Lab Report

Lab 4: Enzymes

Objective: To analyze several conditions that affect enzymatic rates and to measure the activity of an enzyme as it degrades gelatin.

Supplementary Materials on Blackboard:

- “Enzymes (Updated)” Video

- “How Enzymes Work” Video

- “Enzymes Cells Biology” Video

- Presentation

4.1 Introduction

Your cells are like miniature cities, teeming with activity. These little cities are run with millions of chemical reactions, constructing new macromolecules, recycling old ones, making ATP for energy from glucose, and much more. Cells regulate all this activity using enzymes, a subset of the proteins you worked with last week.

Recall that proteins including enzymes consist of one or more polypeptide, chains of amino acids with side groups that can be charged, polar, or nonpolar. The polypeptides fold into secondary, tertiary, and quaternary shapes, which determine the function of the protein. The shape resulting from this folding is critical to the function of the protein. If the protein loses its shaped (is denatured), it may lose its function as well.

In the case of enzymes, that function is to lower energy input required for reactions, thus increasing the rate of reaction. The enzyme is specific to the reaction; an enzyme that breaks down glucose does not also break down synthesize proteins. The reactants in enzymatic reactions are called substrates. Enzyme activity is critical for life. Enzymes catalyze reactions to occur incredibly quickly that would be nearly impossible naturally. Consider, for example, the synthesis of DNA: about 50 nucleotides per second.

In order to function, substrates must fit into the active site where the reaction occurs (Figure 1) and certain amino acids on the enzyme must be in proximity to certain regions of the substrate. The model of alcohol dehydrogenase, the enzyme which detoxifies the ethanol that humans love so much, demonstrates this (Figure 2). You can see that the enzyme must hold at least three molecules, and certain amino acids must be next to these molecules to interact with them. You cannot, for example, have several positively charged argenine next to a nonpolar section of NAD. More clearly, you can see in the left hand image the bonds formed between zinc and the side chains, and the close interaction with ethanol. This is critical for its function.

Many toxic compounds inhibit the action of enzymes, and many diseases stem from abnormal enzymes.

Some enzymes can be harvested from genetically modified bacteria at industrial levels. These enzymes are used in molecular biology to copy DNA and clone genes, sequence DNA samples, analyze gene expression, analyze responses to environmental conditions, and more. Some of these enzymes are used for DNA profiling in crime scene investigations, which you will learn about later this semester. Enzymes also help produce and process foods like cheese, baby food, fruit preserves, and alcohol. You can purchase cleaners with enzymes, meat tenderizer containing enzymes, or supplemental digestive enzymes.

Figure 1: Enzyme activity overview.

Source: Biology, LibreTexts

Figure 2: A model showing ethanol nestled in the active site of alcohol dehydrogenase from two different angles.

Alcohol dehydrogenase uses two molecular "tools" to perform its reaction on ethanol. The first is a zinc atom, which is used to hold and position the alcoholic group on ethanol. The second is a large NAD cofactor (constructed using the vitamin niacin), which actually performs the reaction. PDB entry 1adc , shown here, contains ethanol molecules bound to the two active sites. A slightly-modified version of NAD was used in the structure analysis, so that the enzyme would not immediately attack the ethanol. Notice how the zinc atom is cradled by three amino acids from the protein: cysteine 46 to the left, cysteine 174 to the right, and histidine 67 above. The ethanol binds to the zinc and is positioned next to the NAD cofactor, which extends below the ethanol molecule in this illustration. For more information and to interact with this structure, read the article and visit the entry at the Protein Data Bank.

Source of image and caption: “Alcohol Dehydrogenase” at PDB 101.

Enzyme activity

For enzymes to have maximum activity, they must maintain certain interactions between their amino acids, the substrate, and the environment such as the solvent in which they are dissolved. As the environment changes, enzymes often have some ability to adapt and maintain some function, but the efficiency diminishes.

Consider baking bread, which relies on enzymes present in the yeast: If you allow bread to rise at the optimum temperature (75 F/24 C), it will rise quite quickly. Bread will still rise at lower temperatures, but the process will take much longer (please feel free to try this at home!).

