The Effect of Substrate Concentration on Enzyme Activity

Question

What is the consequence of increasing the substrate concentration, as measured by thining the concentration of 3 % H peroxide in an aqueous solution ( 0.6 % , 1.2 % , 1.8 % 2.4 % and 3.0 % ) , on the rate of enzyme activity of the enzyme catalase, obtained fromBos primigenius ( bovine ) liver, measured by utilizing a stop watch ( ± 1 sec ) to obtain the clip it takes for the decomposition of H peroxide to force the filter paper phonograph record to the surface of the H peroxide solution?

Hypothesis

If the concentration of H peroxide is increased, so the rate of enzyme activity will besides increase until a certain point, so go changeless. This is because as the substrate concentration is increased, there are more possibilities for the substrate and enzyme to clash and adhere, doing an addition in the activity of the enzymes and hence increasing the rate of the decomposition of H peroxide ( making more O and H2O at a quicker gait, which would force the filter paper to the top of the H peroxide solution faster, diminishing the clip observed ) . However, since the enzyme concentration is unchanged, the substrate concentration can be increased to a point where all active sites of the present enzymes are already occupied with substrates, ensuing in impregnation of the enzymes, such that increasing the substrate concentration will no longer increase the enzyme activity.

Variables

Theindependent variableis the per centum concentration of the 3 % H peroxide in the aqueous solution. This will be measured by thining the H peroxide utilizing a graduated cylinder ( ±0.5mL ) . The five per centums will be 0.6 % , 1.2 % , 1.8 % , 2.4 % and 3.0 % . Refer to Postpone 1 for the concluding composing of the 5 conditions.

Table 1.The sum composing, in milliliter, of 3 % H peroxide and distilled H2O to make the concluding per centums for the five chosen conditions.

The dependant variablei s the clip it takes for the O bubbles to force the filter paper phonograph record to the surface of the H peroxide solution. This will be measured by utilizing a stop watch ( ±1 second ) and so recorded.

The controlled variables for this probe will include:

1. The temperature of the solution and the changeless milieus. This will be monitored by remaining in the same room for the continuance of the experiment and measurement and entering the temperature of the solution before every test. This is done because increasing the temperature of the solution ( or of the milieus ) causes an addition in the kinetic energy of the atoms, and hence increases the possibility of enzyme-substrate complex’s forming. Higher temperatures accordingly increase enzyme activity to a certain point, or until the optimal temperature is surpassed  ; commanding the temperature therefore minimizes the opportunity of obtaining skewed consequences. Toptima the temperature will corroborate that it has non been exceeded.
2. The pH and concentration of H peroxide. This will be done by utilizing the same H peroxide solution for every test of each status ( will be taken from the same bottle, 3 % H peroxide ) and mensurating the pH before every test utilizing a pH metre. No extra acids or bases will be added. This will be controlled because enzyme activity is decreased if the pH is increased or decreased from the optimal pH degree of the enzyme, hence doing denaturation and diminishing enzyme activity³. It is of import to observe that altering the concentrations of the H peroxide will change the pH somewhat, but non plenty to dispute the truth of the experiment. In add-on, different initial concentrations of H peroxide will take to different diluted concentrations (different conditions ) , so maintaining it changeless, at 3 % , is necessary.
3. The enzyme concentration. The enzyme catalase will be obtained from intermixing bovine liver and H2O. 15 g of bovine liver will be used to make the enzyme rich mixture, and will be used for each test. This is done because increasing the sum of enzymes for different conditions will accordingly increase the figure of active sites that the substrates can adhere to³, therefore increasing enzyme activity until the enzyme concentration surpasses the substrate concentration, where the enzyme activity will level out.
4. The volume of the combined solution. Each concluding solution will incorporate a different sum of H peroxide and distilled H2O ( based on which status is being performed and hence what per centum of H peroxide must be used ) . However, the combined volume of each status will be 20.0 ±0.5mL. This is done so that the filter paper phonograph record will go the same distance every clip ( from the underside to the top of the solution ) .
5. The trial tubings used will be the same size ( medium sized trial tubing ) . This is done to maintain the distance that the filter paper phonograph record must go consistent for each test. Increasing the size of the trial tubing will alter its dimensions. More significantly, it will increase the diameter of the trial tubing, thereby diminishing the distance the paper phonograph record must go to make the top of the solution ( because volumes of the solution are to be kept changeless ) which would later diminish the times obtained.
6. The filter paper phonograph record. The dimensions of the filter paper discs must be the same for every test. This will be done by utilizing the same filter paper to cut the phonograph record ( to maintain thickness invariable ) , and each disc will hold a diameter of 6mm, which will be ensured by utilizing a hole cowboy to cut the discs ( doing them perfect circles each clip ) .
7. The pureness of H2O. The H2O obtained will be from a packaged distilled H2O bottle, and will be used for the continuance of this experiment. This is done because different H2O beginnings may incorporate different mineral concentrations , hence could take to error in the information collected.

