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:

	Blank	Test
L-Cys Reagent	0.75	0.75
Brij 35 Reagent	0.90	1.00
Enzyme Reagent	0.10	----

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:

	Blank	Test
Arg-Arg-7-AMC Reagent	0.75	0.75

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:

	STD1	STD2	STD3	STD4	STD5	STD BLANK
L-Cys Reagent	0.75	0.75	0.75	0.75	0.75	0.75
Brij 35 Reagent	1.55	1.35	1.15	0.95	0.75	1.75
Standard Reagent	0.20	0.40	0.60	0.80	1.00	----

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.

References

1. Barret AJ, Kirschke H. 1981. Methods in Enzymology 80.535-538.

2. Cathepsin B from human placenta, Product Information, C0150.