Bio 103 Catalase Enzyme Ð Set-up & materials
instruction
Wil Franklin
October 2005
Solutions:
á
2 L of 7.4 buffer from
Fisher packets.
á
Catalase ÒAÓ = 1.0g
Catalase/1000 ml 7.4 pH buffer (make fresh at a minimum every other day, for
best results every day). (500ml for Part I)
á
Catalase ÒBÓ = dilute
catalase A 1/200 with 7.4 pH buffer (1 L for all)
á
Catalase ÒCÓ = 1/5 of B diluted with 7.4 pH buffer
(200 ml - 40 of B dilute up to 200)
á
Catalase ÒDÓ = 1/10 of B
diluted with 7.4 pH buffer (200 ml
Ð 20 of B dilute to 200)
At student tables for 12
total groups:
á
Test tube rack
á
Volumetric test tube
á
Large test tube that
fits over volumetric tt
á
15 fiberglass filter
paper (whatman 934-AH, 2.5cm)
á
Forceps
á
Paper towels
á
Timer
á
Marking Tape & Pen
á
(3) 50 ml beakers for
trials in Parts II-IV
á
100 ml beaker (or
culture vials) for 40 ml of Catalase solutions
á
100 ml beaker for 3% H2O2
use by students
á
100 ml beaker for pH
buffer
á
2-3 paramecium culture
vials
á
(1) 3ml Grad. Pipette or
1 ml grad. Syringe for H2O2 in Part I
At lab station for all to
use:
á
3% H2O2
á
Chilled H2O2 (on ice)
á
Hot H2O2
á
Chilled Catalase ÒBÓ
á
Hot Catalase ÒBÓ Just
boiled then remains in warm water
á
500 ml 2.0 pH buffer
á
1 L 10.0 pH buffer
á
1 L pH 7.4 Buffer
á
400 ml Catalase ÒAÓ
concentrated solution
á
400 ml Catalase ÒBÓ 1
units
á
400 ml Catalase ÒCÓ 1/5
á
400 ml Catalase ÒDÓ 1/10
á
(8) 50 or 100 Grad.
Cylinders to measure solutions
á
Tub of Ice for student
experiments
á
(6) - 500 ml beakers to
hold ice, then hot water
á
hot plate with 1 L warm
water
PART I: SUBSTRATE
LEVEL EFFECTS ON RATE & END PRODUCT
Methods:
Note - rinse thoroughly all beakers to hold solutions and be sure not to mix pipettes or syringes in different solutions.
1) Add 0.5 H2O2 to volumetric test
tube (*Note:½ the class use 1.0 ml instead of 0.5 ml H2O2)
2) Fill volumetric test tube with 7.4 pH buffer
3) With forceps, dip filter disc in catalase A for 20
seconds
4) Dry on paper towel for 10 seconds
5) With forceps, pick up filter disc, fold and stuff in
the top of volumetric flask
6) Place large test tube over volumetric test tube.
7) With finger slide volumetric test tube tightly against bottom of large test tube in order to seal
8) Invert twice to mix, then place inverted to trap gas
in test tube rack, remove finger and start timer
9) Read initial volume of gas trapped in volumetric test
tube
10)
Read gas (O2)
level every 30 seconds until no new gas is produced Ð until you obtain the same
volume for three consecutive readings. Shake test tube to dislodge gas bubbles just before taking measurement.
PART II: ENZYME CONCENTRATION EFFECTS ON
RATE
Methods:
Note Ð rinse thoroughly all beakers to hold solutions and be sure not to mix pipettes or syringes in different solutions.
1) Fill 50 ml beaker with ~ 40 ml of stock 3% H2O2
2) With forceps, dip filter disc ¾ in catalase B
for 20 seconds
3) Dry on paper towel for 10 seconds
4) Drop in 50 ml beaker and start timing immediately
5) Stop timer when filter disc rises completely to top of
H2O2
6) Record the time - This will be considered one trial of
the rate of catalase activity at concentration B
7) With forceps remove the filter disc and discard
8) Repeat two more trials for a total of three trial with
catalase B
9) Repeat steps 1-8 for catalase C & D solutions
PART III: EFFECTS OF pH ON ENZYME ACTIVITY
Methods: Note Ð rinse thoroughly all beakers to hold
solutions and be sure not
to mix pipettes or syringes in different solutions.
1) Fill 50 ml beaker with 35 ml of pH 2.0 buffer solution
2) Add 5 ml of 3% H2O2
3) With forceps, dip filter disc ¾ in catalase B
for 20 seconds
4) Dry on paper towel for 10 seconds
5) Drop in 50 ml beaker and start timing immediately
6) Stop timer when filter disc rises completely to top of
H2O2
7) Record the time - This will be considered one trial of
the rate of catalase activity at pH 2.0
8) With forceps remove the filter disc and discard
9) Repeat two more trials for a total of three trial with
pH 2.0 (do not change solution in beaker until new set of trials at new pH)
10)
Repeat steps 1-9 for pH
7.4 & pH 10 buffers
PART IV: EFFECTS OF TEMPERATURE ON ENZYME
ACTIVITY
Methods: Note Ð rinse thoroughly all beakers to hold
solutions and be sure not
to mix pipettes or syringes in different solutions.
1) Fill a clean 50 ml beaker with chilled Catalase ÒBÓ solution and place on ice. (Change the
catalase ÒBÓ solution to match the temperature condition of your trials, ie use
hot Catalase ÒBÓ and place
in hot bath during your hot trials and room temp Catalase ÒBÓ for the room temperature trials.)
2) Fill 50 ml beaker with 40 ml of chilled 3% H2O2
3) Place beaker in ice bath (or hot bath in next set of
trials)
4) With forceps, dip filter disc ¾ in catalase B
for 20 seconds
5) Dry on paper towel for 10 seconds
6) Drop in 50 ml beaker of 3% H2O2 and start timing
immediately
7) Stop timer when filter disc rises completely to top of
H2O2
8) Record the time - This will be considered one trial of
the rate of catalase activity at chilled temperature (or hot temperature in
next series of trials)
9) With forceps remove the filter disc and discard
10)
Repeat two more trials
for a total of three trial at this temperature (do not change solution in
beaker until new temperature trials are set up)
11)
Repeat steps 1-9 for
room temperature & hot conditions (use H2O2 and
catalase ÒBÓ solutions that match the temperature condition)