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.
CONDITIONS: T = 40 °C, pH = 6.0, Excitation = 348 nm, Emission = A440nm, Light path = 1 cm
METHOD: Fluorometric Rate Determination
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.
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:
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.