Factors That Affect Pepsin Investigation.

Zohaib Khan

Factors That Affect Pepsin Investigation

Aim: is to find out what factors affect pepsin

Introduction:

What It Is & How It Works

Enzymes are proteins that speed up chemical reactions in our bodies Enzymes control how much and when a reaction occurs. Enzymes prevent reactions from occurring spontaneously.

Pepsin is a protease enzyme that breaks down food or further breaks down a product and is the main protease found in the mucosal lining of the stomach ( which protects it) and is called the chief cells. It also is used to break down proteins into smaller molecules called polypeptides. The enzymes are inactive when there is no food in the stomach and when there are the proteins of the food enter and it turns them on. It works best at body temperature or slightly higher.

The pepsin enzyme breaks down protein into polypeptides and then is released as amino acids which can be absorbed by the intestinal lining. This process can only work if it is active, therefore acidic and wet conditions are ideal. It is non-active in alkaline conditions. To keep the acidity level stable hydrochloric acid is produced by the gastric mucosa and maintains the acidity level for pepsin to work. Once the food and gastric juices have reached the intestines the enzymes are neutralised.

An enzyme works first by a substrate entering the active site (as shown in diagram 2) which is the protein and there are specific substrates that fit each and every individual active site e.g. only one key can fit one hole.

Diagrams

1) 2)

3) 4)

If an enzyme is denatured then substrate won’t fit into the active site because of the change in shape.

Factors

PH Scale Factor: A factor that can affect the ph is by altering the charge state of the substrate. If an enzyme is at an extreme of pH there are defence systems that automatically neutralize the enzyme which can disrupt the bonds that hold the enzyme in its structure, denaturing the protein and change of shape.

Temperature Factor: Increase in temperature can speed up reactions until it gets to the point where it denatures but when it decreases in temperature it slows down the enzyme dramatically. Most enzymes work well only in narrow ranges of temperature ...

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Factors

Amount of Enzyme (Pepsin): This affects how quickly the process takes by slowing it down because there are fewer molecules to bond together.

Amount of Food: This also affects the speed of the process as the more food there is the longer the digestion and conversation process.

I will choose to investigate the PH factor at tw0o different ph levels – acidic and neutralized.

Method

Diagram

Please Turn Over For Diagram

Instruments

1x Safety Glasses

2x Test Tubes

1x Test-tube rack

1x 10ml of Pepsin

1x 10ml Albumin

1x Hot Water Bath

1x Water at 37*C (Body Temperature)

1x Stop Watch

1x Thermometer

1x Liquid Measurer

1x Bunsen Burner

1x Tripod

1x Heat Proof Mat

1x Metal Wire Mesh (Lay On the Tripod)

1x Tongs

1x Small Piece of Paper

1x Pen

1x Pipette

This is the appropriate amount of equipment that is required to do the experiment.

Instructions

There are six easy steps to do to complete the practical. I have explained why I have done certain things in this method.

1. The first step is to gather equipment above, then take a thin test tube and pour 10ml of pepsin in it using the measurer. Once you have poured 10ml into a test tube place it in a test tube rack so there is no danger of breaking the test tube.

2. The second steps are to get 10ml of Albumin and also pour that into a same type of test tube, then place it next to the test tube full of pepsin in the test tube rack and don’t forget to use the measurer. Once you have both test tubes in a test tube rack and have filled them with the right amount of liquid you can carry on with the experiment.

3. The Third step is to find a wide normal beaker and pour some tap water into it and fill a third of it. Now you have to heat the water so take a Bunsen burner, heat proof mat (placing it under the tripod), a tripod and place a wire mesh over it and set the Bunsen burner on the blue flame making sure you haven’t heated the beaker yet.

Then take a thermometer and find out the temperature of the water inside the beaker, it should already be around 20*C (room temperature). Keep the thermometer still in the beaker and carefully place the Bunsen burner on the heat proof mat so it should be directly under the beaker. Stay on the alert as the thermometer might rise quickly and exceed 37*C so as soon as the thermometer reads 40 or 41 (heat it a bit more so it doesn’t cool down by the time you put the test tubes in) take the Bunsen burner away. The reason why we heat the water inside the beaker to 37*C is because pepsin works well in body temperature. It would be too slow at room temperature.

