Effect of Temperature on Activity of Salivary Amylase
Purpose:
To determine the effect of varying temperatures on the activity of salivary amylase.
Materials:
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5 Erlenmeyer flasks
A water bath for each temperature: 5, 15, 30, 45, and 70 degrees Celsius.
Test tube rack
Five rows of 10 to 12 colorimeter tubes
Thermometer
Starch solution, Enzyme solution
Iodine
Colorimeter
Procedure:
1. Into each of five Erlenmeyer flasks (125ml), pour 50ml of the starch solution. Immerse these
containers in large beakers of water or water baths adjusted to temperatures of 5, 15, 30, 45, and
70 degrees Celsius. This may be accomplished by adding ice water or hot water to the beakers.
The temperatures in the beakers should be maintained throughout the experiment and should not
vary more than ±3℃.
2. While waiting for the temperature to equilibrate in the beakers, prepare a test-tube rack
containing five rows of 10 or 12 colorimeter tubes each. Label the first tube in each row to
correspond to the five temperature values used.
3. When the starch solution in the flasks has reached the temperature of the hot water bath (verify
this by placing a thermometer in the starch solution), add 1 ml of enzyme solution to each flask,
starting with that at 5℃. Note the time you add the enzyme to each flask. Mix each flask
thoroughly as soon as the enzyme has been added, and put it back in the water bath.
4. Two minutes after the addition of the enzyme, quickly transfer 3ml of the contents of each flask
to the corresponding first colorimeter tube in each series. Immediately test for the presence of
starch by adding three drops of iodine to each colorimeter tube. Mix thoroughly. (At the 70℃
temperature, cool the tube with tap water before adding iodine.) Repeat this procedure at 2minute intervals for the remaining tubes of each series. Be prepared to test for starch longer or
shorter, intervals if necessary.
5. Record time and plot data.
Results:
As the temperature increased the solution turned darker color. It was a red-brown color at 45℃. At that
temperature there was some enzyme activity but the higher temperatures hardly had any activity.
Conclusion:
As temperature increases, the % T decreases and the absorbance increases. At the higher temperatures
(around 70℃) the enzyme denatures and the activity stops.
Effect of Substrate Concentration on Activity of Salivary Amylase
Purpose:
To determine the effect of altering the amount of substrate (starch) available to the enzyme (salivary
amylase).
Materials:
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Test tube rack
Four rows of 10 of colorimeter tubes (or could be 2 rows of 4-what they used in experiment)
Four Erlenmeyer flasks (125 ml)
Distilled water
Starch solution
Stock enzyme solution
Iodine
1-ml pipet
Calorimeter
Procedure:
1. Prepare a test tube rack containing four rows of 10 colorimeter tubes each. Label the first tube in
each row 1:2, 1:4, 1:8, and 1:16, corresponding to their respective dilutions of starch.
2. Label four Erlenmeyer flasks (125ml) to correspond to the dilutions indicated, and add 50ml of
distilled water to each flask. To the first flask, add 50ml of starch solution. Mix thoroughly, and
transfer 50ml of this mixture to the second flask. Mix thoroughly, and transfer 50ml of mixture to
the third flask. Mix thoroughly and transfer 50ml of the mixture to the last flask. Mix thoroughly.
Remove and discard 50ml of the last mixture.
3. Using a 1-ml pipet, add 0.2ml of the stock enzyme solution to each flask.
4. Two minutes after adding the enzyme, transfer 3ml of the contents of each flask to the
corresponding colorimeter tube. Add three drops of iodine to the tubes to determine the presence
of starch. Mix thoroughly and then place each tube in the colorimeter. Determine the percent
transmittance, and record this value. Repeat this procedure at 2 minute intervals for each flask in
the series. (for a reading of 90%T or higher will indicate that hydrolysis is complete.) Plot your
data. Interpret results.
Results:
The tubes with the most substrate and fewer enzymes did were darker in color and took longer to change
to iodine color. The tubes with the lower concentration of substrate and the higher concentration of
enzymes turned iodine color much faster.
Conclusion:
As the amount of substrate (starch) concentration decreases, the % Transmittance increases, and the
absorbance decreases. As the amount of substrate concentration increases the % T decreases and the
absorbance increases.
Effect of Enzyme Concentration on Activity of Salivary Amylase
Purpose:
To determine the effect of altering the amount of enzymes available to a substrate.
Materials:
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Test tube rack
4 rows of 10 colorimeter tubes
4 Erlenmeyer flasks (125 ml)
Distilled water, enzyme solution, starch solution
Iodine
Colorimeter
Procedure:
1. Prepare a test tube rack containing four rows of 10 colorimeter tubes each. Label the first tube in
each row 1:5, 1:25, 1:125, and 1:625 to correspond to the enzyme dilutions that will be used in
this experiment.
