Labortaory 6: Lactate Dehydrogenase Activity:  Effects of Enzyme and Substrate Concentrations

 Background:

Enzyme and substrate concentrations are important in determining the catalytic rate of unregulated enzyme catalyzed reactions.  In addition, the experimental determination of enzyme activities for given substrate concentrations can be used to quantify the Km and Vmax of enzymes, and assess qualitative changes in enzyme function in the presence of activator and inhibitor molecules.

 Experimental Procedures:

You will work with stock solutions of the enzyme lactate dehydrogenase and lactic acid to assess the change in catalytic rate for the LDH enzyme at different substrate and enzyme concentrations.

 1. Prepare your initial reagent as for the lactate assay, but for now, do not add any LDH enzyme.  I recommend making a 25 mL sample of the reagent.  To make sure you have excess NAD⁺, double this amount for this kinetic assay.

 2. Prepare the LDH solutions by adding 10 uL of the LDH stock (record which stock Units/mL you used) to 1 mL of distilled water.  From this, add 10 uL to 1 mL of distilled water (high LDH sample), and again to 2 mL of distilled water (low LDH sample).  Calculate the LDH activity in each of your final (1 and 2) samples.

 3. Prepare the lactate (La) solutions by adding 10 uL of the La stock to 1 mL of distilled water (La-1).  From this sample, pipette 250 uL to 1 mL of distilled water (La-2).  Calculate the concentration of La in each of your final (1 and 2) samples.

 4. You then need to assay for enzyme activity for a given La concentration, but different enzyme amounts, as well as for a given enzyme amount and different La concentrations.  To do this, follow the sample conditions of the table below.

 5. Prepare the spectrophotometer for kinetic assays as shown in the lab demonstration.

 6. Pipette 2 mL of reagent into a cuvette for use as the blank sample.  Keep this sample for latter use for the blank in repeat kinetic assays.

 7. Pipette 2 mL of reagent into another cuvette.  When ready to run the assay, add your desired LDH sample, and then your desired lactic acid sample.  Rapidly cover the cuvette with a piece of parafilm, invert, and then place into the spectrophotometer.  Press the "Measure Sample" button.

 8. The kinetics program will now run, and provide an absorbance data point every 10 s for 1.5 min.  Make sure the program is running by listening for the grinding sound each 10 s sample.  Despite a slight delay in graphing, you will see each successive data point graphed in the display.

 9. After the time course of the kinetic assay has expired, press the "tabular" button to see the numeric data.  Use the up and down triangular arrow keys to scroll forward and backward through the tabular data.  Record this data for latter entry into Excel to perform linear regression to derive the slope (delta Abs/s).  You should be able to convert this to a mmol/L/s expression based on Beers Law.

 10. Repeat this procedure for the remainder of the conditions. 

 Data Analyses:

* Graph the time responses and linear regressions for each condition.  I suggest that you overlay the data for the two conditions of lactic acid on one graph, and then overlay the two LDH conditions on another graph.

* Even though you only have 2 data points, compute the data necessary to complete reciprocal plots for LDH for enzyme catalytic rate and substrate concentration.

* From the reciprocal plot, estimate the Km and Vmax of LDH.

* Is LDH an effective enzyme based on your enzyme kinetic data and knowledge of the in-vivo pyruvate concentrations (1 to 15 mmol/kg wet wt) inside skeletal muscle?

 References

Consult any Biochemistry text that contains enzyme kinetics content.