Catalase Assay Flashcards
How do enzymes increase the rate of a reaction?
By lowering the activation energy barrier
Which region on an enzyme will bind to a substrate?
The small region called an active site
What 3 letters do most enzymes end with?
ase
What can extreme temperature do to an enzyme?
Can cause denaturing of an enzyme which renders it useless and can no longer catalyze the reaction
Part 2 of this lab experiment tested the effects of what environmental factor on the activity of the enzyme?
pH
The dependent variable should be plotted on which axis? What about the independent variable?
Dependent = y
independent = x
What were the 2 objectives for this lab?
To describe the function of the enzyme catalase and state where catalase production occurs in the cell
To analyze experimental data to determine the effects of pH on catalase activity
Why are enzymes necessary for the essential reactions for life to occur?
The essential reactions to maintain life occur at a rate too slow to sustain life, so enzymes are required to speed the reaction rate up without increasing temperature
Define enzymes
Proteins that speed up the rate of reactions without being consumed by the reactions
What does an enzyme act on?
The reactant, aka the enzyme’s substrate
What forms when an enzyme binds to its substrate?
An enzyme-substrate complex
How does catalysis work?
When an enzyme and substrate are bonded, the enzyme enables the conversion of the substrate(s) into the product(s) of the reaction
What happens after the reaction has occurred?
the enzyme will release the product(s), making it available to bind with another substrate molecule
How quickly does catalysis occur?
So fast that a typical enzyme molecule acts on ~1000 substrate molecules per second
T or F: enzymes can catalyze any reaction because they can bind with any substrate -why/why not?
False. Enzymes can only catalyze specific reactions because they can only recognize and bind to its specific substrate(s)
How come enzymes can only bind to specific substrates?
Enzymes have unique 3D structures that are determined by its linear sequence(s) of amino acids (primary structure) and its active site will be compatible to fit with specific substrates
What determines the 3D structure of enzymes?
their linear sequence(s) of amino acids (aka primary structures)
What is the specificity of enzymes a result of?
the compatible fit between the shape of the enzyme’s active site and the shape of the substrate(s)
What function do the rest of the amino acids of the enzymes have?
they are involved in creating the framework that allows for the formation of the active site
What is the purpose of this experiment?
To conduct assays to measure the enzymatic activity of the enzyme catalase
Describe catalase
An enzyme that accelerates the decomposition of hydrogen peroxide into water and oxygen
What is hydrogen peroxide?
A harmful by-product of some metabolic reactions
Where is catalase located in cells?
the peroxisomes of eukaryotic cells
Where is catalase found in high concentrations?
Liver
What is the formula for the reaction of hydrogen peroxide?
H2O2 —> H2O and 1/2 O2
How was the reaction rate of catalase breaking down hydrogen peroxide be measured in this experiment?
by collecting the oxygen gas released as a product of the reaction
what will the assay of oxygen gas be used to determine?
How the rate of hydrogen peroxide breakdown is affected by changes in pH
What were the main materials for this experiment?
hydrogen peroxide
distilled water
pH buffers
catalase stock solution
What were the 7 pH levels tested?
1, 3, 5, 7, 9, 11, 13
What species was the catalase stock solution taken from? Common & latin name
Beef liver, Bos taurus
How was the stock solution of catalase prepared?
By blending 50g of beef liver with 100mL of distilled water then
diluting the solution with buffer (pH 7) until an enzyme solution within desired range was obtained
What was the purpose of diluting the catalase solution with a buffer?
to keep the pH constant
How was the reaction rate measured?
By recording the rate at which the water level fell in the graduated cylinder as hydrogen peroxide was injected into the flask and oxygen gas was being produced
What was the control in this experiment?
the catalase solution with the buffer (pH 7)
What was the purpose of the control in this experiment?
to rule out extraneous variables that could influence the results by ensuring the oxygen gas produced is actually the effect of the catalase enzyme
Also used as a comparison to determine whether pH has an effect on the activity
What was the reaction rate for the control solution?
0.00 mL/second = no reaction occurred
What percentage was the solution of hydrogen peroxide?
3%
Briefly describe the steps of the procedure for testing the control (pt B)
- pipette 4mL of pH 7 buffer and 1 mL of distilled water into rxn flask
- fill syringe with 5mL of 3% hydrogen peroxide
- attach masking tape (~16 cm long) to front of graduated cylinder
- fill graduated cylinder with water
- mark level of water meniscus on tape
- inject H2O2 into flask containing control and gently swirl flask at constant speed - start timer as soon as it is injected
- record water level every 5 seconds until the level did not change for 15 seconds or 2 minutes has passed
- measure volume of displaced water for each time interval from start of assay
what was the total volume of water displaced during the assay?
5mL
Briefly describe the steps of the procedure for testing the effects of pH on catalase activity (Pt C)
- added 4mL of pH buffer and 1mL of catalase solution into rxn flask
- fill syringe with 5mL of 3% H2O2
- put masking tape on graduated cylinder
- fill graduated cylinder with water
- mark water meniscus at the top
- inject H2O2 into the flask containing catalase solution and gently mix and constant speed - begin timer as soon as H2O2 is added
- record water level every 5 seconds until no change for 15 seconds or 2 minutes has passed
- measure volume change for each time interval
- repeat the steps until all 7 of the pHs have been tested
What did each assay differ in?
the pH of the buffer used
Why was the catalase paper important?
- previous studies could not accurately measure catalase activity
- previous studies focused only on catalase interactions with H2O2
- previous studies relied on slower and less advanced techniques for measuring the formation of enzyme complexes
how was catalase activity measured in the study from the paper?
rapid spectrophotometry and polarography
What was rapid spectrophotometry used to measure in the paper?
the decay of hydrogen peroxide
What was the platinum polarography used to measure in the paper?
production of oxygen
From the paper, what was the stable pH range for catalase enzyme activity?
pH 4.0-8.5
At what pH range was binding between catalase and substrate (methyl hydrogen peroxide) stable in the paper?
all pH values tested were stable, from 2-11
Did the authors of the paper attribute the loss of catalase enzyme activity to a change in the enzyme-substrate binding? why?
No because the binding between catalase and substrate was stable at all pH levels
What 4 substrates of catalase were studied in the paper?
- hydrogen peroxide
- ethanol
- formate
- nitrite
from the paper, what was the effect of pH on the ability of catalase to form complexes with the formate and nitrite substrates?
from pH 4.3-5 it was constant and readily detectable, but from pH 5-8, it dropped significantly
What did the researchers of the paper contribute reduced catalase activity below pH 4 to?
the formation of secondary inactive complexes between catalase and hydrogen peroxide
