BIOC 2014 Bioenergetics and Metabolism

LDH

Name: Jahnika Blair

I.D Number: 620131434

Date of experiment: 04/11/20

Subject/Title: Affinity Chromatography of LDH

Aim/Objectives: [4 marks]

To use a group-specified Ligand, Procion Red or Blue, to purify lactate dehydrogenase from rat
liver supernatant to determine the amount of interacting proteins that can be retained by the column.

Method
[3 marks]

Four grams of rat liver were homogenized in 40 ml ice cold 10 mM K + phosphate buffer, pH 7.0
(buffer A) and centrifuged for 30 min at 25,000 rpm. 38 ml of supernatant (S) were recovered.

Set up the micro-column as follows:

1. Lightly tamp a small plug of glass wool into the pasteur pipette with the fine
glass rods provided. If the plug is too hard, you will get a very slow flow rate - start again.

If the plug is too loose, the gel will elute as well as buffer - start again.
2. Attach tubing to the pipette and gently grip the tubing with the clamp.

3. Clamp the pipette into a burette stand.

4. Fill the column with water and adjust the flow rate with the tubing clamp.

5. Introduce gel slung (slurry) into the column until the bed is just below the constriction in the
glass.

6. Wash the column with at least 3 bed volumes of buffer A (WITHOUT THE 1 M NaCl !!!)

NEVER LET THE GEL BED RUN DRY. If it does, start again.

Now apply 0.2 mls (S) to the column allowing it to just run into the surface of the bed. Allow it to
equilibrate for about 5 minutes.
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LDH

Place a measuring cylinder under the outlet.

Wash the column with 6.0 mls of buffer A (NO NaCl), collecting three 2.0 ml fractions (F1, F2,
F3).

Wash the column with 6.0 mls of buffer A containing 1 M NaCl collecting three 2.0 ml fractions
(F4, F5, F6).

After appropriate dilutions you will now determine protein content and LDH activity in (S) and in
fractions F1, F2, F3, F4, F5 and F6.

Lactase Dehydrogenase Assay

Pyruvate condenses with dinitrophenylhydrazine to form a DNP hydrazone, and when this is made
alkaline a colour develops. A standard solution of pyruvate is used to construct a calibration curve,
against which to determine the amount of pyruvate formed enzymically from the lactate.

The calibration curve and enzyme assays should be performed simultaneously AND IN
DUPLICATE !!!

This is the time consuming part of the experiment, prepare as much as possible while the column is
running.

Calibration (all volumes in ml)

Pyruvate standard
Substrate solution
Distilled water
DNP hydrazine
________________________
1 2 3 4 5 6
________________________
0.0 0.1 0.2 0.3 0.4 0.5
1.0 0.9 0.8 0.7 0.6 0.5
0.3 0.3 0.3 0.3 0.3 0.3
1.0 1.0 1.0 1.0 1.0 1.0
________________________

Mix well, incubate at 37°C for 20 min, then stop reaction with 0.4M NaOH
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LDH

10 10 10 10 10 10
________________________
Mix well, leave at room temperature for 10 min.

Read OD at 440 nm.

Enzyme assay (all volumes in ml)

Dilute (S) 1:20 and (F1→ F6) 1:10

________________________________________________
Control (S) (F1) (F2) (F3) (F4) (F5) (F6)
(S) 20 10 10 10 10 10 10
________________________________________________

Substrate
solution 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Sample 0.1 (S) 0.1 0.1 0.1 0.1 0.1 0.1 0.1
20
Distilled
water 0.2 - - - - - - -

NAD+
Solution - 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Mix well, incubate at 37°C for 15 min, then add

 BIOC 2014 Bioenergetics and Metabolism
LDH

DNP
hydrazine 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Mix well, incubate at 37°C for 20 min, then stop reaction with 0.4 M NaOH

10 10 10 10 10 10 10 10
_________________________________________
Mix well, incubate at room temperature for 10 min. Read OD at 440 nm.

Draw a calibration curve of OD vs ml pyruvate solution.

Subtract OD of control from all assays.

Read off the unknown values in mls pyruvate.

Assuming that the relationship between mls pyruvate and moles pyruvate formed/min is linear, and
that 0.3 mls pyruvate = 0.5moles/min, calculate the activity of LDH in each fraction.

