For BMB 443 Students

Determining Aldolase Activity

Aldolase is an enzyme used in glycolysis to break down glucose products into

energy. This enzyme specifically breaks down fructose 1,6-bisphosphate (FBP) into
glyceraldehyde 3-phosphate (Ga3P) and dihydroxyacetone phosphate (DHAP).
Aldolase is usually found in either the muscle or brain, in which to determine the
activity it must be extracted from. Once the cells are lysed, the aldolase enzymes
must be isolated from all other proteins and anything else inside the cells. It isn’t
until the aldolase is purified that it can be used in an aldolase activity assay in which
the absorbance rate can be recorded. The absorbance rate can be used in Beer’s Law
to calculate Units of activity. In this procedure, some of the reagents can be
dangerous if in contact with skin or swallowed so it is imperative that gloves are to
be worn at all times.

Reagents
 Rabbit muscle
 0.03 M KOH
 0.001 M EDTA
 0.1 N KOH
 100% saturated ammonium sulfate
 Solid ammonium sulfate
 1.0 N H2SO4
 Equilibration buffer (10 mM Tris HCl, pH 7.5; 5 mM EDTA)
 Dialysis tubing
 Phosphocellulose resin
 Elution buffer (2.5 mM FBP [B-grade]: 50 mM Tris  HCl, pH 7.5; 5mM EDTA)
 dH2O
 Histidine  HCl (0.2 M, pH7.2)
 NADH (1 mg/ml = 1.3 mM in His  HCl buffer pH, 7.2)
 αGP (glycerol phosphate) dehydrogenase (~40-50 IU/ml)
 FBP (0.1 M, pH 7.2)

Instruments/Glassware
 Centrifuge with GSA rotor
 Glass wool
 Centrifuge with SS34 rotor
 Column
 Stand
 Tubing
 Pinch and screw clamps
 Graduated cylinder
 Ice filled gloves
 Genesys 5 Spectrophotometer *for all absorbance readings use 3ml dH2O for
the blank*
  dH2O rinsed out plastic container
 Eppendorf tubes
 Micropipettes
 Glass pipettes
 Oakridge tubes
 200 ml flask
 Two 15 ml tubes
 Ten 4.5 ml cuvettes

*For this procedure all volumes under 1000 μl are measured out with
micropipettes; volumes larger are measured out with glass pipettes*

Solubilization/Extraction
In this step the proteins will be released from the cells

1. Obtain 80 g of ground rabbit muscle

2. In a 500 ml beaker hand stir the rabbit muscle in 150 ml of cold 0.03 M KOH,
0.001 M EDTA for 20 minutes
3. Separate the mixture into two centrifuge tubes and centrifuge at 10,000 x g,
4°C for 10 minutes in a GSA rotor
4. Decant the supernatant solution, leaving the solid in the tube to be disposed
of, and filter the supernatant through a funnel plugged with pre-moistened
glass wool
5. Adjust the pH of the filtered supernatant to 7.5 with cold 0.1 M KOH
6. Measure and record the volume of the solution using a graduated cylinder
and then remove a 1 ml aliquot to be saved in an eppendorf tube labeled
Fraction I for later analysis

Salt Fractionation
In this step salt is added to disrupt the protein-water interactions and allow the
protein to precipitate out of solution

1. Slowly add an equal volume of 100% saturated ammonium sulfate solution

and stir slowly on ice for 1 hour using a magnetic stir bar
2. Repeat steps 3 and 4 to centrifuge and filter the supernatant again
3. Measure and record the volume of the solution using a graduated cylinder
and then remove a 1 ml aliquot to be saved in an eppendorf tube labeled
Fraction II for later analysis
4. Gradually add 65 g of solid ammonium sulfate per liter of solution with slow
magnetic stirring to the filtered supernatant
5. Measure the pH of the solution with a pH meter and record its value
6. Adjust the pH to 7.5 using 1.0 N H2SO4
7. Store the solution in the cold room for a week

