The Effect Of Enzyme Concentration On Enzyme Activity

The Effect Of Enzyme Concentration On Enzyme Activity
The pancreatic duct in individuals who have cystic fibrosis frequently becomes blocked, reducing or preventing the release of pancreatic enzymes into the small intestine. The aim of this activity is to investigate the effect of a reduction in enzyme concentration on the rate of reaction, in this case the breakdown of protein by protease enzymes. Aim ? Milk powder contains a white protein called casein. A white suspension of milk powder clears on the addition of the enzyme trypsin. The aim of this experiment is to investigate the effect of enzyme concentration on enzyme activity. We will observe how fast the milk turns transparent by changing the concentration of the enzyme on milk. Hypothesis ? The higher the concentration of enzymes, the faster the protein will be broken down, and therefore the egg white will turn transparent faster.
Appropriate observations have to be made to validly test a hypothesis or idea. The term ?observations? is taken to include measurements. Validity is the measure of confidence that can be placed in a conclusion. After deciding upon a dependant variable to vary within the experiment, the method of measurement should also be known, the measurement taken should be valid e.g. the volume of oxygen collected in 30 seconds is a valid measurement of rate of catalase activity. Valid results are derived through precise, repeatable measurements and observations, made with apparatus and experimental procedures that are suitable for the task. Ideally a large number of replicates (repeat measurements) should be taken, and any results that vary considerably from the others should be repeated or discounted. A mean can be calculated to be representative of the set of results. The pressure of time usually puts a limit on the number of replicates that can be taken. To be reliable, measurements should also be accurate. To be accurate a measurement should be close to the true value. Precision involves the choice of apparatus and the skill with which it is used. I had to also be aware of the different types of error that could occur in my experiment, systematic, random, and human errors. To overcome systematic problems I will need to know how to use apparatus correctly, to avoid human error I will have to give my full attention and concentration towards the experiment. To avoid random errors, I have to control changes in the material used or the conditions in which they are carried out in, as this will all affect the final result we produce. If we follow this we should be able to produce valid results. Apparatus ? To carry out this experiment, the following apparatus are needed: ? Standard alkali trypsin solution ? Milk powder ? Standard laboratory glassware and apparatus ? Stop clock ? Ruler ? Thermometer Safety ? An important aspect of good practical work is safety. To work safely during practical work, I made sure I followed each of the following rules. 1. Do not bring non-essential materials into the lab. Keep bags and coats outside of the lab as it could get in the way, and cause injury. 2. No food or drink is allowed in the lab at any time. We will be working with some fairly nasty chemicals and some exercises involve bacteria. It can be dangerous if the chemicals r ingested. 3. The eyes are particularly sensitive to chemicals, and provide a good portal of entry for microorganisms; the best way to avoid contact is to keep your hands away from your face as much as possible and to wear safety goggles at all times. Gloves may also have to be worn when handling hazardous chemicals. 4. We will be working with stains in some exercises that will not wash out of cloth, and so lab coats are needed. 5. Keep long hair tied back and ties tucked in, as we will have open flames in some exercises. 6. Dispose of waste material properly. There are specific, clearly labeled containers for glass and contaminated materials, they have to be disposed of correctly otherwise this could cause problems later on. We are also required to maintain a clear surface worktop. 7. Alert the instructor to any spills or breakage. These accidents need to handled correctly; we are not authorized to clean it up ourselves. Variables ? The independent variable is the factor that is being varied. A rang of values for the independent variable can be chosen. The dependant variable depends on the value of the independent variable. The dependant variable is the one that is measured in some way. Any other variables that may affect the dependant variable should be controlled (kept constant) in order to produce results that are valid and reliable. ============== The independent variable within this experiment is the concentration of the trypsin. The dependent variable within this experiment is the time taken for the milk to digest. The constant/control variables of this experiment are the substrate (milk) concentration, the temperature and the pH. ================= To keep the temperature constant in this experiment, we could have put the test tubes in a water bath, and set the temperature to 