The Effect Of Copper Sulphate On Pepsin Activity.

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By Rohin Butani
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The Effect Of Copper Sulphate On Pepsin Activity

Introduction

My coursework is investigating the way copper sulphate affects pepsin activity. Pepsin is an enzyme. Enzymes are globular proteins, which have a specific 3D structure, which is determined by its tertiary or quaternary structure. In spite of their size only a small area is functional, the active site, which binds with the substrate as shown below in Fig.1.

Enzymes act as biological catalysts which mean they are used to speed up reactions which otherwise would take longer to complete. They work by lowering the activation energy, the minimum amount of energy, required by the substrate, casein, for it to hydrolyse.

Key

A.        The amount of activation energy required without the use of an enzyme.

B.        The amount of activation energy required with the use of an enzyme

As you can see from Fig.2 the amount of activation energy which is to be overcome for a successful reaction is considerably lower with the help of an enzyme.

The way in which an enzyme works is similar to that of a lock and key. The substrate temporally attaches itself to the active site to create an enzyme substrate complex. Once together the enzyme hydrolyses the substrate and breaks it up. It then releases the products. If we relate this to the lock and key theory it is evident that the enzyme’s active site, ‘the lock’, is specific to the substrate, ‘the key’. However this is not entirely true as the enzyme can change shape slightly to fit other similar fitting substrates; this is known as the ‘induced to fit theory’.

Pepsin is one of three proteolytic, protein digesting, enzymes in the digestive system; the other two are trypsin and chymotrypsin. Pepsin is found in the gastric glands of the stomach. It is secreted into the stomach in the form of gastric juices along with hydrochloric acid, rennin and mucus. It acts to catalyse the hydrolysis of proteins; it does this by severing links between particular types of proteins, making them break down into smaller components including peptides or amino acids.

There are several factors that affect the rate at which enzymes work: pH, temperature, concentration, surface area, volume and the presence of an inhibitor. The optimum pH for pepsin is between 2-4, which is considerably acidic. This is because when it is secreted along with the hydrochloric acid it is still able to work efficiently without denaturing. The enzyme works best at 35-40oC due to the fact that it is used at body temperature, however can still work up to 50oC.

Copper sulphate is an inhibitor of enzymes such as pepsin. Due to this, I will now explain how inhibitors affect enzyme rate of reaction in more detail.

An inhibitor basically means that it can ‘inhibit’ or slow down the speed at which enzymes work so that they are no longer active. There are two main categories of enzymes inhibitors that work in two very different ways:

  • Competitive inhibitors

Competitive inhibitors, like substrate, fit the active site of the inhibitor. Due to this they are also known as active site directed inhibitors. By binding with the enzyme like this, the enzyme cannot bind with the substrate therefore cannot carry out hydrolysis. These types of inhibitors are only temporarily attaches to the enzyme however they still are competing with the substrate for the enzyme, hence the name competitive inhibitors.

  • Non-competitive inhibitors

Competitive inhibitors do not compete for the active site with the substrate unlike competitive inhibitors, but instead bind at a site away from the active site, known as the allosteric site. This causes the tertiary structure to change, consequently changing the shape of the active site, so that the substrate no longer fits. These types of inhibitors are permanent making the enzyme completely inactive. Copper sulphate and other heavy metal ions such as silver or mercury are effective non-competitive inhibitors. Copper sulphate will attach itself permanently to the pepsin causing the active site of pepsin to change. This means that it will be unable to hydrolyse casein, as it will not be able to form the enzyme substrate complex.

Hypothesis

As the concentration of copper sulphate increases the time taken for pepsin to catalyse the hydrolysis of casein will increase.

Reason for hypothesis

I believe that if you increase the concentration of copper sulphate, the inhibitor, it will slow down the rate of reaction between the casein and pepsin, as more pepsin molecules will be made inactive faster. As a result it will take longer for pepsin to catalyse the hydrolysis of casein with the remaining pepsin molecules that are not inhibited.

Variables

There are several variables that may affect my experiment they include:

  • Independent Variable:
  • Concentration of copper sulphate used
  • Dependant Variable:
  • Time take for casein solution to go clear
  • Absorbency readings from colorimeter every 30 seconds
  • Control Variables
  • Volume of copper sulphate
  • Concentration of pepsin
  • Volume of pepsin
  • Concentration of casein
  • Volume of casein
  • PH of casein, copper sulphate and pepsin solution
  • Temperature of the casein, copper sulphate and pepsin solution
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In order to carry out a fair, accurate and reliable investigation to find the effect of copper sulphate on pepsin activity I have decided to keep all of the variables constant and only alter the concentration of copper sulphate solution. I have described why I intent to keep the following variables constant below:

Volume of copper sulphate: If I increase volume of copper sulphate solution more copper sulphate molecules will inhibit more pepsin particles therefore slowing down the rate of reaction making the test unfair and altering my results.

Concentration of pepsin: If I increase the concentration of pepsin more ...

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