Results:
Table 1.: Absorbance obtained for the calibration of protein utilizing BSA at 595 nm, using
different volumes and concentrations of BSA
Volume of BSA/µl Concentration of Absorbances/OD Average
BSA (mg/ml) Units595nm Absorbances/OD
1 2 Units595nm
100 0.332 0.221 0.277
80 0.274 0.291 0.283
60 0.113 0.107 0.110
40 0.147 0.159 0.153
20 0.076 0.076 0.076
15 0.111 0.116 0.114
10 0.048 0.056 0.052
5 0.044 0.042 0.043
0.00 0.000 0.000 0.000
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LDH

0.1 ml of standard was removed twice to create duplicates and 5 ml of Bradford reagent was
added to each tube and absorbance readings taken at 595nm.

Table 1.1 Absorbances of the fractions obtained from affinity chromatography with Bradford’s
reagent at 595 nm for fractions obtained from the red and green gel.
Absorbance/ OD Units595nm
Sample
Red Green
S 0.375 0.299
F1 0.026 0.167
F2 0.023 0.105
F3 0.033 0.100
F4 0.039 0.112
F5 0.003 0.124
F6 0.035 0.125
0.1 ml of the diluted supernatant and fractions were used, and 5 ml of Bradford reagent added
to each to be assayed.
The concentration above indicates the amount of protein (LDH) in each fraction obtained from
the chromatography using Sepharose red and green. Each sample was however diluted, so the
actual volumes are therefore obtained by multiplying by the dilution factor.

Table 2. Absorbances at 440 nm of the duplicate solutions containing pyruvate standard, DNP
hydrazine and substrate solution used for calibration curve
Test Tube Volume of First Absorbance/ Second Average
pyruvate OD Units Absorbance/ OD absorbance/OD
solution/ml Units Units
1 0.00 0.000 0.000 0.000
2 0.1 0.040 0.046 0.043
3 0.2 0.062 0.058 0.060
4 0.3 0.106 0.105 0.1055
5 0.4 0.153 0.145 0.149
6 0.5 0.183 0.175 0.179
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LDH

Table 3. showing the absorbances of the fractions obtained using Sepharose red and Sepharose
green gels at 440nm wavelength after conducting the test for pyruvate using DNP hydrazine.
Absorbance Readings (OD Units)
Sample Red Green
Average Average
1 2 1 2
Control 0.000 0.000 0.000 0.000 0.000 0.000
S 0.193 0.189 0.191 0.009 0.271 0.140
F1 0.052 0.054 0.053 0.006 0.107 0.057
F2 0.073 0.075 0.074 0.037 0.088 0.063
F3 0.057 0.050 0.054 0.062 0.047 0.055
F4 0.083 0.080 0.082 0.035 0.107 0.071
F5 0.047 0.044 0.046 0.058 0.037 0.048
F6 0.042 0.043 0.0425 0.043 0.034 0.039

1. Table showing absorbance vs BSA concentration [5 marks]

2. Table showing pyruvate calibration absorbance reading [5 marks]
3. Table showing absorbance reading for LDH assay. [5 marks]

Calculation
 Sample calculation of dilution of BSA and samples + tabulate the rest

[5 marks]

 Volume all fractions and S [4 marks]

 Sample Calculation of protein mg/mL (Extrapolate value from graph, factor in D.F and find how
much is in 1 mL) + tabulate other protein conc. [5 + 5 marks]

 Sample Activity Calculation mmol/min/mL (Extrapolate mLs of pyruvate from calibration then
use ratio (0.3 mls pyruvate= 0.5 moles/min) to find moles/min of pyruvate. Then convert the
activity to mmol/min/ml. + tabulate other activities [5 + 5 marks]
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LDH

 Sample Specific Activity Calculation (divide Activity by protein) + tabulate other specific
activities [5 + 5 marks]

 Sample calculation of Purification factor (divide specific activity of fraction by that of

supernatant) + tabulate other purification factor [5 + 5 marks]

 Sample calculation for Total activity (activity multiplied by total volume) + tabulate other total
activities [5 + 5 marks]

 sample calculation of recovery of activity (total activity of fraction divided by total activity of S
multiplied by 100) + tabulate recovery of other fractions [5 + 5 marks]

Graphs

 Bradford Calibration Curve (absorbance versus BSA concentration) [5 marks]

 Pyruvate calibration Curve (absorbance vs volume of pyruvate mls) [5 marks]
 Bar chart comparing protein concentration, activity and specific activity for each fraction using
both green and red gel [10 marks]

Discussion [20 marks]

 Is there a specific difference between the two gels based on protein concentration, activity and
specific activity.
 Suggestion an explanation as to why you recovered more activity than that applied.
 Give a method to investigate why you recovered more activity than that applied.
 How else can you express recovery?

Conclusion [4 marks]

Sources of Error/Limitation [5 marks]

References (at least 3 - APA) [5 marks]

****TOTAL MARKS = 150****