The Effect of pH on Enzyme Activity- Salivary Amylase and Starch Digestion.

Introduction: (Initial Observation)

Enzymes are biological catalysts. The term enzyme comes from zymosis, the Greek word for fermentation, a process accomplished by yeast cells and long known to the brewing industry. Like all catalysts, enzymes accelerate the rates of reactions while experiencing no permanent chemical modification as a result of their participation. Enzymes can accelerate, often by several orders of magnitude, reactions that under the mild conditions of cellular concentrations, temperature, pH, and pressure would proceed imperceptibly (or not at all) in the absence of the enzyme.

Today, thousands of enzymes are produced for their medical applications as well as industrial applications. By knowing the factors affecting enzyme activity, we can control the activities of enzymes in the direction that is more beneficial for us. In this project we investigate the effect of pH on the activity of Amylase Enzyme on digesting starch.

Information Gathering:

The efficiency of an enzyme’s activity is often measured by the turnover rate, which measures the number of molecules of compound upon which the enzyme works per molecule of enzyme per second. Carbonic anhydrase, which removes carbon dioxide from the blood by binding it to water, has a turnover rate of 106. That means that one molecule of the enzyme can cause a million molecules of carbon dioxide to react in one second.

Most enzymatic reactions occur within a relatively narrow temperature range (usually from about 30°C to 40°C), a feature that reflects their complexity as biological molecules. Each enzyme has an optimal range of pH for activity; for example, pepsin in the stomach has maximal reactivity under the extremely acid conditions of pH 1–3. Effective catalysis also depends crucially upon maintenance of the molecule’s elaborate three-dimensional structure. Loss of structural integrity, which may result from such factors as changes in pH or high temperatures, almost always leads to a loss of enzymatic activity. An enzyme that has been so altered is said to be denatured .

Consonant with their role as biological catalysts, enzymes show considerable selectivity for the molecules upon which they act (called substrates). Most enzymes will react with only a small group of closely related chemical compounds; many demonstrate absolute specificity, having only one substrate molecule which is appropriate for reaction.

Numerous enzymes require for efficient catalytic function the presence of additional atoms of small nonprotein molecules. These include coenzyme molecules, many of which only transiently associate with the enzyme. Nonprotein components tightly bound to the protein are called prosthetic groups. The region on the enzyme molecule in close proximity to where the catalytic event takes place is known as the active site. Prosthetic groups necessary for catalysis are usually located there, and it is the place where the substrate (and coenzymes, if any) bind just before reaction takes place.

The side-chain groups of amino acid residues making up the enzyme molecule at or near the active site participate in the catalytic event. For example, in the enzyme trysin, its complex tertiary structure brings together a histidine residue from one section of the molecule with glycine and serine residues from another. The side chains of the residues in this particular geometry produce the active site that accounts for the enzyme’s reactivity.

Identification and Classification

More than 1,500 different enzymes have now been identified, and many have been isolated in pure form. Hundreds have been crystallized, and the amino acid sequences and three-dimensional structure of a significant number have been fully determined through the technique of X-ray crystallography. The knowledge gained has led to great progress in understanding the mechanisms of enzyme chemistry. Biochemists categorize enzymes into six main classes and a number of subclasses, depending upon the type of reaction involved. The 124-amino acid structure of ribonuclease was determined in 1967, and two years later the enzyme was synthesized independently at two laboratories in the United States.

The enzyme amylase will catalyze the hydrolysis of starch to maltose when the pH is near 7.0. But when the HCl is added to the solution the amylase will be denatured which results in the enzyme being deactivated. The iodine serves as an indicator for the presence of starch. Iodine (I2) will reach with iodide ion to produce the I3- ion. This ion will form a dark blue complex with the starch molecule.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

The purpose of this investigation is to know the effect of pH on the activity of Amylase Enzyme in digesting starch.

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

Independent variable or manipulated variable is the pH.

Dependent variable is the rate of Enzyme activity.

Controlled variables are temperature, light and all other environmental factors that may affect the activity of enzyme.

