Enzymatic Assay of Cathepsin B

1. Objective

To standardize a procedure for determining the enzymatic activity of Cathepsin B.

2. Scope

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.

3. Definitions

3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

3.2 CBZ – carbobenzoxy.

3.3 Arg-Arg – arginylarginine

3.4 7-AMC – 7-amino-4-methylcoumarin.

3.5 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.

4. Discussion

4.1 Cathepsin B is a lysosomal cysteine proteinase which 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

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

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:

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

7.2 METHOD:

7.2.1 Fluorometric Rate Determination

7.3 REAGENTS:

7.3.1 352 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, such as product number P5379; 6.8 mg/ml of Sodium Phosphate Dibasic, such as product number S0876; 1.7 mg/ml of Ethylenediaminetetraacetic Acid, such as product number ED4SS. Adjust the pH to 6.0 at 40 °C using 1N HCl or 1N KOH.

7.3.2 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 1N NaOH.

7.3.3 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, such as product number B4184.

7.3.4 0.02 mM Nα–CBZ–Arg–Arg–7–amido–4–methylcoumarin (Arg-Arg-7-AMC).
Prepare a fresh solution in Dimethyl Sulfoxide such as product number D5879 using 7.1 mg/ml of Nα–CBZ–Arg–Arg–7–amido–4–methylcoumarin, such as product number 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.

7.3.5 5.0 μM 7–amino–4–methylcoumarin (Standard).
Prepare a solution in Dimethyl Sulfoxide, such as product number D5879, using 1 mg/ml of 7–amino–4–methylcoumarin such as product number A9891. Dilute to a final concentration of 5.0 μM with Reagent 7.3.3 (Brij 35). Protect this solution from light.

7.3.6 Cathepsin B Enzyme Solution (Enzyme).
Immediately before use, prepare a solution containing 5-10 units/ml of Cathepsin B in cold Reagent 7.3.3 (Brij 35).

7.4 PROCEDURE

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

7.4.2 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.

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

7.4.4 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.

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

7.4.6 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.

7.5 CALCULATIONS

7.5.1 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.

7.5.2 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

7.6 FINAL ASSAY CONCENTRATION :

7.6.1 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.

8. References

9. Approval

Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.