25?C, so that it would not affect the overall results. We did not do this, as we discovered, from the preliminary trial, that the experiment only lasted about five minutes maximum, and so temperature would not change dramatically in such a short space of time. We did however, close all windows and switch of radiators, we kept the experiment going in one area. This variable could have affected the end results if it had not bee controlled. If there were dramatic changes in room temperature, results would not have been reliable. As temperature gets higher, enzyme activity increases ? enzymes work at its optimum at about 37?C, if temperatures get too high, they become denatured and no longer work. As temperatures decrease, enzymes become less active and stop working if temperatures are too low. So it is important to keep the temperature constant in order to see the effects of enzyme concentration on enzyme activity. Method ?First, I will take the pipette and put the solution in the beaker of water and then use the stopwatch to time how long it takes to turn clear. I will know when it turns clear, as I will be able to see through it and read the writing. I will record the time it takes to turn transparent, in a results table. ================ Enzyme Concentration Volume of 1% trypsin (cm3) Volume of water (cm3) 0 0 5 0.2 1 4 0.4 2 3 0.6 3 2 0.8 4 1 1.0 5 0 A solution was made that meant that we could judge what the outcomes were compared to that. This was made up of 5cm3 of milk, 5cm3 of distilled water and no trypsin. This as I expected showed no transparency, which means that then the enzyme, actually works. Once all the solutions were prepared, 5cm3 of enzyme was added into a solution; by repeating a few more times I was able to get the next few results. Results ? ===== Enzyme concentration 1 ? Time taken for milk to Digest (min/sec) 2 ? Time taken for milk to Digest (min/sec) 0 - - 0.2 4:43 4:13 0.4 2:33 2:51 0.6 1:56 1:14 0.8 1:34 0:59 1.0 1:12 0:51 The results show that as the enzyme concentration increases, the time taken for the milk to digest decreases. Therefore the time taken for the protein to be broken down by the enzyme decreases, as the concentration of enzymes increased. ================= Evaluation ? ==== When I look at my results I can see that the increase in concentration actually has a faster reaction of the enzyme and the casein so the more of this enzyme there is the faster this can be broken down and so the rate of digestion is directly proportional to the amount of concentration of enzyme there is. I.e. the higher the concentration the faster the reaction and then the lower the concentration the slower the reactions are. In this case I can say that my hypothesis was right as I predicted that this would happen. ============== When looking at any of the results in biology we have to see that they are reliable and accurate. Reliability is when we take a look at the possible errors of the experimenter and see if he stopped the stop watch in time. Also we have to look at the accuracy e.g. if the glassware has got a big percentage error. If so how we need to reduce them. Also in the stopwatch as it can only go to seconds perhaps it would be better if we used milli seconds. I believe that the reliability of the results is low in this case when we look at all the possible errors. We could only take the person who did the experiments word in that the point in which he stopped the watch was the exact point that he could see the writing on the other side. A person?s judgement may be different to another person and therefore we will never be sure because of the accuracy of the human eye. For example one may have recorded the time when the solution was misty however another person may have recorded the time when the solution was completely clear. Although the results were good, I could see a few anomalous results that may not be that accurate. I can identify that there is a significant difference between some of the results, for example the 0.8 and 0.2 enzyme solution. The variance of this suggests that there is an error within the results. The reason for this error may be identified as error systematic error and random error. An example of systematic error is if the solution was made up incorrectly. For example too much enzyme may have been added and too little water, if the solutions were made up incorrectly than the true result is not correct at anytime. Random error can also been the reason for the error within the result. An example of this is tiredness of the determining the end point of the solution. Also, as time was limited many of the recorded results may have been rushed. As a result of this, the accuracy of the results may have been affected. Like I said before the end point of each solution may not be correct it may always be different. To gain a more reliable result, more repeats should have been done and an overall average should have been worked out. Also other variable such as the concentration of the substrate i.e. the milk is to be kept constant and the temperature is also to be kept constant.

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