Investigate what effects the duration of exposure to a high temperature has on enzyme denaturation.

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Biology Individual Investigation- Enzymes

Aim: To investigate what effects the duration of exposure to a high temperature has on enzyme denaturation.

Introduction: Living cells carry out many biochemical reactions. These reactions take place rapidly due to the presence of enzymes. Enzymes are biological catalysts that speed up the rate of reaction without being used or destroyed. Enzymes also have the ability to control reactions. The ability of an enzyme to function depends on the specific shape of the protein molecule. The intricate shape created by the polypeptide folding is a key factor in the theories of enzyme action, in which temperature has a great effect.

Trypsin

   Trypsin is the enzyme that will be used in this investigation. Trypsin is an endopeptidase enzyme, or pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. Its optimum pH is 8.0. Trypsin is a constituent of pancreatic fluid, splitting complex proteins into simpler components that can be absorbed and used in reconstructing body proteins.

   Although Trypsin can begin the digestion of a wide variety of foods rich in protein; for example eggs, pork, chicken and Soya, it is specific. The hydrolysis of Trypsin is limited to bonds formed by either lysine or arginine residue. Most proteins have these two amino acids next to each other at some points in their polypeptide chain, and so Trypsin is able to partially digest it.

Substrate-Casein

   Organophosphorus compounds such as diisopropylflurophosphate and natural inhibitors from the pancreas inhibit trypsin. Soybean, lima bean and egg whites are also sources of natural inhibitors including Casein, a protein precipitated when milk is mildly acidified. When hydrolysed, the suspension begins cloudy, but becomes clearer as the products dissolve. This reaction is catalysed by Trypsin, producing amino acids as the reaction product. The effect of the duration of exposure to a high temperature on Trypsin denaturation can be noted by the changes in clarity of the casein suspension.

Hypothesis; an increase in duration of exposure at a particular temperature will increase the time of clarification of casein.

Preliminary Experiments

I will conduct a series of trail experiments to establish:

  • What volumes and concentration of enzyme and substrate to use
  • What range of temperatures to investigate

This will also enable me to specify what quantities and concentration of reactants give me the most favourable results. Additionally, I will be able to standardise a basic procedure, which needs to be less time consuming yet efficient, enabling me to work in a restricted time frame, which is a common problem in a classroom environment.

Apparatus used in preliminary experiments

  • Casein suspension 4%
  • Trypsin solution0.5%
  • Distilled water
  • Test tubes and rack
  • Syringes and pipettes
  • Water baths (40oC and 60oC)
  • Thermometers
  • Black card
  • Stopwatch

Method

  • Set up water bath at 60oC
  • Pipette 5cm3 of Trypsin into test tubes
  • Incubate in water bath
  • Once the enzyme has reached 60oC, immediately begin the stop clock and take the test tubes out at regular timed intervals of 5 minutes up to 10 minutes
  • Leave the enzyme to cool back down to room temperature, 25oC
  • Mix the enzyme with 5cm3 of casein in a beaker and begin the stopwatch immediately observe the changes in clarity of the casein suspension by checking against the black card to judge the end point
  • Record the time taken for the casein suspension to clear

Results

   At 60oC, there seemed to be little or no change in clarification of the casein suspension.

This may have been due to two factors:

  • The substrate concentration was too high so there is more competition between the substrate molecules for the active site.
  • The enzyme had denatured

   It is important in any investigation, that apart from the variables being investigated, other external factors remain constant to ensure fair testing is maintained.

   For this investigation to work successfully I need to find a suitable volume ratio which is a compromise between enzyme and substrate to establish which gives the most favourable rate of reaction. The concentration of Trypsin is a fixed amount and so the concentration of casein can only be changed.

Therefore;

  • I will make up different concentrations of substrate to establish which gives the most favourable rate of reaction.
  • I will also test the enzyme at 40oC to see if this is the optimum temperature of the enzyme

I made up the concentration of casein as follows: (Distilled water will be added in correct proportions to make up a total volume of 5cm3 of the casein suspension)

Results

At 40oC

Rate at which casein suspension cleared increased with exposure time.

At 60oC

   Rate at which casein suspension cleared decreased with exposure time. This may suggest the enzyme had denatured. However, at the most dilute ratio of the casein suspension, the rate at which it cleared increased with increased duration of exposure. (See * in above table).

Concentration of the casein suspension

   The results indicate that the rate of reaction was fastest when the casein was diluted using the ratio of 1.5:1, which is 3cm3 casein and 2cm3 distilled water. I addition, it increased with duration of exposure. When considering the rate of an enzyme-catalysed reaction, the proportion of enzyme to substrate molecules should be considered. If there is more substrate molecules than enzyme molecules then the number of active sites available is the limiting factor and the enzyme is working at Vmax. When there are fewer substrate molecules than enzyme molecules, the reaction will take place more quickly as there is less competition for active sites.

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Main Investigation

   The focus of this investigation is to investigate the effect of duration of exposure at high temperatures on Trypsin denaturation.

Prediction

   As the duration of exposure increases, the time of clarification will increase. However, I predict that although there will be a net increase in the time of clarification, it will be lower at the highest temperatures in comparison to the lowest temperature in the experimental range

Reasons

Duration of exposure

   The duration of exposure to a high temperature and its effect on Trypsin denaturation is one of the variables being investigated. Suitable ...

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