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ENZYME QUANTITATIVE ANALYSIS

The purpose of this experiment is to study the enzyme amylase which is found in saliva.
Amylase breaks down starch into the simple sugar glucose and is one of the first steps in
digestion. Starch can be detected with a simple chemical stain. In the presence of starch, a
solution of potassium iodide/iodine (KI/iodine) will turn blue/black in color. This is a
quantitative reaction – the darker the blue/black color, the more starch is present. As amylase
digests the starch, the intensity of the blue/black solution of KI/iodine decreases. We will
quantify the reaction using a spectrophotometer.
Amylase can also be studied by looking at the simple sugars generated as starch breaks
down. Reducing sugars such as glucose can be detected using Benedict's reagent, a solution of
copper (II) sulfate, sodium carbonate, and sodium citrate. Benedict's reagent turns from a
soluble blue color into a brick red precipitate. It is also a quantitative reaction, and we will use a
desktop scanner and a free imaging program (ImageJ) developed by the National Institutes of
Health to measure the intensity of the signal in the red and blue channels. We will then use a
standard curve (similar to the curve for starch) to calculate the amount of reducing sugar present
as amylase digests starch.

This lab has four parts:

Part I: Making a standard curve using known amounts of starch and KI/iodine solution.
Part II: Determining the activity of amylase present in a given sample of saliva using KI/iodine
reagent.
Part III: Using Benedict’s reagent to detect the amount of glucose in a given sample.
Part IV: Determining the activity of amylase present in a given sample of saliva using Benedict’s
reagent

Materials and Reagents


The iodine reagent is made by dissolving 6.0 g of KI in 100 mL of 2% tincture of iodine.
Benedict's reagent is made by dissolving 30.0 g of Cu(II) sulfate in 60 mL of hot water (may
require extensive heating to fully dissolve), and in a second flask dissolving 20.0 g of sodium
carbonate and 34.6 g of sodium citrate in 120 mL of hot water. While still warm, these two
solutions are mixed slowly as some bubbling will occur, and then diluted to 200 mL with
distilled water. Starch solution is made by dissolving 0.5g soluble starch in 100 mL of cold
water (0.5% starch solution) followed by gentle boiling. A 0.1% starch solution should be made
by diluting the 0.5% stock into distilled water. A 0.5% dextrose (ie. glucose) solution is made by
dissolving 0.5g dextrose in 100 mL distilled water. The concentrations of these reagents are
important, as volumes are chosen to optimize spectrophotometry readings and to avoid saturating
conditions.

Hazards
Iodine is often obtained as a solution in alcohol, which is flammable. Iodine also stains
clothing and skin, so special care should be taken with this reagent. The Benedict's reagent assay
requires that samples be boiled for at least 5 minutes. Tubes should be boiled in a slightly
bubbling water bath and appropriate precautions against burns from the boiling water must be
taken. Do not heat test tubes directly over a flame. Benedict's reagent should NOT be mixed
with acids.
Part I- Starch Standard Curve
Materials Needed:
Spectronic 20 spectrophotometer 5 spectrophotometer test tubes Dropper
KI/iodine reagent Regular test tube Tissue paper
0.5 mg/mL (0.05%)starch solution Deionized water Pipettes
Test tube rack PBS

Procedure:

1) Turn on the spectrophotometer and allow to warm up for at least 15 minutes. Set the
wavelength on the spectrophotometer to 620 nm.

2) Make sure all spectrophotometer test tubes are clean and dry. Label each test tube 1-5,
then insert into test tube rack

3) Obtain KI/iodine reagent (pipette into regular test tube), PBS, and starch solution.

4) Each test tube will contain 3 mL total volume to enable them to be read by the
spectrophotometer. Fill Tube 1 with 3 mL distilled water – it will be the “blank.” Fill
each subsequent tube according to the following chart:
Starch water Starch Concentration Absorbance
1) 0 mL 3.0 mL
2) 0.1 mL 2.9 mL
3) 0.3 mL 2.7 mL
4) 0.6 mL 2.4 mL
5) 0.9 mL 2.1 mL

5) Add one drop of KI/iodine reagent to each tube and mix completely. The iodine solution
will tend to settle at the bottom of the tubes and must be evenly distributed.

