PRACTICAL 3

Digestive enzymes, specificity and pH: Methods

3.3 Experiment A: Determination of the pH optimum of trypsin

Reagents:

1 mM p-nitroaniline (p-NA)

2 mM N-a-benzoyl-DL-arginine p-nitroanilide (BAPNA)

0.1 mg/ml trypsin (in 20 mM CaCl₂) *KEEP THIS STOCK ON ICE*

0.2 M various assay buffers, pH range 1 to 10

Procedure:

First, it is necessary to prepare a calibration curve. Label six test tubes with a magic marker then, using the appropriate automatic pipette, pipette either 2.0 ml, 1.6 ml, 1.2 ml, 0.8 ml, 0.4 ml or 0 ml of the yellow 1 mM p-nitroaniline (p-NA) standard into each of the tubes in a test tube rack. In each case make up the total volume to 2.0 ml with the appropriate amount of water to give six calibration standards. Check that all tubes have the same final volume by eye: if not, you've made a pipetting error. Calculate the p-NA concentration (mM) AND the number of micromoles (µmol) in each tube then measure the absorbance of each in the colorimeter with the 430 nm filter using a water blank (see Appendix for instructions). Complete the table below then plot absorbance (y-axis) against the number of µmol p-NA (x-axis), labelling the graph appropriately.

Volume (ml)	2.0	1.6	1.2	0.8	0.4	0
Concentration (mM)
No. of µmol per tube
Absorbance

(11 tubes in all). Prepare 10 labelled assay tubes as follows: Pipette 1.0 ml of each of the 10 assay buffers (from pH 1 to 10) into 10 separate test tubes then add 0.9 ml of the enzyme substrate, 2 mM BAPNA to each and mix well. To an 11^th control tube, add 1.1 ml water and 0.9 ml 2 mM BAPNA. Then, quickly but carefully, add 0.1 ml trypsin to each of tubes 1 to 10 (but NOT tube 11), and mix well. Note that thorough mixing of the contents of the tubes is essential. Again, check that all tubes appear to have the same final volume.. Finally, place all 11 tubes in the 37 degree water bath for 15 min.

After 15 min incubation, measure the absorbance of each of the 10 assay tubes at 430 nm using the control tube as a blank. Do this quickly but carefully so that all tubes will have been incubated for about the same time – 15 min. Using the calibration curve, convert the absorbance values into µmol p-NA and complete the table below. Plot enzyme activity (µmol p-NA produced per min, y-axis) against pH (x-axis).

pH	1	2	3	4	5	6	7	8	9	10
Absorbance
µmol p-NA/min

3.4 Experiment B: Determination of the pH optimum of chymotrypsin
Reagents:
2 mM N-succinyl-L-phenylalanine p-nitroanilide (NSLPN)

5 mg/ml a-chymotrypsin (in 20 mM CaCl₂) *KEEP THIS STOCK ON ICE*

0.2 M various assay buffers, pH range 1 to 10

Procedure:

Note: The same calibration curve can be used for this experiment so you don't need to prepare another one.

Repeat the procedure exactly as described for trypsin substituting NSLPN as substrate and chymotrypsin as enzyme. Calculate the data as before and plot on the SAME piece of graph paper as the trypsin curve using a different symbol for the chymotrypsin points.

pH	1	2	3	4	5	6	7	8	9	10
Absorbance
µmol p-NA/min

3.5 Experiment C: Determination of the substrate specificity of trypsin and chymotrypsin
Reagents:
As above

Procedure:

Prepare a set of four labelled tubes containing all combinations of 0.1 ml trypsin or chymotrypsin with 0.9 ml BAPNA or NSLPN according to the table below. Use 1.0 ml pH 8 buffer in all four tubes. Add the enzyme LAST, mix well, then incubate at 37 degrees for 15 min. Measure the absorbance at 430 nm using a water blank, calculate the amount of product in each case and enter the values into the table.

	Trypsin	Chymo-trypsin	BAPNA	NSLPN	Absorbance	µmol product/min
1	+	-	+	-
2	+	-	-	+
3	-	+	+	-
4	-	+	-	+

3.6 Experiment D: Determination of pH optimum of pepsin
Reagents:
0.2 % (w/v) N,N-dimethylated haemoglobin (Hb) (in 10 mM NaCl)

1 mg/ml pepsin (in 10 mM NaCl)

0.2 M various assay buffers

stop solution (0.2 M Tris-HCl, pH 8.5)

ninhydrin reagent

Procedure: (12 tubes in all)

Prepare 10 labelled assay tubes as follows: Pipette 0.20 ml of each of the 10 assay buffers (from pH 1 to 10) into 10 separate test tubes then add 0.25 ml of the enzyme substrate, 0.2 % Hb, to each and mix well. Then, quickly but carefully, add 0.05 ml (50 µl) pepsin solution to each of the tubes, mix well, and place all tubes in the 37 degree water bath for 30 min.

Prepare two control tubes as follows: to one (tube 11), add 0.45 ml water and 0.05 ml pepsin and to the other (tube 12) add 0.25 ml water and 0.25 ml 0.2% Hb. Incubate these with the other tubes.

N.B. FROM THIS POINT ON WEAR DISPOSABLE GLOVES AS THE NINHYDRIN REAGENT WILL STAIN YOUR HANDS BRIGHT BLUE!

After 30 min, remove the tubes from the water bath and add 0.2 ml "stop solution".

Place in the 80 degree water bath for 5 min, then remove.

Next, add1.0 ml ninhydrin reagent. Mix carefully by tapping the sides of the tubes (don't spill any) then place in the 80 degree water bath for a further 10 min to develop the colour.

Remove the tubes, allow to cool, then observe the colour that has developed.

You should notice that the tubes in which pepsin is inactive contain a precipitate of undigested haemoglobin that has been denatured by heating at 80 degrees. In order to measure the absorbance of the blue colour in the colorimeter, you would need to remove this precipitate by centrifugation. Instead you will simply score the absorbance visually using the scale (+++) — strong blue, (++) — medium blue, (+) — light blue, (-) — no colour. Score the "volume" of the precipitate in the same manner as it gives a complementary indication of digestion.

pH/tube	1	2	3	4	5	6	7	8	9	10	11	12
Blue colour
Precipitate

3.7 Conclusions:

For future reference, you should note your conclusions here. Some of these can be worked out directly from the results, others will require some literature searching. Examples of what you should be thinking about are:

Over what pH range are trypsin and chymotrypsin active and does this reflect their working environment in vivo?

What is the molecular basis for the demonstrated substrate specificity of trypsin and chymotrypsin. What might the relevance of this be in vivo?

Over what pH range is pepsin active and does this reflect its working environment in vivo?

Is the digestion of protein in the stomach by pepsin directly important for protein breakdown per se or might there be some other purpose/benefit in the production of gastric amino acids and peptides?

Why do the proteases stored in the gastric mucosa and pancreas not digest these tissues?

Why do proteins aggregate and precipitate when heated in solution?

Assuming that not all the haemoglobin in the tubes where pepsin is active has been completely digested to amino acids (true), why is there no precipitate of undigested Hb in these tubes?

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