Dialysis
In this step the protein solution is desalted

1. Centrifuge at 10,000 x g, 4°C in a GSA rotor for 30 minutes

2. Remove supernatant and measure and record the volume using a graduated
cylinder and then remove a 1 ml aliquot to be saved in an eppendorf tube
labeled Fraction III for later analysis
3. Measure and record the pH of Fraction III
4. Dissolve the precipitate from the previous centrifugation in as little volume
of very cold equilibration buffer as possible
5. Transfer the solution into two Oakridge tubes and centrifuge at 10,000 x g,
4°C for 15 minutes in a SS34 rotor
6. Measure and record the volume of the supernatant and remove a 0.1 ml
aliquot and dilute it to 1 ml with equilibration buffer in an eppendorf tube
labeled Fraction IV for later analysis
7. Prepare the dialysis tubing by soaking it in distilled water
8. Tie a knot at one end and fill the tubing with water to check for leaks
9. Empty the tubing and place the supernatant inside and tie the other end
10. Place the filled dialysis tube in a bucket of cold equilibration buffer to dialyze
for one day

Affinity Column Chromatography

In this step everything except the aldolase protein that is bound to the resin is
washed out of the column to result in a purified product

1. Remove the sample from the dialysis tubing and place into Oakridge tubes to
be centrifuged at 10,000 x g, 4°C for 15 minutes in a SS34 rotor
2. Measure and record its volume with a graduated cylinder and remove one
0.1 ml aliquot and dilute to 1 ml with equilibration buffer in an eppendorf
labeled Fraction V for later analysis
3. Clamp the column to a stand and attach the tubing to the outlet and run it
into a discard flask
4. Use pinch and screw clamps to close the tubing
5. Suspend the prepared phosphocellulose resin by gently swirling and stirring
it
6. Slowly pour the resin slurry into the column
7. Open the screw clamp just enough to achieve an approximate flow rate of 1.5
ml/min using a graduated cylinder to adjust the flow rate
8. Equilibrate the column by allowing 75 ml of equilibration buffer to run
through it *Never let the column run dry*
9. Attach ice filled gloves to the column to keep it cold and obtain a 1 ml/min
flow rate
10. Let the buffer run to the surface of the resin bed and stop the flow with the
pinch clamp
11. Gently apply the aldolase solution to the top of the resin
12. Allow equilibration buffer to run through the column and collect this “wash”
in a cold flask
 13. Continually check the absorbance of the “wash” using the Genesys 5
Spectrophotometer at 280 nm until the absorbance falls below 0.1 Abs
14. Stop the flow with a pinch clamp
15. Measure and record the final volume of the “wash” using a graduated
cylinder and save 1 ml in an eppendorf tube labeled “wash” for later analysis
16. Add 100 ml of elution buffer to the column and collect 40 1 ml fractions
17. Measure and record the absorbance of each fraction at 280 nm after it is
collected
18. Remove 0.3 ml from the fraction with the highest absorbance reading and
dilute to 1 ml with equilibration buffer in an eppendorf tube labeled “peak”
for later analysis
19. Remove another 0.1 ml from the fraction with the highest absorbance
reading and dilute to 1 ml with equilibration buffer in a eppendorf tube
labeled “peak 1/10” for later analysis
20. Pool together the fractions whose absorbance readings have indicated that
they are part of the peak in a 15 ml tube
21. Pool together the fractions whose absorbance readings are lower and have
indicated that they are part of the tail end of the peak in another 15 ml tube
22. Measure and record the volumes of each tube
23. Remove 0.1 ml from both the pooled peak fractions and the pooled tail
fractions and dilute to 1 ml with equilibration buffer in separate eppendorf
tubes labeled VIA and VIB respectively for later analysis

Enzyme Activity Assays

In this step the absorbances of the saved fractions are measured and recorded

1. Create a master mix of reagents for the nine saved aliquots that includes:
o ml dH2O
o 13.0 ml Histidine HCl
o 3.0 ml NADH
o 2.0 ml FBP
o ml αGP dehydrogenase
2. Measure out 2.01 ml of master mix into nine 4.5 ml cuvettes labeled with the
fraction that they will contain
3. One at a time, add 10 μl of fraction into its respective cuvette, mix, and
immediately read and record the change in absorbance at 340 nm using the
Genesys 5 spectrophotometer

Enzyme Activity Calculations

For each fraction:
1. Multiply the absorbance rate by 0.48 to get μmoles/minute, which is also
known as Units
2. Multiply the amount of Units by the dilution factor to get Units/ml
3. Multiply Units/ml by the ml of fraction solution recorded to get total units of
enzyme activity