Table1.
This table shows how I will vary my independent variable-concentration of trypsin. I will keep the concentration of trypsin in a test tube controlled by measuring the volume of each trypsin and distilled water with different measuring syringe (to avoid contamination) and then adding both to the test tube simultaneously. This will bring about more accurate result and less % error.
Apparatus:
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Distilled water (room temperature) 15cm3
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Blank white sheet with large black X indicator
Method:
- Set out 6 test tubes ready for use when needed on a test tube rack
- Use a pencil/pen to label your test tubes directly on the glass.
- Dilute trypsin according to the different ratios shown in the table. Use distilled water at room temperature and a syringe to achieve the required dilution in test tube.
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Measure out 5cm3 of the casein substrate with a syringe and add it to the test tube containing the diluted trypsin and lightly swill.
- Start stopwatch as soon as solutions touch.
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Place the card with a large black X behind the test tube rack containing the test tube. The card with the X will give me an indication of when the suspension clears (when the X is clearly visible)
- Stop stopwatch and record results
- Repeat this process for the other five different concentrations of trypsin with the substrate casein and record time taken for suspension to clear.
- This whole process will then be repeated two more times to calculate an average, and to increase the reliability of the data collected of how long the suspension takes to clear at different concentrations.
Safety:
Only very few enzymes present hazards, because of their catalytic activity, to those handling them in normal circumstances but there are several areas of potential hazard arising from their chemical nature and source. These are, activity-related toxicity, residual microbiological activity, and chemical toxicity.
All enzymes, being proteins, are potential allergens and have especially potent effects if inhaled as a dust. Once an individual has developed an immune response as a result of inhalation or skin contact with the enzyme, re-exposure produces increasingly severe responses becoming dangerous or even fatal. Because of this, dry enzyme preparations have been replaced to a large extent by liquid preparations, sometimes deliberately made viscous to lower the likelihood of aerosol formation during handling.
- Tuck in ties and tie hair back and remove dangling jewellery
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Wear goggles and lab coat when handling solutions
- Trypsin is an irritant and should be handled with care
- Lab work top should be clear before experiment begins (e.g. bags, coats work books)
- Equipment should be set out quickly and correctly before any experiment begins this ensures maximum safety.
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Do not eat food, drink beverages, or chew gum in the laboratory
- Read all procedures thoroughly before entering the laboratory. Never fool around in the laboratory. Horseplay, practical jokes, and pranks are dangerous and prohibited.
- . Always work in a well-ventilated area.
- Work areas should be kept clean and tidy at all times.
- Be alert and proceed with caution at all times in the laboratory. Notify the teacher immediately of any unsafe conditions you observe.
- Dispose of all chemical waste properly. Never mix chemicals in sink drains. Sinks are to be used only for water. Check with your teacher for disposal of chemicals and solutions.
- Know the locations and operating procedures of all safety equipment including: first aid kit(s), and fire extinguisher. Know where the fire alarm and the exits are located.
Rate of Reaction
Reactions with enzymes are up to 10 billion times faster than those without enzymes. Enzymes typically react with between 1 and 10,000 molecules per second.
Fast enzymes catalyze up to 500,000 molecules per second.
Substrate concentration, enzyme concentration, Temperature, and pH affect the rate of enzyme reactions.
Temperature
Enzymes have an optimum temperature at which they work fastest. For mammalian enzymes this is about 40°C, but there are enzymes that work best at very different temperatures, e.g. enzymes from the arctic snow flea work at -10°C, and enzymes from thermophilic bacteria work at 90°C.
Up to the optimum temperature the rate increases geometrically with temperature (i.e. it's a curve, not a straight line). The rate increases because the enzyme and substrate molecules both have more kinetic energy so collide more often and also because more molecules have sufficient energy to overcome the (greatly reduced) activation energy.
The rate is not zero at 0°C, so enzymes still work in the fridge (and food still goes off), but they work slowly. Enzymes can even work in ice, though the rate is extremely slow due to the very slow diffusion of enzyme and substrate molecules through the ice lattice.
