Dependence of activity of α-amylase on pH

Introduction:

Most of the enzymes are strongly pH-dependent in their activity. This will be

demonstrated on α-amylase which is present in the saliva of oral cavity and in the pancreatic

juice. It hydrolyses starch and glycogen to maltose, maltotriose, and a mixture of branched

oligosaccharides, and non-branched oligosaccharides. At pH 4 the enzyme is completely

inactivated.

Procedure:

1. Prepare a set of 7 test tubes and mark them. Pipette the buffers according to the

table:

Test tube Na2HPO4 (ml) Citric acid (ml) pH Colour

1 2.9 2.1 5.6

2 3.1 1.9 6.0

3 3.5 1.5 6.4

4 3.8 1.2 6.8

5 4.3 0.7 7.2

6 4.7 0.3 7.6

7 4.9 0.1 7.8

2. Add 1 ml of 1% starch solution (not containing buffer!) into each tube as substrate.

3. About 0.5 ml of saliva dilute with 6 ml of water (not buffer). Add 0.5 ml of diluted

enzyme solution to each tube and leave in water bath (37°C) for 10 minutes.

4. After 10 minutes of incubation pour approximately half of the reaction mixture into

another set of tubes and the remaining half leave on the water bath for another 5

minutes.

5. To each of the samples taken of the incubation add 5 ml of water and 1 drop of

iodine solution (Lugol). Register the colour that develops.

6. After the second incubation is completed take the samples of the bath, add again 5

ml of water to each tube and again 1 drop of iodine. Register the colour again.

7. Evaluate the results:

What pH is the most suitable for α-amylase?

Enzyme specificity

Introduction:

Most of the enzymes are highly specific for the reaction to be catalyzed and for the

substrate to be selected. This property will be demonstrated in reaction with adequate

substrate. We shall demonstrate also the thermolability of enzymes. We shall use α-amylase

from saliva and sucrase of baker´s yeast (Saccharomyces cerevisiae). Sucrase hydrolyses β-

glycosidic bond of sucrose to glucose and fructose. This enzyme is different from sucrose in

the intestinal juice of mammals which hydrolyses α-1 glycosidic bond.

Procedure:

1. Take approximately 0.5 g of yeast and suspend in 2 ml of distilled water. This is the

sucrase solution.

2. Take diluted saliva from previous experiment as amylase solution.

3. Prepare a set of 6 test tubes and pipette according to the table. Perform the

described experiment:

Test tube number 1 2 3 4 5 6

Amylase (ml) 0.5 0.5 0.5

Sucrase (ml) 0.5 0.5 0.5

Boil (ml) yes yes

Starch (ml) 2.0 2.0 2.0

Sucrose (ml) 2.0 2.0 2.0

Place on the water bath (37°C) for 30 minutes. Then divide the content of each tube into two

parts. The first portion will be examined with Fehling reagent (mix 2 ml of Fehling I + 2 ml of

Fehling II, add 0.5 ml of this solution to each tube and boil it) and second with iodine (1 drop

of iodine solution + 5 ml of water).

Recorde the result:

With Fehling r.

With iodine

4. Evaluate the result in each test tube and make conclusions in your protocol:

Tube

No

Explanation

1

2

3

4

5

6

Estimation of alpha-amylase in urine by Wohlgemuth´s test

Procedure:

1. Into a series of ten test tubes pipette 1 ml of 0.9% NaCl with the exception of the

first test tube.

2. Into tube No 1 and 2 place 1 ml of urine.

3. Mix the content of tube 2 and take over 1 ml to the third test tube. Mix, measure

off again 1 ml and in this way continue the dilution of urine in the following test

tube. The last 1 ml volume from tube No 10 should be discarded.

4. To each test tube add 2 ml of the buffered 0.1% starch solution. Allow incubate in

a water bath at 45°C for 15 min.

5. Cool test tubes and add into each tube 3 drops of iodine solution. Examine the

coloration of the content. Record the number of the last test tube which gives no

blue colour with iodine.

6. Calculate the activity in Wohlgemuth units corresponding the volume (in ml) of

0.1% starch digested by 1 ml of urine under conditions described.

For example: if ¼ ml of urine (dilution 4x) digested 2.0 ml of starch, the enzymic

activity equals 2 x 4 = 8 U. The Wohlgemuth units correspond very roughly to

µkalt/l.

Complete urine dilution:

1x 2x

Conclusion:

Estimation of alpha-amylase in urine by the test – BIOSYSTEMS- EPS

Principle of the method:

Amylase catalyzes the hydrolysis of synthetic substrate (4- nitrophenyl-maltoheptaoside-

ethylidene) to smaller oligosacharides which are hydrolyzed by α-amylase liberating 4- nitrophenol.

The amount of arising 4-nitrophenol correspond with the catalytic concentration of alpha-amylase.

Procedure:

1. Switch on photometer ECOM E 6125 and the printer. Deprres the key ABSORBANCE and

with arrow keys set the wavelength to 405 nm. Rinse several times the cuvette in phofometr

with distilled water. The cuvette is emptied with the pump.(Ask for the demonstration how

to use it.)

2. Stop the pump and fill the cuvette with distilled water as blank. Check the display of the

photometr. It should show ABSORBANCE 405 nm BLANK. If not, used the key BLANK to

obtain this parameter. Depress the key ENTER- MEASURE, the absorbance on the display

should be 0,000.

3. Sample measurement: Mix into Eppendorf tube - 20µl of urine and 1000µl of the “Reagent

solution “, immediately transfer into cuvette and start the stop-watch. Measure the

absorbance of sample exactly after 2,3,4 and 5 minutes. The measurement is achieved by

depressing the key ENTER-MEASURE. The result of measurement is always printed out.

4. Rinse several times the cuvette with distilled water and switch of the pump, photometer and

printer.

5. From the values obtained, calculate the average ΔA / min. and substituting in formula

concentration α- amylase in the urine sample.

Calculation: Urine measurements at 37⁰C (reaction temperature)

∆A1= A2 – A1

∆A2= A3 – A2

∆A3= A4 – A3

mathematical average ∆A /min = ∆A1+ ∆A2+ ∆A3 / 3

µkat/l = ∆A /min x 105,9 (conversion factor)

U/L = ∆A /min x 6384 (conversion factor)

Benchmarks at 37⁰

µkat/l = 0,26-8,15 U/L = 16 - 491