Quantitative Measurement of Mucolytic Enzymes in Fecal Samples

Materials and Reagents

1. 1.7 ml Posi-Click^TM Tubes (Denvelle, catalog number: C2170 )

2. 96-well flat bottom plate (Thermo Scientific, catalog number: 12565136 )

3. Aluminium foil (Fisher Brand, catalog number: 01-213-100 )

4. Micropipette barrier tips (from 10 μl to 1,000 μl) (Genesee Scientific)

5. 8-10 weeks-old C57Bl6/j mice

6. 4-nitrophenol (Sigma-Aldrich, catalog number: 241326-50G )

7. 4-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich, catalog number: N9376 )

8. 4-nitrophenyl α-D-galactopyranoside (Sigma-Aldrich, catalog number: N0877 )

9. 4-nitrophenyl β-D-glucopyranoside (Sigma-Aldrich, catalog number: N7006 )

10. Fresh or -80 °C stored feces pellets

11. Acetone (EM Science, catalog number: AX0120-8 )

12. DNases (Sigma-Aldrich, catalog number: 11284932001 )

13. Lysozyme (Fisher BioReagents^TM, catalog number: BP535-1 )

14. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M-0250 )

15. Methanol (Fisher Chemical, catalog number: A433P-4 )

16. p-nitrophenyl α-L-fucopyranoside (Sigma-Aldrich, catalog number: N3628 )

17. p-nitrophenyl β-D-xylopyranoside (Sigma-Aldrich, catalog number: N2132 )

18. Pierce^TM BCA protein assay kit (Thermo Scientific, catalog number: 23227 )

19. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541-500G )

20. Protease inhibitor cocktail tablet (Roche, catalog number: 26733200 )

21. Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )

22. Trizma^® hydrochloride (Sigma-Aldrich, catalog number: S8045-1KG )

23. 4-Nitrophenyl (4NP) standard curve (see Recipes)

24. Mucolytic enzyme buffer (see Recipes)

25. Nitrophenyl-linked substrates and their corresponding mucolytic enzymes (see Recipes)

Equipment

1. A pair of sterile forceps

2. -80 °C freezer (Thermo Scientific)

3. Centrifuge (Thermo Scientific, Legend Micro 21R )

4. Ice making machine (Hoshizaki American Inc.)

5. Micropipette (from 10 μl to 1,000 μl) (Labnette)

6. Multi-channel pipette (300 μl) (Fisher Brand)

7. Sonicator with 3 mm tapered microtip (Branson Digital Sonifier, Model: 102C )

8. Spectrophotometer (TECAN, SPARK 10M)

9. Vortex Genie 2 (VWR Scientific Products)

10. Weighing balance (ADAM Equipment, PW124 )

Procedure

1. Collect 2-3 fecal pellets (approximately 50 mg) from each mouse into a tube.

   Note: Feces can be stored at -80 °C until measurement.

2. Add 0.5 ml of ice-cold mucolytic enzyme buffer (Recipe 1) into the tube containing fecal pellets and gently vortex.

   Note: Vertexing is not necessary if feces are not solid.

3. Sonicate samples using ultrasonic processor for 5 s with 35% amplitude and 3 mm tapered microtip on ice. Repeat ultrasonication following a 10 s interval for a total of 9 cycles or 45 s.

4. Centrifuge sonicated samples at 10,000 × g for 10 min at 4 °C.

5. Transfer ~400 μl supernatant into fresh 1.5 ml tubes and measure protein concentration.

   Note: Supernatant can be stored at -80 °C, until measurement.

6. Adjust the protein concentration to 1 mg/ml by adding ice-cold mucolytic enzyme buffer.

7. Transfer 5 μl (5 μg protein) supernatant of a particular fecal sample into three wells (5 μl each) of a 96-well flat-bottom plate. Add 150 μl of a specific 10 mM nitrophenyl-based substrate (Recipe 2) prepared in ice-cold mucolytic enzyme buffer. For each enzyme tested, corresponding substrate is added into triplicate wells containing the same sample. These procedures are repeated when multiple samples are used.

8. Measure the absorbance at 405 nm in a plate reader at 37 °C at every 30 min interval.

9. Using a known concentration of 4-nitrophenol standard curve (Recipe 3), determine the individual mucolytic enzyme activity (Figure 1).

   
   

   
   Figure 1. Time-dependent mucolytic enzymatic activity in mouse fecal samples. Fecal samples collected from healthy mouse were homogenized and processed as described in the protocol. The activity of indicated enzymes was measured in the fecal lysate following the procedure described in the protocol.

Recipes

1. Mucolytic enzyme buffer (pH 7.25)

   Stock solution Working solution For 100 ml

   1 M Tris            50 mM 

   1 M KCl            100 mM 

   1 M MgCl₂       10 mM 

   Lysozyme                                        5-10 mg

   12% Triton X-100                           100 μl

   DNases                                            5-10 mg

   Protease inhibitor                         One tablet

2. Nitrophenyl-linked substrates and their corresponding mucolytic enzymes

   Nitrophenyl-linked substrates	Mucolytic enzymes
   4-nitrophenyl α-D-galactopyranoside	α-galactosidase	Plant glycans
   4-nitrophenyl N-acetyl-β-D-glucosaminide	β-N-acetylglucosaminidas	Mucin
   4-nitrophenyl β-D-glucopyranoside	β-glucosidase	Plant glycans
   p-nitrophenyl α-L-fucopyranoside	α-fucosidase	Mucin
   p-nitrophenyl β-D-xylopyranoside	β-xylosidase	Plant glycans

   Note: We mentioned above nitrophenyl-linked substrates, which we use in the Figure 1.

3. 4-Nitrophenol (4NP) standard curve

   4-Nitrophenol (4NP) is an enzymatic product of p-nitrophenyl-linked substrates. The amount of 4NP produced during reaction of mucolytic enzymes with its substrate corresponds to the mucolytic enzyme activity (Recipe 2). 4NP provides yellow color and can be measured spectrophotometrically at 405 nm. Thus, a standard curve of 4NP can be used to measure mucolytic enzyme activity in a reaction mixture of mucolytic enzymes and its p-nitrophenyl-linked substrates.

   1. Prepare a 100 mM 4NP (MW = 139.11) stock solution by dissolving 0.0139 g (13.9 mg) of 4NP in 1 ml of methanol.

   2. Prepare a working stock solution of 1 mM 4NP (from 100 mM 4NP) for standard curve with mucin enzyme assay buffer.

   3. Prepare a series of 4NP concentration ranging from 0 to 1,000 µM (or 0 to 1 mM) from the working stock (1 mM) prepared in mucin enzyme assay buffer.

   4. Add 150 µl of each standard solution into a 96-well plate including blank and read the absorbance with a spectrophotometer at 405 nm at 37 °C.

   5. Plot the standard curve with known standard concentration on the X-axis and absorbance on the Y-axis.

Acknowledgments

This protocol was adapted with minor modification from previous study published by Desai et al. (2016) and Khan et al. (2020). We would like to thank the UT Southwestern Animal Resource Center (ARC) for maintenance and care of our mouse colony. This work was supported by The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institute of Health (NIH) under Award Number R01DK125352, and Cancer Prevention and Research Institute of Texas (CPRIT) Individual Investigator Awards (RP200184), and UT Southwestern funding given to H.Z.

Competing interests

The authors declare no competing interests.

Ethics

This study was approved by the Institutional Animal Care and Use Committee (IACUC; approval No. 2016-101683), and was conducted in accordance with the IACUC guidelines.

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