Above the optimum temperature the rate decreases as more and more of the enzyme molecules denature. The thermal energy breaks the hydrogen bonds holding the secondary and tertiary structure of the enzyme together, so the enzyme (and especially the active site) loses its shape to become a random coil. The substrate can no longer bind, and the reaction is no longer catalysed. At very high temperatures this is irreversible. Remember that only the weak hydrogen bonds are broken at these mild temperatures; to break strong covalent bonds you need to boil in concentrated acid for many hours.
pH
Enzymes have an optimum pH at which they work fastest. For most enzymes this is about pH 7-8 (physiological pH of most cells), but a few enzymes can work at extreme pH, such as protease enzymes in animal stomachs, which have an optimum of pH 1. The pH affects the charge of the amino acids at the active site, so the properties of the active site change and the substrate can no longer bind.
is found in the , and therefore, its optimum pH is in the neutral range to match the pH of the duodenum.
Enzyme concentration
As the enzyme concentration increases the rate of the reaction increases linearly, because there are more enzyme molecules available to catalyse the reaction. At very high enzyme concentration the substrate concentration may become rate-limiting, so the rate stops increasing. Normally enzymes are present in cells in rather low concentrations.
Substrate concentration
The rate of an enzyme-catalysed reaction shows a curved dependence on substrate concentration. As the substrate concentration increases, the rate increases because more substrate molecules can collide with enzyme molecules, so more reactions will take place. At higher concentrations the enzyme molecules become saturated with substrate, so there are few free enzyme molecules, so adding more substrate doesn't make much difference (though it will increase the rate of E-S collisions).
Results table:
Table2.
Hand drawn graph goes here
Interpretation of results
The aim of this experiment was to investigate the rate of enzyme activity with varying enzyme concentration. Different concentrations of trypsin enzyme were combined with 5cm3 of casein (2.0% concentration). I repeated the test three times with six different concentrations as shown in the results table and tested the rate of reaction by looking at the time taken for the milky suspension to clear, and finally calculate an average.
From my results I can interpret the following:
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When the concentration of trypsin enzyme is 0.0% is mixed with 5cm3 of casein nothing occurred. Different concentrations were obtained by diluting trypsin with distilled water as shown in table1. Therefore no trypsin was present at that particular dilution.
Collision theory explains how chemical reactions occur and why rates of reaction differ. For a reaction to occur, particles must collide. If the collision causes a chemical change it is referred to as a fruitful collision. Increasing the concentration of a solution leads to more collisions so the rate of the reaction goes up. In a less concentrated solution of trypsin, the number of collisions is low, so the rate of the reaction is slower. In a higher concentration of trypsin, more molecules are present; therefore more enzyme-substrate complexes; products formed, thus the faster rate of reaction.
- The table of results and the graph shows an obvious trend; as there is an increase in concentration of trypsin the faster the suspension clears. The graph illustrates this by the downward sloping curve. This is said to be proportionate.
Evaluation
During this experiment I encountered a few problems. The trial preliminary test a problem had arisen; the suspension did not seem to clear after a period of time, this occurred at all different concentrations. After a few trials I came to the conclusion that the substrate casein was not prepared properly. When the experiment begun I overcame this problem by continuously mixing the casein, this was to reduce the chance of any precipitate forming whilst not in use.
At 0.4% concentration of casein an anomaly occurred- the time taken for the suspension to clear was 21.61s, as shown in the computer generated graph and the hand-drawn diagram a kink is present, disrupting the smooth flow of the curve. There are number of reasons why this anomaly occurred but none I can define as 100% correct. When diluting- calculating distilled water to trypsin ratios was correct but measuring the content is different all together. An error could have happen when drawing in the solutions from the beaker with burette, as a (mm) too much can cause this error. A similar error could have occurred with the inaccurate syringes that were supplied to measure the casein; this was a lack of professional resources/equipment.
Improper cleaning of equipment- sterilisation of burettes and test-tubes may have lead to residues of previous equipments remaining in the medium and thus affecting the result.
The use of large scale syringes and burettes leads to inaccuracy as the scale doesn’t show the measurements in greater detail, e.g. In the process of measuring the casein with a 10ml syringe instead of a 5ml.
Extracts taken from Encarta reference library