6) Adjust the left knob on the Spectrophotometer to zero. Clean the outside of Tube 1 (the
“blank”) with tissue paper to remove finger prints. Insert into spectrophotometer and
adjust the transmittance control knob (right knob) to 100 percent. Record Tube 1’s
Absorbance as zero.

7) Insert each Tube 2 – 5, (making sure to clean the outside of each tube) into the
spectrophotometer, read and record the Absorbance for each on the chart under Step 4.

Calculations:

1) Plot on a graph Absorbance for each tube versus the Concentration of starch in each tube.
(Absorbance versus Concentration) This is the Standard Curve.

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2) Using excel, plot a trendline using the obtained data points and calculate the linear regression.
Part II – Amylase in Saliva

This part of the experiment will determine the activity of amylase present in a given
sample of saliva. It will be necessary to take time points of the reaction. Absorbance will need to
be measured and recorded at each given time point. KI/iodine solution should be made fresh!

Materials Needed:

Spectronic 20 spectrophotometer 8 spectrophotometer test tubes Dropper


4 large test tubes fresh KI/iodine reagent (6g, 2g I2 per 100ml)
0.5 mg/mL (0.05%) starch solution Deionized water Pipettes
Test tube rack(s) Tissue paper Stop watch

Procedure:

1) Turn on the spectrophotometer and allow to warm up for at least 15 minutes. Set the
wavelength on the spectrophotometer to 620 nm. Adjust the left knob on the
spectrophotometer to zero. Clean the outside of a “blank” test tube (filled with deionized
water) with tissue paper to remove finger prints. Insert into spectrophotometer and adjust
the transmittance control knob (right knob) to 100 percent. Remove blank and set aside.

2) Ensure that all test tubes are clean and dry. Label the spectrophotometer test tubes 1-7
and insert them into test tube rack

3) Obtain KI/iodine reagent (pipette into regular test tube), deionized water, and starch
solution.

4) From a volunteer, collect at least 1 mL of saliva in a large test tube.

5) Pipette 1 mL of the saliva into a second large test tube. Add 9 mL distilled water to the 1
mL saliva sample, mix well. This is the 1:10 saliva dilution. Make sure to label your test
tubes!

6) Take 1 mL of the 1:10 saliva dilution and pipette into a fresh tube and add 9 mL of
distilled water to make a 1:100 saliva dilution.

5) Remove 1 mL of the 1:100 saliva dilution and pipette into the 4th large test tube.

6) Fill each of the 7 spectrophotometer tubes with 2 mL deionized water and add 1 drop of
KI/Iodine reagent to the tubes. This will speed up sample processing.

7) Prepare to time your experiment – you will be taking time points every 2 minutes, and
recording your results. Note that the first time point is CRITICAL- you should make sure

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to have everything ready to go and be ready to collect the first sample at the earliest
possible time point.

8) You will add 9 mL of starch solution to the 1 mL of 1:100 saliva dilution in the 4th test
tube and mix well. As soon as you begin adding the starch to the dilution, start timing
with the stop watch. (This is time = 0). Record the time on the following chart:

Target time Actual time Absorbance starch concentration


1) 0 min.
2) 2 min.
3) 4 min.
4) 6 min.
5) 8 min.
6) 10 min.
7) 12 min.

9) Immediately pipette 1 mL of the starch/saliva dilution mixture into the first


spectrophotometer test tube (test tube 1) with KI/iodine reagent, mix quickly and well.

10) Insert the tube into the spectrophotometer, quickly read and record the Absorbance.

11) At 2 minutes, pipette another 1 mL of the starch/saliva dilution mixture into the second
tube. Add 1 drop of KI/iodine reagent, mix well, and read and record the Absorbance on
the chart under Step 8.

12) Repeat these steps at every 4, 6, 8, 10, and 12 minutes.

Calculations:

1) Using the standard curve determined in part I, calculate the concentration of starch using the
determined absorbance.

2) Graph the concentration of starch vs. time. What is the shape of this graph?

3) Plot the reaction rate ([starch]/∆ t) vs. time. How does the reaction rate change over time?

4) Plot the reaction rate vs [starch] (Michaelis-Menten plot). What does this graph look like?

5) Make the double reciprocal plot (1/reaction rate) vs 1/[starch] (Lineweaver-Burke plot).
What is the shape of this graph?