2. Next, set up four Erlenmeyer flasks (125ml) each containing 20ml of distilled water. Label each
flask to correspond to the dilution series. Add 5ml of the enzyme solution to the first flask. Mix
thoroughly, and transfer 5ml to the second flask. Repeat this procedure for the third and fourth
flask.
3. Next add 25ml of starch solution to each flask, starting with the first flask. Note the time.
4. Two minutes after adding the starch, remove 3ml of solution from each flask, transfer it to the
first colorimeter tube in each series, and immediately test for starch by adding three drops of
iodine to each tube. Determine the %T for each tube and record data.
5. Repeat this procedure at 2 minute intervals for remaining tubes in each series.
6. Plot data and interpret results.
Results:
The tubes with the most enzyme concentration turned iodine color faster than the tubes with less
concentration of enzymes. The tubes with the smaller concentration of enzymes remained a blue black
color longer. The longer it sits the more it breaks down into maltose.
Conclusion:
As the enzyme concentration increases, the %T increases, and the absorbance decreases. As enzyme
concentration decreases, the %T decreases, and the absorbance increases.
Effect of pH on Activity of Salivary Amylase
Purpose:
To test the effect of decreasing pH on salivary amylase.
Materials:
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Test tube rack
4 rows of 10 colorimeter tubes
Pipet
Buffer solutions: pH of 4.5,7,8,10 OR 2,4,7,10
Enzyme solution, Starch solution
Iodine
Procedure:
1. Prepare a test tube rack containing 4 rows of 10 colorimeter tubes each. Label the first tube in
each row to correspond to pH values of 4.5, 7, 8, and 10, respectively. To each of four
Erlenmeyer flasks (125ml), add 25ml of buffer solutions at a pH of 4.5, 7, 8, and 10, respectively.
Into each flask, pipet 0.5ml of enzyme solution. Mix thoroughly by swirling each flask.
Beginning with the tube of lowest pH, add 25ml of starch solution to each flask. Note the time.
Thoroughly mix the contents immediately after adding the starch.
2. Two minutes after adding the starch to the first flask, quickly transfer 3ml from each flask to the
first tube in each corresponding series. Test for the presence of starch, using three drops of iodine.
Record colorimeter readings as quickly as possible after the addition of iodine.
3. Repeat this procedure at 2 minute intervals for the remaining test tubes in each series.
4. Record these times and plot data.
Results:
At the lower pH the blue black color remained and did not turn iodine color. The more normal pH of 7 did
have some enzyme activity as the color was more iodine color.
Conclusion:
As the pH decreases the concentration of starch is not broken down by the enzymes (salivary amylase) as
they are denatured and the %T decreases, and the absorbance increases. Amylase can break down starch
at the normal pH level in your mouth.
RNA Detection by Orcinol Reaction
Comments:
The strong acids in the reagent hydrolyze the purine nucleotides to sugars, bases, and phosphoric acid to
produce color.
Purpose:
To quantitatively determine the concentration of RNA by measuring the green color that is formed when
ribose reacts with Bial’s orcinol reagent.
Materials:
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Test tube rack with 6 test tubes
1mg of commercially prepared yeast RNA
0.1 HCl
Distilled Water
Acid-orcinol reagent
Alcohol-orcinol reagent
Hot water/Ice Water Baths
Colorimeter tubes
Procedure:
1. Prepare a test tube rack containing six test tubes numbered 1-6. Tubes 1-4 will be used to prepare
a standard RNA concentration curve, Tube 5 will serve as a “blank,” and Tube 6 will contain the
extracted RNA solution of unknown concentration.
2. Dissolve 1 mg of commercially prepared yeast RNA in 6ml of distilled water. The dissolution of
RNA can be aided by adding several drops of 0.1 N HCl. This will be your stock RNA solution
containing 0.166mg of RNA per milliliter.
3. Pipet 3ml of distilled water into Tubes 2, 3, 4, and 5. Pipet 3ml of the stock RNA solution into
Tubes 1 and 2. Mix the contents of Tube 2 thoroughly and then pipet 3ml of this solution into
Tube 3. Mix the contents thoroughly and transfer 3ml to Tube 4. Mix the contents and discard
3ml of solution from Tube 4. Into Tube 6, pipet 3ml of your extracted RNA preparation.
4. Pipet 6ml of the acid-orcinol reagent and 0.4 ml of the alcohol-orcinol reagent into each of the six
test tubes. Place all tubes in a boiling water bath for 20 minutes. Then cool all of the tubes by
immersing them in an ice bath.
5. Determine the absorbance (A) for each of the tubes at 660nm following the procedure used for
the DNA measurements. Record data. Prepare standard RNA curve and determine concentration
of RNA in the spleen extraction.
