the Effect of Copper Ions on a

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Introduction

Aim

Copper ions are heavy metals therefore copper sulphate (CuSO4) is likely to be an inhibitor. This is since heavy metals are known to be inhibitors having an effect on enzyme functioning. In this experiment I will investigate how a range of copper sulphate concentrations affects the ability of the enzyme amylase to hydrolyse starch into maltose.

Hypothesis

I predict that as I increase the concentration of CuSO4  the more time it will take for the enzyme amylase to hydrolyse starch into maltose thus the rate of reaction will decrease. This is since the Cu2+ is likely to alter the shape of the active site of amylase hence reducing the enzyme’s ability to catalyse the reaction.

I know that as I double the concentration of CuSO4 twice the volume of copper ions will be present for the same given volume. This therefore means that the chance of the copper ions colliding with amylase will increase by a scale factor of 2. The chance of a collision is random but doubling the concentration of CuSO4 means that the average chance of meeting copper ions will increase by a scale factor 2. Thus I quantitatively predict that as the concentration of CuSO4 doubles then the time for my reaction to take place will also double. Thus the concentration of CuSO4 will be proportional to the time of the reaction (see graph A) hence the rate of reaction will be inversely proportional the CuSO4 concentration (see graph B).

Predicted Graph A

 

        

CuSO4 Concentration (mol dm-3)

Predicted Graph B

Scientific Background

Amylase is a digestive enzyme which speeds up the rate of a reaction by reducing the activation energy required for the enzyme to hydrolyse starch into maltose, however at the end of the reaction the enzyme remains chemically unchanged. The enzyme hydrolyses the polysaccharide starch by breaking the glycosidic bonds converting it into the disaccharide maltose. This enzyme may be derived from animal, fungal and plant resources. (1)

Enzymes are biological catalysts which speed up chemical reactions that would normally happen very slowly. Enzyme molecules have a complicated three-dimensional shape due to the particular way the amino acid chain that makes up the protein is folded. This  gives the enzyme its catalytic ability. A few of the amino acids on the surface of the molecule fold inwards to make a specific indentation, called the active site, into which a particular substrate can fit. Once the enzyme and the substrate are joined they form an enzyme-substrate complex. The formation of an enzyme-substrate complex makes it possible for substrate molecules to be brought together to form a product. The product is released and the enzyme is free again to take part in another reaction.

Enzymes function by the ‘induced fit’ method. The substrate does not simply bind with the active site. It has to bring about changes to the shape of the active site to activate the enzyme and make the reaction possible. So small molecules may enter the active site, but they cannot induce the changes in shape to make the enzyme behave like a catalyst. The method suggests that when the enzyme's active site comes into contact with the right substrate, the active site slightly changes or moulds itself around the substrate for an effective fit. This shape adjustment triggers catalysis and helps to explain why enzymes only catalyse specific reactions.

The functioning of an enzyme is influenced by a number of factors such as temperature, pH, enzyme concentration, substrate concentration and the addition of inhibitors and activators.

Inhibitors are any substances which interfere with the formation of enzyme substrate complexes. They can act directly on the active site (competively) hence competing with the substrate molecule for the active site, or they can act indirectly on another area of the enzyme other than the active site called the allosteric site (non competitively) thus disrupting the enzyme’s tertiary structure hence preventing any substrate molecules binding with the enzyme. Competitive inhibitors are reversible however, non competitive inhibitors are irreversible.

Figure 1.1: Diagram to show non competitive inhibition (2)

Figure 1.2: Diagram to show Competitive Inhibition (3)

CuSO4 is likely to be non competitive inhibitor. This is since the Cu2+ ions present in CuSO4 is a heavy metal and heavy metals tend to be non-competitive inhibitors. This means that they inhibit the action of the enzyme-controlled reactions by attaching themselves to the enzyme molecule to an area other than the active site preventing the formation of enzyme substrate complexes. This therefore means that with fewer successful collisions fewer enzymes substrate complexes are produced thus the time period for the hydrolysis of starch into maltose increases. In this case, the extent of the inhibition depends entirely on the concentration of the inhibitor i.e. the copper sulphate and cannot be varied by changing the amount of substrate present i.e. the amount of starch. In this investigation the effect of varying concentration of CuSO4 is indicated by measuring the effect on a solution of amylase and starch using iodine.               

Preliminary Experiment

Apparatus

  • Bacterial amylase solution 0.2M, 100cm3 
  • Starch solution 0.1M, 100cm3
  • Copper sulphate solution 0.1M, 25cm3
  • Iodine solution 1.0M, 25cm3
  • 3 10cm3 syringes
  • 9 test tubes (3 for amylase, 3 for starch and 3 for the CuSO4 concentrations)
  • 1 test tube rack
  • Water bath set at 60oC since this is the optimum temperature for amylase. A water bath will be used to provide the solutions with a constant temperature.
  • Spotting tiles to monitor the changes in the reaction mixture with iodine.
  • Stop clock
  • Thermometer
  • Buffer solution pH 7.0

Spotting tiles will be used in this experiment since this will enable me to time my endpoint at which amylase is no longer hydrolysing the substrate starch. Comparisons in any colour change between each spotting well can also be observed when using a spotting tile thus my end point decision making can be carried out thoughtfully providing me with more accurate results.

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Risk Assessment

  1. CuSO4 is harmful if swallowed and may cause vomiting. Therefore hands must be washed after the experiment preventing the salt coming in contact with any food. If vomiting ensues then wash out the mouth and have a glass or two of water. If further vomiting takes place then seek medical attention. (4)

  1. CuSO4 may also be irritating to the eyes and the skin thus must be handled with care. If any solution comes into contact with the eye then flood the eye with gently running tap water for 10 ...

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