Enzymatic Measurements for the Detection of Trypsin and Carboxypeptidase A Inhibitory Activity.
Lab/Group: Aviles Lab (Universitat Autònoma de Barcelona)
Related Journal & Article Information
Journal: Nature Protocols
Introduction
This method can be used to measure the inhibitory activity of a biological extract against the target protease. In the associated Nature Protocol this method was used to determine the activity of C18 HPLC fractions before assaying non-convalent protein interactions by ‘intensity fading’ MALDI-TOF mass spectrometry.
It is advised that the inhibitory activity assay of each fraction should be performed in triplicate for each RP-HPLC fractionation to ensure reproducibility.
Materials
Reagents
Trypsin gold, MS grade (Promega, cat. no. V5280): 100 μg lyophilized powder
Synthetic trypsin substrate BAEE Prepare aliquots of 1.06 mg ml-1 BAEE in 0.05 M Tris buffer, pH 8.0.
CRITICAL Store at -20 °C to avoid degradation.
Bovine carboxypeptidase A (Sigma-Aldrich, cat. no. 21940): approximately 60 units per mg protein (approximately 20 mg ml-1); alternatively, recombinant human carboxypeptidase A (hCPA) can be used
Synthetic carboxypeptidase substrate: N-(4-methoxyphenylazoformyl)-Phe-OH-potassium salt (Bachem AG, cat. no. M-2245) Prepare aliquots of 3.65 mg ml-1 in 2% DMSO.
CRITICAL Store at -20 °C to avoid degradation.
Equipment
Procedure
Trypsin inhibitory activity assay
1. Incubate in a quartz cell 10 μl of biological sample (from each re-dissolved chromatographic fraction) with 50 μl trypsin (33 μg ml-1 in 20 mM CaCl2, 0.1 M Tris, pH 8.0) and 790 μl activity buffer (20 mM CaCl2, 0.1 M Tris, pH 8.0) for 3 min at room temperature.
2. Repeat Step 1 in another quartz cell and add 10 μl activity buffer (20 mM CaCl2, 0.1 M Tris, pH 8.0) instead of biological sample in order to obtain the control sample.
3. Add 150 μl of the substrate N-benzoyl-arginine ethyl ester hydrochloride to each quartz cell (see REAGENTS).
4. Gently mix all the quartz cells and measure the absorbance change at 254 nm and 25 °C over 2 min, producing 240 time points (signal averaging time = 0.5 s).
5. Control slopes should be 0.050 ± 0.003 units of absorbance (U.Abs.) per min.
6. Biological samples yielding slopes below 0.035 U.Abs. per min are taken for intensity fading MS assays.
Carboxypeptidase A inhibitory activity assay
1. Incubate in a quartz cell 10 μl biological sample (from each re-dissolved chromatographic fraction) with 2 μl carboxypeptidase A (35 μg ml-1 in 20 mM Tri-HCl buffer, 0.5 M NaCl, pH 7.5) and 978 μl activity buffer (20 mM Tri-HCl buffer, 0.5 M NaCl, pH 7.5) for 3 min at room temperature.
2. Repeat Step 1 in another quartz cell and add 10 μl activity buffer (50 mM Tri-HCl buffer, 100 mM NaCl, pH 7.5) instead of biological sample in order to obtain the control sample.
3. Add 10 μl of the substrate N-(4-methoxyphenylazoformyl)-Phe-OH to each quartz cell (see REAGENTS).
4. Gently mix all the quartz cells and measure the absorbance change at 350 nm and 25 °C over 3 min, producing 360 time points (signal averaging time = 0.5 s).
5. Control slopes should be 0.12 ± 0.02 U.Abs. per min.
6. Biological samples yielding slopes below 0.060 U.Abs. per min are taken for 'intensity fading MS' assays.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
inhibitory activity assay, trypsin, carboxypeptidase A