Results:
With the heavier concentration of RNA a darker green color was present.
Conclusion:
As the concentration of RNA increases the %T decreases and the absorbance increases.
DNA Detection by Dische Diphenylamine Reaction
Comments:
 In the presence of acid, deoxyribose is converted into a molecule which then binds to
diphenylamine to form a complex that turns a blue color. The intensity of the color is proportional
to the concentration of the dissolved nucleic acid.
Purpose:
 To quantitatively determine the concentration of DNA by measuring the blue color that is formed
when deoxyribose reacts with Dische diphenylamine reagent.
Materials:
 Test tube rack with 6 test tubes
 Commercially prepared DNA
 Distilled water
 Dische Diphenylamine Reagent
 Hot Water bath/Ice Water Bath (500ml Beaker)
 Colorimeter
Procedure:
1. Prepare a test tube rack containing six test tubes numbered 1-6. Tubes 1-4 will be used to prepare
a standard DNA concentration curve, Tube 5 will serve as a “blank” control, and Tube 6 will
contain the extracted DNA solution of unknown concentration.
2. Dissolve exactly 5mg of commercially prepared DNA in 5ml of distilled water. This will be the
stock DNA solution containing 1mg of DNA per milliliter.
3. Pipet 2ml of the stock DNA solution into Tubes 1 and 2. Pipet 2ml of distilled water into Tubes 2,
3, 4, and 5. Mix the contents of Tube 2 thoroughly and then transfer 2ml to Tube 3. Thoroughly
mix the contents of Tube 3 and transfer 2ml to Tube 4. Mix well and then discard 2ml from Tube
4. Pipet 2ml of your unknown DNA solution in Tube 6. All six tubes should now contain 2ml of
solutions.
4. Pipet 4ml of the Dische diphenylamine reagent into each of the six test tubes and mix thoroughly.
Place all tubes in a boiling water bath for 10 minutes. While these tubes are being heated, prepare
an ice bath by placing crushed ice in a 500ml beaker and adding water until the beaker is about
two thirds full. After heating for 10 minutes, transfer the six test tubes to the ice bath and agitate
them gently for 5 minutes to cool the contents rapidly.
5. Turn on the colorimeter and allow 5 min. for the instrument to warm up. Check to make sure the
sample holder is empty. Adjust the dial so that it reads 0% T at a wavelength of 500nm. Place the
“blank” tube (Tube 5) in the tube holder, close the cover, and adjust the light control until the dial
reads 100% T. Remove the tube, then determine the %T of tubes 1,2,3, and 4. Convert to
absorbance. Record data.
6. Prepare standard DNA curve (plot absorbance). Determine concentrations of DNA in each tube.
Results:
 The darker blue color was in the tubes where the DNA concentration was the highest and lighter
blue in the tubes where concentration was low.
Conclusion:
 As the concentration of DNA increases, the absorbance increases and the %T decreases. As the
concentration of DNA decreases the absorbance decreases and the %T increases.
Enzyme Cytochemistry
Purpose:
To identify the sites of respiration, starch storage, and the peroxidase in the germinating corn seed.
Materials:
 Corn seed (germinated for 1-2 days before lab)
 Tetrazolium
 Potassium Iodide
 Peroxidase Substrate solution (hydrogen peroxide, chloronapthol, and Tris Buffer)
 Large Transfer Pipets
 Reaction Plates
 Razor blades, hand lens, distilled water, forceps or tooth picks, a water bath at 37 degrees
Procedure:
1. Prepare the germinating seed: 1-2 days before lab by soaking 5-6 seeds in a petri dish with paper
towel and 5ml of water
2. Obtain reaction plate and label 4 wells 1-4 with a pen.
3. Use a large transfer pipet, place 1ml of the following into the four well:
a. 1-Water, 2-iodine, 3-Tetrazolium solution, 4-Peroxidase solution
4. Use a razor blade, cut 4 seeds in half as described above, and rinse the seed halves in water.
5. Place two halves in the solutions in each well, cut sides down. The seeds should be transferred
with forceps or by piercing them with toothpicks.
6. After 3 minutes remove seeds from well 1, rinse, and note stain areas.
7. After 20-30 minutes, use forceps to remove seeds from well 2, 3, and 4. Note the placement of the
color stains.
Results:
In well 1 there was no reaction with the water. In well 2 there was a black stain for the starch. In well 3
with the Tetrazolium there was a pinkish-red stain, and in well 4 with the peroxidase there was a purple
stain.
Conclusion:
The iodine stained the place where the starch is stored. The tetrazolium stained where the cellular
respiration occurred (pinkish-red). The peroxidase reacted as the enzyme to produce a purple stain
(stained the aleurone, lower activity of embryo).