Obtain the maximum Δ Intensity/min using the maximum linear rate for both the test and the blank.

For standard curve determination, pipette (in milliliters) the following reagents into fluorometric cuvettes:

Mix by inversion and equilibrate to 40 °C. Measure the fluorescence intensity at the excitation wavelength of 348 nm and the emission wavelength of 440 nm for all standards and standard blank.

CALCULATIONS:

Correct standard intensities versus the standard blank.

    Δ Intensity Standard = Intensity STD – Intensity STD blank

Obtain the linear regression of the standards by plotting the Δ Intensity Standard versus nanomoles of 7–amino–4–methylcoumarin for each standard.

Determine the nanomoles of 7–amino–4–methylcoumarin liberated using the linear regression obtained from the standard data:

where:
    DF = dilution factor
    0.100 mL = volume of enzyme used

FINAL ASSAY CONCENTRATION:

In a 2.50 mL reaction mix, the final concentrations are 105.6 mM potassium phosphate, 14.4 mM sodium phosphate, 1.2 mM ethylenediamine tetraacetic acid, 2.4 mM L-cysteine, 0.07% (v/v) Brij 35, 0.006 mM Nα–CBZ–Arg–Arg–7–amido–4–methylcoumarin, 0.0525% (v/v) dimethyl sulfoxide, and 0.2 – 0.4 units of Cathepsin B.