Bio 103 Catalase Enzyme Set-up & materials instruction

Wil Franklin

October 2005

 

Solutions:

      2 L of 7.4 buffer from Fisher packets.

      Catalase A = 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)

      Catalase B = dilute catalase A 1/200 with 7.4 pH buffer (1 L for all)

      Catalase C =  1/5 of B diluted with 7.4 pH buffer (200 ml - 40 of B dilute up to 200)

      Catalase D = 1/10 of B diluted with 7.4 pH buffer  (200 ml 20 of B dilute to 200)

 

At student tables for 12 total groups:


      Test tube rack

      Volumetric test tube

      Large test tube that fits over volumetric tt

      15 fiberglass filter paper (whatman 934-AH, 2.5cm)

      Forceps

      Paper towels

      Timer

      Marking Tape & Pen

      (3) 50 ml beakers for trials in Parts II-IV

      100 ml beaker (or culture vials) for 40 ml of Catalase solutions

      100 ml beaker for 3% H2O2 use by students

      100 ml beaker for pH buffer

      2-3 paramecium culture vials

      (1) 3ml Grad. Pipette or 1 ml grad. Syringe for H2O2 in Part I


 

At lab station for all to use:


      3% H2O2

      Chilled H2O2 (on ice)

      Hot H2O2

      Chilled Catalase B

      Hot Catalase B Just boiled then remains in warm water

      500 ml 2.0 pH buffer

      1 L 10.0 pH buffer

      1 L  pH 7.4 Buffer

      400 ml Catalase A concentrated solution

      400 ml Catalase B 1 units

      400 ml Catalase C 1/5

      400 ml Catalase D 1/10

      (8) 50 or 100 Grad. Cylinders to measure solutions

      Tub of Ice for student experiments

      (6) - 500 ml beakers to hold ice, then hot water

      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 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 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 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 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 B solution and place on ice. (Change the catalase B solution to match the temperature condition of your trials, ie use hot Catalase B and place in hot bath during your hot trials and room temp Catalase B 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 B solutions that match the temperature condition)