12) Gas syringe (100cm3)
I will use a gas syringe to measure the amount of oxygen released in 30 seconds when I react the hydrogen peroxide with the liquidised liver
13) Hydrogen peroxide
I will be using hydrogen peroxide as the substrate which the liver enzymes will catabolically breakdown.
14) Distilled water
I will add distilled water to the blender when liquidising the liver for dilution purposes.
15) Chemical waste bowl
I will use a chemical waste bowl to dispose of my chemicals such as H2O2 and liver as these chemicals could cause problems if disposed of carelessly.
16) Plastic disposable gloves
Hydrogen peroxide is an irritant, so when covering the top of the funnel stop the oxygen escaping out of the top I will put plastic disposable gloves on to protect my hands
Choice of equipment
I have chosen to use a 500cm3 volumetric flask, gas measuring syringe and measuring cylinder because they are highly accurate in their measurements.
Safety issues
Hydrogen peroxide is an irritant and corrosive in its concentrated form and should be handled when working with and around it and in its disposal. It is also poisonous if swallowed. Safety goggles should be worn at all times to protect our eyes from contact with the liver and especially the H2O2. Also when dealing with hot water in the water baths I must be careful not to burn myself.
Background information on enzymes in relation to this experiment
Enzymes are biological catalysts made from amino acid polymers, which are present in every living organism. Enzymes have the ability to speed up the rate of reaction considerably without having to be used in the process; this is done by reducing the activation energy required by giving a surface area for the substrate to bind its complementary enzyme.
By increasing the temperature of the substrate and catalyse it will speed up the rate at which particles move and therefore increasing the number of successful collisions. The optimum temperature for which liver enzymes will work would be around 37ºC based on the humans constant body temperature, controlled by homeostasis.
After certain temperatures enzymes tend to denature and not fit with their complementary substrate. In the case of liver the temperature at which denaturing occurs is between 40-50ºC.
When the hydrogen peroxide binds to the liver enzyme’s active site a catabolic reaction takes place the
In this investigation, I intend to explore the one of the factors that affect the rate of enzyme catalysis. My research from textbooks and the Internet suggests that this depends on several factors; temperature, pressure, pH and concentration. After research and careful consideration, I have decided to first look at how a change in temperature could affect the rate of reaction.
In order to design a suitable experiment and make a credible prediction, I must first explore more closely how temperature is likely to affect the rate of catalysis. Enzymes are specific - they only control one type of reaction; therefore I must use one specific enzyme in my experiment, in order to find a clear way of measuring the rate of reaction. Although they are specific, all enzymes work in a very similar way and have similar properties. They are all globular proteins and are all biological catalysts, they increase the rate of a given reaction without being used up and their presence does not change the nature of the reaction or the end product. Enzymes work by having an active site, made from amino acids. Here, substrate molecules will bind with the enzyme (and other substrate molecules if necessary) and a reaction takes place. The enzyme itself is not affected and releases the new chemical after the reaction. After release of the end product, more substrate molecules can bind with the active site.
Enzymes can catalyse anabolic reactions or catabolic reactions (involved in breakdown). The diagram above shows an anabolic reaction. In a catabolic reaction, the reverse would happen.
Using the information gained here together with my knowledge of kinetic theory, it is possible to understand how temperature affects the rate of reaction. Kinetic theory states that when a substance is heated, energy is given to the particles and they speed up. Therefore when heat is applied to an enzyme and substrate, the particles speed up, increasing the rate at which they bind with each other. This would suggest that the rate of reaction should increase as the temperature is increased. This is not quite true, as there is a limit to the temperature at which an enzyme can work because excessive heat causes an enzyme to become denatured and stop working. Also, there is a minimum temperature at which an enzyme can function. Every chemical reaction requires activation energy in order to get started. Although enzyme catalysis greatly reduces this, some energy is still required. Because of this the reaction is still unable to happen below a given temperature (this varies depending on the type of enzyme and reaction, as does the maximum temperature). If warmed to above the activation temperature, an enzyme will work again as normal. A denatured enzyme, however, is damaged and will not work again even if cooled below the optimum temperature.
Increasing reliability and accuracy of results and minimising errors
In this experiment due to the vast amount of variables, so there is a high chance of errors occurring in measurement and therefore causing inconsistent and conflicting results. I will use various checks and procedures to minimise the chances of these occurring.
From preliminary test, I have found that the measurements can sometimes be hard to judge, as there is a thick layer of foam formed from when it was blended which is hard to remove. To bypass this predicament, as the thickness of the foam is consistent I will include the layer of foam in volume of liver measurement.
Another problem, which I realised in the preliminary tests, was the fact that the liver and hydrogen peroxide slowly readjusted to room temperature after removed from the water bathes. I will not be able to prevent this, but it will be minimised by its effect by carrying out this processes of moving it to the conical flask then adding the hydrogen peroxide as fast as possible so the temperate fluctuation will be vastly reduced.
Method
- Blend liver with 200ml of distilled water in a blender
- Pour blended liver into a measuring cylinder, measuring 10cm3 then pour into a clean test tube(repeat 5 times)
- Pour H2O2 into a measuring cylinder, measuring 2cm3 (repeat 5 times)
- Put three of each of tests tubes of H2O2 and liver in a test tube rack in a different temperature water bath (30, 40 or 50) and leave it there until the substrate and liver catalyst adjust to the temperature.
- Place 2 of the remaining test tubes (1 H2O2 and 1 liver catalyst) in a beaker full of ice till the temperature adjusts to 10°C
- Leave the remaining 2 test tubes in a test tube rack so they maintain their temperature of room temperature.
- Once one temperature set of liver/Hydrogen peroxide have been allowed to adjust to the temperature required pour the liver into a conical flask and put the bun on then pour the H2O2 in through the funnel and cover the top with your hand. Then start the stopwatch timing 30 seconds. (see problem A)
- Once the 30 seconds has passed, move my hand from the top of the funnel and note down the volume of oxygen collected in the gas syringe. (See Problem B and C). (repeat stage 3 for each set of temperatures)
Results
Analysis
Upon analysis of the table of results I can determine that my predictions were reasonably accurate and that, as predicted the most gas was released in the 30 seconds time period when the liver and the hydrogen peroxide were adjusted to the temperature of 40°C this is because it is closes to the body temperature of 37°C maintained by homeostasis. However there were minor flaws in my prediction as there was small amount of oxygen released when the temperature was at 50°C, this could nevertheless have been caused by the problem of displacement of oxygen when the hydrogen peroxide was added to the liver.