4. The forth step is pick up both of the test tubes which are full of Albumin and Pepsin and place them side by side in the beaker, this should be done quickly as the water inside the beaker might cool down. Once the test tubes are in the beaker then take out the thermometer from the beaker and place it in one of the test tubes to see the temperature. Keep the thermometer in there and wait till it reads 37*C in the test tube. Then take out the thermometer from one of the test tubes and place it in the other and when that test tube is at 37*C then can carry on to the next step. We heated the water first to make it accurate in temperature conditions because if we used a Bunsen burner to heat up a test tube on a blue flame full of enzyme it would denature it very quickly leaving us with little time to take it away from the Bunsen.

5. The fifth step is to get a small piece of paper (at least 3cm long and wide) and draw a round small dot in the centre of it. This is to see when the test tube goes clear and the two liquids are mixed to formed bonds. You have to now verify that they are both at 37*C and carefully pour the albumin protein into the pepsin enzyme test tube. Now you will see very cloudy liquid in the test tube. That cloudy liquid is like lots of keys floating around looking for the right hole to fit into.

To help the reaction add 3ml of hydrochloric acid using a pipette so the pH is acidic. This is because in the stomach works in acidic conditions. Now take the piece of paper that you have drawn a dot on and place that against the test tube to see if it has gone clear yet. If it has gone clear and you can see the dot quite sharply then the bonds are formed and the conversion process from the protein into amino acids is completed.

6. The sixth step is to repeat the whole experiment using different test tubes or washed equipment. Repeating the experiment is necessary because you have to get an average time of how long it took for the two liquids inside the test tubes to go clear, you need at least 3 results.

Predictions

I predict that it will take a short period of time for the conversion process to be completed. I also predict that if I added alkali into the pepsin it would slow down the process and eventually denature the enzyme.

I also predict that the optimum ph level is acidic and will work best and most efficient at helping the enzyme convert the substrate to a product. I based this upon the idea that the stomach works in acidic environment from the facts I know.

Results

Time How Clear Words

4:23:53 quite clear Four minutes, twenty three seconds

4:14:22 very clear Four minutes and fourteen seconds

4:26:04 extremely clear Four minutes and twenty six seconds

Average: 4:21:38

Fair Test

The practical was a fair test because we placed both of the test tubes in a beaker so they both were at the same temperature when the conversion took place. We weren’t accurate when we had to make the decision that the liquid was clear or not. We put the same amounts of protein and enzyme so that was a fair test. It wasn’t really fair though when we had to put both the test tubes in a beaker and by that time the beaker would have cooled down.

Modifications

I would modify lots of different things as there is a lot to change either to make the experiment fair or to simply make the experiment easier to do. First I would get a machine that keeps the temperature at the same level when something like a beaker will be placed on it.

The second thing I would do is to shake the test tube (at the start of the reaction when the pepsin is mixed with the protein) as it will speed up the reactions or you could heat it very slightly to speed it up rapidly. This will make the substrate move around the test tube quickly and also by shaking it.

The third thing I would do is instead of having the conversion process in a thin tiny test tube were you can’t see much, you could just mix both the pepsin and the amylase in big beaker so it is clear enough and spread out to make those vital decisions on how clear it actually is.

Conclusion

I found investigating the ph quite straight forward as there wasn’t much in it to investigate in my opinion. I made a couple of alterations as I was going through my method I changed it by recording one result when the pepsin in the test tube goes clear instead of recording a result every 5 minutes because there isn’t any point as it is much easier to get one result every time you do the experiment. I was right in my prediction that the conversion process was short and that If I put an alkali in (when the bonding process is activated) it would slow it down and gradually denature it.

I found out that the enzyme mostly works well only at limited ranges of temperature and pH. I also found out that extremes in pH can disrupt the hydrogen bonds that hold the enzyme in its three-dimensional structure, denaturing the protein.

I was right in my prediction that the optimum acidic of normal gastric juice was conditions that were the most suitable for the conversion. The enzyme is neutral when there is enough that has been taken away from the substrate. I found out that the temperature can get up to 50*C and not denature.

Overall pepsin is a specific enzyme for converting substrate (which is the amylase protein) into polypeptide that is released as amino acids.