Materials

• 300.0 milliliter of 3 % H peroxide solution
• 220.0 milliliter of distilled H2O
• 15 g of bovine liver
• 25.0 milliliter graduated cylinders ( ± 0.5 milliliter ) ( x2 )
• Mercury thermometer ( ± 1°C )
• pH metre ( ±0.5 pH )
• Stopwatch ( ± 1 second )
• Medium trial tubings ( x5 )
• Test tubing rack
• Electric liquidizer
• 10cm x 10cm filter paper
• Hole cowboy ( 6mm diameter )
• 50 milliliter beaker ( ±5 % )
• Rubber trial tubing stoppers ( x5 )
• Stir stick
• Goggles

Safety Precautions: Sodium hydrated oxide is a caustic substance, and can therefore lead to clamber annoyance and do hurt to the eyes. Wear safety goggles for the continuance of this lab, or until the process has been completed ( solution has been disposed of and equipment has been washed ) . Afterwards, rinse your custodies with soap and H2O. If sodium hydrated oxide comes in contact with tegument, wash with soap and H2O instantly.

Method

1. Find a level tabular array with equal infinite ( around 1m x 1m ) to execute five conditions, each with five tests, and remain at that place for the continuance of the experiment.
2. Gather all needed stuffs ( mention to list of stuffs above ) and make an observation tabular array. This tabular array should include adequate infinite for the measurings of the per centum concentration of H peroxide ( % ) , clip observed for the paper filter phonograph record to make the surface of the solution ( ± 1 sec ) , temperature of the solution before the reaction ( ±1°C ) , pH of the solution before every test ( ± 0.5 pH ) and qualitative observations to be recorded for five conditions ; each with five tests. Give your tabular array a rubric.
3. Take the 15 g of bovine liver and set into the electric liquidizer. Taking a 25.0 milliliter graduated cylinder, step 20.0 ±0.5mL of distilledH2O and pour into the liquidizer. Blend until the substance is wholly assorted together ( approximately 40 seconds ) . If there are any seeable balls of liver, intermix once more for20 seconds, oruntil the consistence is smooth. Pour the mixture into the 50 milliliter beaker. The same enzyme mixture will be used for the continuance of the lab.
4. Take the five medium trial tubings and put them gently onto the trial tubing rack. They will be used throughout the lab.
5. Grab the bottle of 3 % H peroxide solution and maintain it for the continuance of this lab. Make non add any substances to this solution or alter it in any manner. Taking the fresh 25.0 milliliter graduated cylinder, step out 4.0 ±0.5mL of H peroxide. Pour into a trial tubing.
6. Take the 25.0 milliliter graduated cylinder that was used in measure 3, and step 16.0 ±0.5mL of the samedistilledH2O ( use the same H2O beginning for each test ) . Pour the H2O into the trial tubing that already holds the H peroxide. This completed solution now contains 0.6 % H peroxide ( mention to Table 1 ) and should be 20.0 ±0.5mL. Using the quicksilver thermometer, step and record the temperature of the solution ( ±1°C ) . Then step and record the pH of the solution utilizing the pH metre ( ±0.5 pH ) .
7. Repeat stairss 5-6 for the staying four medium trial tubings that were placed onto the trial tubing rack.
8. Take the 10 centimeter x 10 cm filter paper and, utilizing the whole cowboy, cut out a filter paper phonograph record that is 6mm in diameter. Take the filter paper phonograph record and coat it to the full in the enzyme rich liquid that was prepared in measure 3. Chuck the filter paper phonograph record to take any extra liquid, and topographic point it on the terminal of a gum elastic stopper ; it will lodge to the stopper because it is wet.
9. Take one trial tubing from the trial tubing rack, splash with a splash stick foremost to avoid the subsiding of the H peroxide solution, so infix the stopper steadfastly.
10. Simultaneously invert the trial tubing and get down mensurating the clip it takes for the paper phonograph record to make the top of the H peroxide solution in the trial tubing utilizing the stop watch ( ± 1 sec ) . Once it is at the surface of the solution, halt the stop watch. Record the clip shown and reset the stop watch.
11. Repeat stairss 8-10 with the staying 4 trial tubings in order to hold a sum of 5 tests for this status.
12. Take the 5 medium trial tubing, 5 trial tubing stoppers and two 25.0 milliliters graduated cylinders and rinse them exhaustively with soap and H2O. Once clean, glib dry with paper towels so that the stuffs can be reused for the following status.
13. Repeat stairss 4-12, but replace the per centum of H peroxide with a new status until all five conditions have been performed ( 0.6 % , 1.2 % , 1.8 % , 2.4 % and 3.0 % ) . Mention to Postpone 1 for the right volumes of 3 % H peroxide and distilled H2O needed for each status.
14. When finished, dispose of all solutions by blushing them down the drain with H2O and return all equipment to their original locations.