Nadh extinction coefficient at 340 nm

Activity of Lactate Dehydrogenase in Mammalian Tissues

Lactate dehydrogenase (LDH) catalyses the reversible reduction of pyruvate to lactate using NADH as a coenzyme.

CH3.CO.COOH + NADH + H+ CH3.CHOH.COOH + NAD+

The activity of LDH can be measured by continuously following the decrease in absorbance at 340 nm using pyruvate as the substrate. If the pyruvate concentration is varied, in the presence of a saturating concentration of NADH, the kinetic parameters of the enzyme with respect to pyruvate can be determined.

Method:

Prepare SEVEN tubes as shown below, initially omitting the pyruvate solution.

Tube No.

Phosphate buffer (ml)

NADH solution (ml)

Cytosol‡

(ml)

Pyruvate solution (ml)

1

2.6

0.1

0.3

0.0

2

2.5

0.1

0.3

0.1

3

2.4

0.1

0.3

0.2

4

2.3

0.1

0.3

0.3

5

2.1

0.1

0.3

0.5

6

1.9

0.1

0.3

0.7

7

1.6

0.1

0.3

1.0

Results:

Tube n0

Fast

Muscle

Liver

Heart

1

0

0

0.02

2

-0.017

-0.029

-0.042

3

-0.027

-0.045

-0.054

4

-0.030

-0.051

-0.058

5

-0.036

-0.053

-0.043

6

-0.035

-0.057

-0.047

7

-0.039

-0.062

-0.045

You should also attach: (i) A table containing the raw data for all three cytosols (ii) a table containing the calculated data used to create the Michaelis Menten plot and the linearised plot, (iii) the hand drawn Michaelis Meneten plot and (iv) the hand drawn linearised plot.

1. Write the equation for the reaction being studied, SHOWING STRUCTURES, and explain how the rate of the reaction is monitored in this experiment [do not just describe the protocol]. (5 marks)

2. FOR EACH OF THE THREE SETS OF DATA plot the rates of reaction in (A340/min against pyruvate concentration (mmol/l) ([S]) in the final assay volume. Explain your calculations by presenting a sample calculation for [S] below. (10 marks)

3. For each set of data convert the rate data from (A340/min to nmol NADH oxidised/min/mg tissue. The molar extinction coefficient of NADH at 340nm ((340) is 6300 litres/mol/cm, the reaction volume is 3 x 10-3 l, there are 109 nmoles/mole and the diluted cytosols contain 0.25 mg tissue/ml [liver & heart] or 0.125 mg tissue/ml [muscle]. Therefore 1 (A340/min equals 6349 nmol NADH oxidised/min/mg [heart & liver] or 12699 nmol NADH oxidised/min/mg [muscle]. Present an example calculation below, but tabulate each set of data. (10 marks)

4. Transform each set of the above data to obtain an appropriate linear plot [tabulate the manipulated data with correct units] and hence calculate the Km and Vmax values. Present the Km and Vmax for each cytosol below, including units. (15 marks)

5. Compare the Vmax and Km values in the three tissues and explain any similarities or differences in the three values. (10 marks)

6. Explain how you could characterise/determine the specific isozymes present in the three cytosolic preparations. (10 marks) [for this answer you may extend onto the next page].