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.

My hypothesis is that Amylase enzyme will have it’s optimum activity at pH 5 to 7. My hypothesis is based on my gathered information and the source of this enzyme which is human saliva.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”

Introduction:

In this experiment you will test the ability of the enzyme amylase, found in human saliva, to hydrolyze or break down starch molecules in various pH solutions.

Procedure:

Prepare a 1% solution of starch. To do that dissolve 1 gram starch in half a cup of cold water. (One gram starch is about half tea spoon flat.) Then pour it into 900 ml boiling water. Let it boil for 2 minutes. Then let it cold to room temperature.

Add 5ml of 1% starch solution to each tube

Prepare a 2.0M HCl solution. Hydrochloric acid purchased from hardware stores is usually 10M (or 36%). Get 2 ml of that acid and add water to make it 10ml. If you can not find Hydrochloric Acid (Also known as muriatic acid), use vinegar instead.

Don’t add any acid to the first tube (control).

And 1 drop of 2.0M HCl to the second tubes

Add 2 drops of 2.0M HCl to the third tube

Add 3 drops of 2.0M HCl to the fourth tube

Add 4 drops of 2.0M HCl to the fifth tube

Obtain a sample of saliva (2-5 ml). This can be done by chewing a clean rubber band and drooling into a test tube. Place 2 drops (no bubbles) in each of five test tubes.

Allow the tubes to stand for approximately 15 minutes. It is even better to warm them in your hand during this period. Then add 1-2 drops of iodine solution. A black or dark blue color indicates the presence of starch, the yellow iodine color means no starch remains, having been hydrolyzed by the amylase.

Considering the result of this experiment, what do you think about the activity of amylase in the stomach.

All substances can be poured down the sink.

Temperature

At what temperature should we conduct the experiment? Amylase works best near 37 C, the normal internal temperature of the human body. You will use the water bath to achieve this temperature. Water bath can be a cup of warm (37ºC) water.

Iodine is toxic and an irritant and will stain clothing. Handle acids with care, avoid contact with skin or eyes.

Variations:

• You may repeat this experiment with an alkaline instead of acid and compare the results. (Use ammonia solution as an alkaline).
• You may also do the above experiment with 2 acidic test tube, 2 alkali test tube and one as control with no acid or base.
• If you can do this experiment with 10 test tubes, then increment of acid is better to be one drop. So instead of 2, 4, 6 and 8 drops you will use 1, 2,…and 9 drops.
• You can use vinegar to acidify your solution instead of hydrochloric acid. If you do this you don’t need to dilute the vinegar. (Vinegar is 5% acetic acid in water).
• If you have access to a pH meter, measure the pH of each tube and record the actual pH in your results table.
• If you have access to pH paper, measure the pH of each tube and record the estimated pH of each tube in your results table.

Materials and Equipment:

• 5 to 10 test tubes
• 100 ml of 1 % starch (add one gram of soluble starch in a paste to boiling water)
  rubber bands
• 50 ml of iodine test solution (Lugol’s iodine or 1 g iodine to 100 ml 1.0M KI) divided into 5 dropper bottles.
• 50 ml of 2.0 M HCl divided into 5 dropper bottles

Results of Experiment (Observation):

Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.

Sample results

This is a sample result in an experiment with 5 test tubes. Tubes 1 is very acidic, tube 2 is slightly acidic, tube 3 has no added acid or base. Tube 4 is slightly basic, tube 5 is very basic.

Presence of starch in the final result is shown by (++ and+)

Absence of starch in final result is shown by (-)

In this experiment, Iodine solution is added right after adding amylase. Observation and recording continued for 21 minutes. Solution is shacked every minute.

Interpret the above results and explain the influence of pH on the action of the enzyme amylase on starch.

You may use your results to make a graph.

Calculations:

No calculation is required.

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.

References:

John Whitsett, LaCrosse Central HS, LaCrosse, WI 54601

http://botit.botany.wisc.edu/courses/Botany_130/Organic_compounds/enzyme.html