Reference no: EM132860526

Practical - Enzyme Activity

Learning outcome 1: Gain experience with pipetting and use of spectrophotometers
Learning outcome 2: Recognise why initial rates are used when measuring reaction rates
Learning outcome 3: Calculate molarity and a change of molarity over time using the Beer-Lambert law
Learning outcome 4: Calculate the enzyme activity in a crude extract using a series of dilution factors and appropriate units
Learning outcome 5: Produce a Lineweaver-Burk Plot using additional data
Learning outcome 6: Estimate the Km and Vmax of an enzyme using a Lineweaver-Burk plot

Practical component - Assaying for lactate dehydrogenase

In this practical you will be determining the number of enzyme units present in one ml of stock enzyme solution. One enzyme unit is defined as the amount of enzyme that catalyzes the conversion of 1 μmol substrate per minute.

Assay solutions:
50 mM potassium phosphate pH 7.4 (assay buffer) 4 mg/ml NADH
30 mM pyruvate
LDH stock solution from mouse liver

Assay Design:

1. In a microfuge tube dilute your stock enzyme solution in assay buffer as advised in the practical class. Make a total of 500 μl (this is your diluted enzyme solution)

2. Place 2.7 ml assay buffer in the cuvette, place it in the spectrophotometer and press the CAL button.

3. Add 100 μL NADH.

4. Add 100 μl diluted enzyme solution (from step 1) to the cuvette and mix thoroughly with the pipette tip.

5. Read the absorbance of this solution at 340 nm - this is the zero-rate. This reading is due to the NADH present and should be around 0.9 Abs -1.20 Abs. If the absorbance of your reaction mix is outside this range see a tutor for assistance. Leave the cuvette in the spectrophotometer for the next part.

6. Start the reaction by adding 100 μl pyruvate. QUICKLY mix the contents of the cuvette using the pipette tip. Start the timer as soon as you add the pyruvate. Take readings at regular intervals (~ every 15) and record these in a table similar to Table 1 from the tutorial. Plot the results on graph paper. When the reaction has slowed down, remove the cuvette.

7. Measure the initial rate from your graph. You are measuring the decrease in absorbance, so the number you obtain should not be negative. Aim for a rate of 0.05 to 0.3 AU/min (if it is not in this range ask a tutor).

8. Repeat the above steps, adjusting the amount of enzyme solution so that you get a good initial rate reading. (Remember to keep the total volume as 3 ml.) Once you have decided how much enzyme to add, repeat once more so that you have the reading in duplicate.

9. Average the rates obtained from your duplicate readings. Use the Beer-Lambert law (A = εcl) to calculate the concentration of NADH converted per minute. What is the amount (μmol) of NADH converted per minute in the cuvette?

10. How much had the stock enzyme solution been diluted before it was added to the cuvette? What volume of this diluted solution was added to the cuvette? Work out the activity of the stock enzyme solution.

11. If one unit of enzyme activity is the amount of enzyme that catalyses the conversion of 1 µmol substrate per minute, how many units/ml are present in the stock solution?

12. If, rather than working the activity out using the units of per ml, but rather wanted to work it out in enzyme units (mg protein)-1 what other assay would you have to perform?

6. Lineweaver-Burk Plot for lactate dehydrogenase

Additional lactate dehydrogenase assays were performed in order to determine the Vmax and Km of pyruvate for lactate dehydrogenase under these conditions. Remembering from your lectures on enzyme kinetics that Vmax is the rate of a reaction when the enzyme is saturated with substrate, which is the maximum rate of the reaction. The Km is the Michaelis constant, which is the concentration of substrate required for the reaction to proceed at half the maximum rate. To produce a Lineweaver-Burk plot, a minimum of four data points are required.

Table 1: Reaction rates for LDH with varying concentrations of pyruvate

1. Produce a table with the double reciprocal of the Table 1 values required to produce a Lineweaver-Burk plot (be sure to ensure you have the correct units).

2. Produce a Lineweaver-Burk plot for lactate dehydrogenase under these conditions.

3. What are the Vmax and Km of the reaction as catalysed by lactate dehydrogenase?

Attachment:- Enzyme Activity.rar