The effect of pH on the activity of the enzyme Amylase

        pH is a measure of how acidic a chemical is. The acidity of a chemical is determined by how concentrated the chemical is with Hydrogen ions.

This is describe in an example

Loses H                      Gains H

Alkaline solution          Acidic Solution

NH  CHRCOO  ← NH  CHRCOO  ➔ NH  CHRCOOH

               Overall Negative charge          “Zwitterion”           Overall Positive charge

               lost H  so makes solution         No change        gained H  so makes solution

                        more acidic.                                                         less acidic.

As the solution becomes more concentrated with hydrogen ions the more effective it is on taking up the R (residual) group on amino acids: Lys, Arg, His, Asp and Glu. This affects the way in which they bond with each other therefore affect their 3D arrangement. So if the ionic bonds are important to structural stability, then the shape of the enzyme will change which in turn affects the functionality of the enzyme.

As a type of protein, enzymes are easily affected by changes in pH. At their optimum pH the shape of the enzyme is such the active site can fit perfectly with the substrate. As the pH deviates, either decrease or increase from the optimum the acid or base conditions begin to disrupt some of the loops between the loops of the protein chains. If this disruption occurs on the active site the site will become distorted and the substrate will not fit perfectly. Thus not all enzymes will be able to catalyse (break down) their reaction. Further increase or decrease of pH, will result in more enzymes becoming denatured and fewer are able to form the enzyme-substrate complex. At some point all the enzymes are denatured and the reaction rate falls to zero.

Prediction

More specifically to our experiment we are using the enzyme Amylase, this is a enzyme found in the salivary glands, pancreas and small intestine. Amylases, act on starch, breaking down (hydrolysis) to produce glucose and other sugars, other uses are in textile manufacture, in some biological detergents, and baking, to produce lighter loaves. In our experiment we will be measuring how quickly the enzyme Amylase catalyses (breaks down) the substrate starch (a polysaccharide) into the product Maltose (a disaccharide). The Maltose produced will be a reducing sugar and to test this with our final product we will be using Benedict’s solution. With this information I can predict that the optimum pH of Amylase will be of pH 7. I predict this because generally most enzymes work at a neutral pH, where there are least Hydrogen ions for less interference. If amylase was of a high deviation from a neutral pH it would be unlikely that it would be found in the small intestine, the small intestine contains fine glandular tissue (Villi) used for absorbing nutrients from broken down food and any high pH would be likely to destroy the fine tissue.

Also amylase is found in the mouth which denotes a neutral pH I know this due to previous experiments with litmus paper and saliva swabs. The outcome was a neutral green colour.

Chemicals

1. 2% Starch Suspension
2. 1% Amylase Suspension
3. Distilled water - To clean glass wear
4. Iodine - Test for starch
5. Benedict’s solution - Test for sugars
6. Range of buffers - To maintain correct pH (7,10,4 & 9.2)

Apparatus

1. Spot plate - Needed for testing for starch, taken at regular time intervals.
2. Measuring cylinder - For measuring chemicals - graduated to 1 ml for accuracy.
3. Test tubes - Where solutions are contained and to be observed. samples to be taken.
4. Water bath - Set at 37°C - perfect body temperature - optimum temperature.
5. Stop watch - Needed to measure the time taken for the reaction to take place.

Diagram

Method

1. Set up apparatus as diagram
2. Heat amylase solution, starch suspension and distilled water at 37°C
3. Mix 5cm³ of the Starch suspension with 5cm³ of a buffer in test tube ‘A’
4. Add 5cm³ Amylase suspension to test tube ‘B’
5. Heat in water bath until they have both reached 37°C
6. Add 1 drop of iodine to each hollow on the spot plate
7. Add test tube ‘B’ to test tube ‘A’
8. Start stop watch take samples and add to hollow every 20 seconds until iodine remains same brown/orange colour
9. Once completed experiment, test solution for reducing sugars by adding Benedict’s solution:- positive result is a red precipitate.

Fair test

Variables that I will need to remain constant:-

Enzyme Concentration

Temperature

Enzyme concentration:- Must remain constant because as the concentration of enzyme increases the reaction rate does proportionally. It does this because the more enzyme molecules there are more active sites available. More specifically to this experiment, we are controlling the amount of amylase we are using as any increase will create anomalous results and major inaccuracies

Substrate concentration

Temperature:- Must remain constant as deviation from the optimum (37°C) will eventually result in a denature of the enzyme. Specifically to this experiment we must keep the Starch suspension, buffers and amylase suspension at a constant of 37°C. Doing so will keep my results constant and accurate.

Substrate concentration:- Must remain constant because as the concentration increases the reaction rate does also. This is because as the substrate concentration increases the enzymes can bind with the substrate more frequently. I shall keep the volume of substrate the same each time this should produce accurate results.

Variables that I will be altering:- I will be altering the pH in order to see how it affects the reaction rate of enzymes.

Observations and Measurements

        I will use a stop watch to measure the time the enzyme takes to convert starch (a polysaccharide chain)  to maltose (a disaccharide)

        I will test the end solution with Benedict’s solution to see if maltose is definitely produced.

Repeats

        I shall repeat the experiment 3 times this shall produce an accurate set of results. This will insure that any anomalous results obtained will be more spread out.

Accuracy

        In order to achieve a high level of accuracy I have made the following choices:-

1. All glass wear used for measuring e.g. measuring cylinder will be of a grade B or higher.

Safety

        The usual safety precautions must be enforced for example safety goggles and clear walkways. Specific care must be taken when handling the acidic pH as they may burn the skin.