Enzymatic measurements for the detection of trypsin and carboxypeptidase A inhibitory activity

doi:10.1038/nprot.2007.386

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

+4

Enzymatic measurements for the detection of trypsin and carboxypeptidase A inhibitory activity

Oscar Yanes, Josep Villanueva, Enrique Querol, Francesc Aviles

Oscar Yanes

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Josep Villanueva

Protein Center and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA.

Enrique Querol

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Francesc Aviles

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

DOI:

10.1038/nprot.2007.386

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

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.

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.

Trypsin inhibitory activity assay

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.

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.

Add 150 μl of the substrate N-benzoyl-arginine ethyl ester hydrochloride to each quartz cell (see REAGENTS).

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).

Control slopes should be 0.050 ± 0.003 units of absorbance (U.Abs.) per min.

Biological samples yielding slopes below 0.035 U.Abs. per min are taken for intensity fading MS assays.

Carboxypeptidase A inhibitory activity assay

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.

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.

Add 10 μl of the substrate N-(4-methoxyphenylazoformyl)-Phe-OH to each quartz cell (see REAGENTS).

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).

Control slopes should be 0.12 ± 0.02 U.Abs. per min.

Biological samples yielding slopes below 0.060 U.Abs. per min are taken for 'intensity fading MS' assays.

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Subject Areas

Biological techniques

Structural biology

Chemical biology

Drug discovery

Biochemistry

Computational biology and bioinformatics

Associated Publications

Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors
by Oscar Yanes, Josep Villanueva, Enrique Querol & Francesc X Aviles
Nature (04 September, 2007)

Method Article

Biological techniques

Structural biology

+4

Enzymatic measurements for the detection of trypsin and carboxypeptidase A inhibitory activity

Oscar Yanes, Josep Villanueva, Enrique Querol, Francesc Aviles

Oscar Yanes

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Josep Villanueva

Protein Center and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA.

Enrique Querol

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Francesc Aviles

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Version History

CURRENT STATUS: Posted

Version 1

Posted 04 Sep, 2007

MetricsComments: 0HTML Views: ...

Subject Areas

Biological techniques

Structural biology

Chemical biology

Drug discovery

Biochemistry

Computational biology and bioinformatics

DOI:

10.1038/nprot.2007.386

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

It is advised that the inhibitory activity assay of each fraction should be performed in triplicate for each RP-HPLC fractionation to ensure reproducibility.

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.

Trypsin inhibitory activity assay

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.

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.

Add 150 μl of the substrate N-benzoyl-arginine ethyl ester hydrochloride to each quartz cell (see REAGENTS).

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).

Control slopes should be 0.050 ± 0.003 units of absorbance (U.Abs.) per min.

Biological samples yielding slopes below 0.035 U.Abs. per min are taken for intensity fading MS assays.

Carboxypeptidase A inhibitory activity assay

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.

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.

Add 10 μl of the substrate N-(4-methoxyphenylazoformyl)-Phe-OH to each quartz cell (see REAGENTS).

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).

Control slopes should be 0.12 ± 0.02 U.Abs. per min.

Biological samples yielding slopes below 0.060 U.Abs. per min are taken for 'intensity fading MS' assays.

Associated Publications

Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors
by Oscar Yanes, Josep Villanueva, Enrique Querol & Francesc X Aviles
Nature (04 September, 2007)

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Biological techniques

Structural biology

+4

Enzymatic measurements for the detection of trypsin and carboxypeptidase A inhibitory activity

Oscar Yanes, Josep Villanueva, Enrique Querol, Francesc Aviles

Oscar Yanes

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Josep Villanueva

Protein Center and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA.

Enrique Querol

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Francesc Aviles

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

DOI:

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Introduction

Reagents

Procedure

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Version History

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Version 1

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Subject Areas

Biological techniques

Structural biology

Chemical biology

Drug discovery

Biochemistry

Computational biology and bioinformatics

Associated Publications

Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors

by Oscar Yanes, Josep Villanueva, Enrique Querol & Francesc X Aviles

Nature (04 September, 2007)

Method Article

Biological techniques

Structural biology

+4

Enzymatic measurements for the detection of trypsin and carboxypeptidase A inhibitory activity

Oscar Yanes, Josep Villanueva, Enrique Querol, Francesc Aviles

Oscar Yanes

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Josep Villanueva

Protein Center and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA.

Enrique Querol

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

Francesc Aviles

Institut de Biotecnologia i de Biomedicina and Departament de Bioqumica, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

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Declarations

Introduction

Reagents

Procedure

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