The rate of the enzyme activity over a range of temperatures can be visualized well as a graph:

As the temperature increases or decreases, the ability of the enzyme to function decreases and the reaction rate decreases. Why? Heat affects the movement of atoms, and as the temperature changes some bonds become tighter or looser.

What other conditions affect enzyme function? You might be able to predict what might interfere with the interactions of amino acids. You will investigate some conditions in this week’s activity.

4.2 Activity: Prediction of enzyme rates

Do some research and critical thinking and list below 10 conditions that may affect enzymatic functions. You can be specific, but they should not all be one subset of the same condition (for example 10 different temperatures).

Choose three of these conditions and draw graphs of activity, with rate of reaction on the y-axis as the dependent variable and the condition on the x-axis as the independent variable. If possible, find a specific enzyme and mark its specific optimal activity. Use a separate sheet of paper or a program that allows you to draw with a stylus.

4.3 Activity: Analyzing enzyme rate compared to pH

In this lab activity you will investigate the mode of action of enzymes in the degradation of the proteins that make gelatin.

Gelatin is made of proteins from animals. The most abundant protein in gelatin is collagen, which is found in the bones, tendons, muscle, skin and cartilage of many animals. The main amino acids found in the proteins of gelatin are glycine, proline and glutamic acid.

Gelatin is easily dissolved in hot water and solidifies into a gel when it cools. You may have experienced this if you’ve make stock or roasted a chicken, and of course in making gelatin deserts like Jello. This happens because when water is added to the gelatin, long chains of proteins form. Water is trapped in the middle of the proteins and changes the liquid into a semi-solid. If the bonds between the gelatin chains are broken and the proteins in gelatin degraded, the three dimensional matrix returns into a liquid state.

There are many ways to degrade the bonds in gelatin. For example, heat will break those bonds. This is the reason why gelatin melts in our mouths into a liquid. Another way to break the bonds in gelatin is by the use of enzymes.

Nature provides us with a variety of enzymes that can break the bonds of proteins and degrade them. Those enzymes are used in digestive process and in the transformation of protein in waste and other materials. Two important examples of those degrading enzymes are papain and bromelain. Papain is derived from the papaya fruit and bromelain is found in the pineapple plant. Due to their ability to degrade proteins, those enzymes are included in meat tenderizers in order to pre-digest meat and improve its digestion.

In this lab activity, we will investigate the action of the enzymes in meat tenderizer, which contains digestive enzymes, on the degradation of proteins in gelatin. As discussed above, several physical factors can affect the rate of an enzymatic reaction. For this reason, the action of the enzymes in meat tenderizer will be analyzed under different experimental conditions.

Gelatin will be exposed to meat tenderizer alone, meat tenderizer with sodium bicarbonate (creating basic conditions) and meat tenderizer in vinegar (creating acidic conditions)

Materials

2 gelatin boxes (any color, brand or flavor)

Meat tenderizer (powder)

Alternative: fresh pineapple, kiwi, or papaya, blended to a paste or liquid - these contain similar enzymes as meat tenderizer

Sodium bicarbonate (baking soda)

Vinegar

Water

Plastic cups (4)

Plastic tablespoons (reusable is fine)

Protocol

Prepare gelatin in four plastic cups following the manufacturer instructions. Try to fill just half of the volume of the cups with gelatin and leave the remaining volume for the experimental solutions.

As the gelatin solidifies, prepare the following experimental solutions:

Solution 1) Pure Water (this will be the negative control)

Solution 2) Water with one and half tablespoon of meat tenderizer (or pineapple/kiwi/papaya)

Solution 3) Water with one and half tablespoon of meat tenderizer and the tip of a tablespoon of sodium bicarbonate

Solution 4) Water with one and half tablespoon of meat tenderizer and one tablespoon of vinegar

Once the gelatin is solid, label the cups 1, 2, 3, 4. Add 1 tablespoon at a time of the first solution to the first cup, until the liquid is about 1 cm above the gelatin. Record how many tablespoons you added to the cups. Then, wash or exchange the spoon and add the same amount of solution 2 to cup 2. Repeat, adding each solution to the appropriate cup. DO NOT MIX SOLUTIONS.

