Acid and Amylase Enzyme: Denaturing a Protein

SKILL LEVEL

Middle school, High School

SUBJECTS

Biology, Life Science, Cell Biology

Amylase structure

TRY IT!

An enzyme is a protein that is designed to speed up (catalyze) a specific biochemical reaction.

You can think of an enzyme as a tiny molecular robot (a nano-bot!) that, much like a robot on an assembly line, has only one specific job.  Also like a real robot, a protein is like a precision piece of equipment designed to operate only under very specific conditions.

Imagine trying to operate your cell phone in salty sea water, 700 meters below the surface.  Needless to say, your phone would be destroyed in an instant.  Similarly, when an enzyme is exposed to unnaturally high temperatures or extremes in pH (acidity), it is also rendered ineffective.  The useless protein is said to be denatured.

In this experiment,  you will investigate the effect of temperature and pH on the activity of the protein amylase.  Amylase is an enzyme that breaks down starches into simple sugars.  The goal will be to observe whether the amylase activity is reduced as a result of being denatured by exposure to a relatively weak acid or heat.

SUPPLIES, TOOLS, MATERIALS

  • Three plastic disposable cups or three beakers
  • Spoons and/or stir sticks
  • Amylase enzyme*
  • 10% povidone iodine
  • Liquid laundry starch
  • Water
  • Vinegar
  • Microwave or stove

*Amylase enzyme is readily available on amazon.com for use in brewing.  It is sold in powdered form for roughly $10/lb.

INSTRUCTIONS

Prepare starch solutions

  1. Add approximately 100 mL of water to 3 cups.
  2. Add approximately 30 mL of liquid starch to each cup.
  3. Add approximately 10 mL of iodine solution to each cup and stir.  Notice the color change in the iodine when it is added to the starch.  Record your observations.

Prepare amylase solutions

  1. (control) Add approximately 100 mL of water and a level  tablespoon of powdered amylase to a cup and stir.
  2. (acid treated amylase) Add approximately 50 mL of water, 50 mL of vinegar (acid), and a level  tablespoon of powdered amylase to a second cup and stir.
  3. (heat treated amylase) Add approximately 100 mL of water and a level  tablespoon of powdered amylase to a microwavable cup, stir, and microwave until nearly boiling.
  4. Allow the hot solution to return to room temperature before proceeding.

Observe amylase activity

  1. Combine each amylase solution with a starch solution.  Be sure your samples are clearly labeled.
  2. Stir each solution for a few seconds, being careful not to cross contaminate the three samples.
  3. Wait 1-2 hours and record your observations!
Amylase Enzyme

WHAT IS HAPPENING?

You probably noticed when you added the iodine solution to the starch that the iodine turned from a dark orange-red to a deep purple, almost black, color.  This is a result of a unique complex that iodine forms with starch.

When the amylase is working properly, the starch will be chemically broken down into simple sugars.  Iodine does not react with these sugars the way it does with starch.  Thus, the iodine color may be used as a detector for the activity of amylase.

If you observe a dark purple color, the starch remains because the amylase has been denatured and will no longer break it down.  If the dark purple color is absent, the amylase is still functional.

What did you see?  Did the color changes occur at the same time?  Did both of the denatured samples reach the same final color?

QUESTIONS TO CONSIDER

  1. Look up and define the following terms:
    Enzyme
    Starch
    Enzyme Activity
    Denature
  2. Record your observations for each amylase, starch, iodine solution.
  3. Would you expect the same results if the amylase were cooled rather than heated?  If not, why not? Try it!
  4. Your blood is a buffered system, meaning its pH is maintained within very narrow limits.  Why is this so important?  What would happen if your blood experienced large swings in acidity?