Hydrogen peroxide is a common byproduct produced during metamorphosis in life beings. On accretion, H peroxide can hold assorted deductions on populating cells such as tegument upsets ( Schallreuter & A ; Rokos 2006 ) . Decomposition of H peroxide gives out harmless H2O and O, as shown by the equation 2H2O2 ( aq ) A??’A 2H2O ( cubic decimeter ) + O2 ( g ) .
The rate of decomposition of H peroxide is low and it can be increased by an enzyme called Catalase. An enzyme is basically a biological accelerator that can increase the rate of reaction but remains chemically unchanged at the terminal of the reaction ( Pang 1997, p.63 ) . Catalase readily speeds up the dislocation of H peroxide at a rate of 1000000s of H peroxide molecules per second ( Goodsell 2004 ) . It is peculiarly of import in liver cells and kidney cells for remotion of any toxins present in the blood watercourse to keep wellness ( Alberts et al. 2002 ) .
By changing the temperatures utilizing H2O baths and mensurating the clip taken for first bubbling and when bubbling remains changeless, the rate of dislocation of H peroxide can be calculated by the reciprocals of the mensural clip. The temperature at which the reaction rate is the greatest is referred to as optimal temperature ( Pang 1997, p.70 ) . That is the say, enzyme catalase catalyses the dislocation of H peroxide the most efficaciously at this temperature.
Purposes
In this experiment, the influence of temperature on the activity catalase is examined. We aim to happen out how its activity alterations over a scope of temperatures, in order to set up the optimal temperature of this enzyme catalysed reaction.
Methods
- Equipment
- Water bath ( 10oC, 35oC, 45oC, 60oC )
- Ice
- Beakers x2 ( for the ice bath )
- Thermometer ( to guarantee that the ice bath is & lt ; 10oC
- Test tubings x10
- Non-permanent marker
- Timer
- Hydrogen Peroxide x200mL
- Cork Borer
- Scalpels
- Watch spectacless
- Aluminum Foil
- Ruler
- Potato
- Pipettor
- Safety
Hydrogen Peroxide ( H2O2 ) is caustic and therefore safety spectacles must be worn to forestall oculus contact.
The decomposition of Hydrogen Peroxide would bring forth pure O so combustible stuffs must be kept good off.
Experimental process.
Set up the undermentioned setup harmonizing to the undermentioned conditions:
- Test Tube
- Temperature
- A
- & lt ; 10oC
- Bacillus
- 35oC
- C
- 45oC
- Calciferol
- 60oC
- Tocopherol
- Room temperature
Label the trial tubing to be used, harmonizing to the above tabular array.
Cover the stock H2O2 with aluminum foil to forestall decomposition under visible radiation.
Prepare enzyme catalase by infixing through the Centre of the murphy, with assistance of a cork bore bit.
Using the scalpel and swayer, cut 1cm pellets of murphy that was extracted with the cork bore bit.
Half-fill a beaker with tap H2O and add in crushed ice to do H2O bath for A. Wait for several proceedings to let stabilisation of temperature.
Insert trial tubing A, leave for a 3 proceedings before utilizing the 10mL pipette to add 8mL H2O2 to the trial tubing.
Add the 1cm pellet of murphy to the trial tubing.
Get down the timer and enter the clip required for the first bubbling to happen and the clip when the sum of bubbles produced remain changeless. Record all observations.
Repeat stairs 6-8 for trial tubing B to E but without add-on of ice. Put them straight into the H2O baths available in the research lab.
Repeat stairs 4-10 twice, entering all observations and consequences.
Average the consequences obtained for trial tubings A-E in each experiment ( disregarding outliers ) , and plot your consequences against temperature.
Generalize the graph to find the optimal temperature for enzyme activity.
Consequences
During the experiment, colourles gas bubbles were observed around the surface of the murphy. The murphy repeatedly sank to the underside of the trial tubing and so rose to the surface of the trial tubing.
Discussion
To accomplish consequences with greater truth, we have taken several safeguards. First, I was the individual who recorded the clip throughout the experiment and this could avoid disagreement caused by different reaction times among persons. Second, the usage of cork bore bit might guarantee unvarying sizes of murphies so that the sum of catalase would be comparatively the same. Third, stock H2O2 solution was wrapped to cut down unwanted decomposition under visible radiation. Fourthly, trial tubings with murphies were put into the H2O baths for a few proceedings before adding H2O2 and this allowed the temperature of the content to make that of the H2O baths. Last, no temperature was applied to tube E ( at room temperature ) and it acted as a control to demo that the alterations in activity of catalase resulted from alterations in temperatures.
From our consequences, enzyme activity is low at really low ( 0.5oC ) and high ( 61.2oC ) temperatures. At really low temperatures, substrate and enzyme molecules lack energy for hits and therefore adhering to catalase reactions ; and at really high temperatures, the change of the adhering site of enzyme sets in and the denaturized enzyme contact actions reactions with diminishing efficiency ( Pang 1997, p.70 ) . The optimal temperature for catalase activity was about 35.3oC, as indicated by the extremum in figure 2. This agrees with the research conducted by Yumoto et Al. ( 1999, p.67 ) , in which catalase works the best at approximately 30oC. However, this does non hold with our findings from figure 1 ( optimal temperature at around 40oC ) , whereas the extremum activity occurs at 35.3oC and 43.5oC. This might be explained by the fact that first bubbling occurred within a few seconds on add-on of H2O2 to potato and it was hard to mensurate this clip exactly. Therefore, the clip taken for bubbling remained changeless might be a better representation of our experimental results.
As respects the observations, it is apparent that the colorles gas bubbles are oxygen and the ground why murphy sank to the underside might be explained in footings of denseness. As dilute H2O2 solution was used, the denseness of solution can be assumed to be equal to H2O, which is about 0.9970gcm-3 at room temperature ( Aylward & A ; Findlay 2008, p.154 ) . It is sensible to foretell that murphy is basically denser than H2O and therefore it sinks. However, the gaseous O produced on the surface of the murphy can bring forth an upthrust to force the murphy upwards ( Goodwin 2002 ) . Therefore the murphy temporally floats on the surface. When the gaseous O is discharged at the surface, the consequence of denseness takes precedence once more, doing the murphy to drop.
Despite of careful design of our protocol, some experimental mistakes could hold arisen. The 8ml H2O2 was added on a 4ml footing by a pipettor and the timer was started at the first add-on. In other words, the mensural clip could hold differed from the existent one by several seconds. This inaccuracy might be improved by the usage of calibrated pipette so that the 8ml solution could be added via one add-on without any holds. Furthermore, we forgot to dry the trial tubing wholly in some of our tests and this could hold caused dilution of the H2O2. This mistake could be fixed by the usage of long cotton sticks to dry the interior parts of the trial tubing. Furthermore, the opinions of whether or non the sum of bubbles remained comparatively the same might be subjective and this job could be solved by turn toing our focal point on the volume of O evolved alternatively. For illustration, we might roll up the O over H2O and step the volume of it every 30 seconds for 5 proceedings with a graduated syringe ( Morris 2006 ) . In this manner, we might accomplish a better measuring of the reaction rate. From this defect in the design, I realized the importance of confer withing more beginnings instead than trusting on our ain cognition as we lack experience in experimental design.
Decision
In decision, enzyme catalase exhibits low activity at low temperatures ( 0.5oC ) and high temperatures ( 61.2oC ) . Its activity is the greatest at around 35oC. The experimental set-up was by and large satisfactory to minimise mistakes except of some defects such as the methodological analysis in mensurating the rate of the reaction. It is suggested that more research should be done in planing the experimental protocol.