Take photos or write observations of the state of the gelatin and the solutions.

Answer questions 1 -3 of activity 4.4.

Leave the cups for 2-3 hours and then observe the gelatin. (If the weather is too hot, place the gelatin cups in the refrigerator for additional hours (3-4) and then observe the cups).

Any portion of the gelatin that is liquid is an indication of the degradation of the proteins by the actions of the enzymes. Carefully measure the amount of liquid using your tablespoon (or measuring cup if needed). Record the amount of liquid in each solution.

Record the amount remaining solid in each cup with photographs and/or written observations.

Record your results in the table below:

Gelatin with water will create a jell like consistency and less than a tablespoon of liquid will be left. 90% to 100% solution.	Returning amount of liquid, with units (Tbsp, eg)	Rough estimate % of gelatin remaining solid
1) Gelatin with meat tenderizer: Gelatin with water	Gelatin with meat tenderizer create a semi solid and semi liquid solution.	35% to 45%
2) Gelatin with meat tenderizer + vinegar will create a liquid. 5% Gelatin with meat tenderizer + sodium bicarbonate	Gelatin with meat tenderizer + sodium bicarbonate it will create a thick semi solid liquid.	10% to 20%

4.4 Activity: Gelatin degradation by meat tenderizer enzymes

1. Write a hypothesis predicting the results of your experiment. Meat tenderizer acts as an enzyme to break down gelatin. The sodium bicarbonate mixed with the meat tenderizer acts as a binding agent to create a thick semi solid liquid. Meat tenderizer with the vinegar acts as an enzyme and an acid to create a liquid.

2. How many tablespoons of liquid did you add to each cup? Which solution was closest to neutral pH? Which solution was basic? Which solution was acidic? Cup 1 I put 1 tablespoon, Cup 2 put 1 tablespoon, Cup 3 put 1.5 tablespoon, and Cup 4 put 2 tablespoons. Gelatin and water. basic is going to be sodium bicarbonate and acidic is going to be the vinegar.

3. What is the independent variable? What is the dependent variable? What is one controlled variable?

Independent variable is Gelatin. The dependent variable vinegar and the sodium bicarbonate. The controlled variable would be the gelatin and water.

4.5 Questions for Review

Which solutions containing enzyme resulted in the most liquid (or least solid) and the least liquid (or most solid)? Was this in line with your hypothesis? The least solid was the gelatin mixed with the meat tenderizer and vinegar. The most solid is the gelatin and water. I originally thought the water and gelatin would be the most liquid.

What can you infer about enzyme activity under the condition resulting in the least percent of solid gelatin and the greatest amount of liquid? The enzyme is breaking the solid down into a more liquid form if the beginning state is liquid then there is little to know about enzyme activity.

If your results were unexpected, think of at least one explanation. My initial reaction went into how water is fluid.

Write a conclusion for the experiment. We saw how gelatin reacts to different substances like Meat tenderizer, sodium bicarbonate and also vinegar. These substances displayed the different strength of enzymes.

Look up the enzymes in the meat tenderizer. What are they? Where specifically are they naturally found? Can you find a recipe for meat that makes use of fruits containing enzymes? Enzyme tenderization is used to reduce the amount of detectable connective tissue in the meat. Enzymes are proteinases, also known as cysteine endopeptidasas, derived from plants, such as papain, bromelain, and ficin.

What other environmental condition could you alter in this experiment besides pH? Propose a next experiment.

Could change the temperature and concentration.

Eating too much pineapple causes mouth irritation and redness in some people. Could you think of a possible explanation for this? Is there a connection between this reaction and the results of this experiment?

Because we don’t enzymes to break down pineapple.

Submit the answers to the questions and written observations (if any) of activities 4.2, 4.4, and 4.5 on one word processor document (.doc, .docx). If possible, add the graphs from 4.2 and photos of results. Otherwise, submit photos and graphs separately.