Lactate Dehydrogenase Kinetics and Inhibition: Laboratory Analysis
It is well-known that enzyme kinetics and inhibition is a difficult but mandatory topic for any biomedical scientist to clearly know about. That is why we -medical students- have to understand this topic well-enough as soon our second year of studies. In order to comprehend it, to the necessary extent, we have to execute a laboratory experiment in which we study the properties and activity of enzymes and their inhibitors.
For the reasons above, an experiment was developed that allowed us to collect enough information and data, during our 3-hour laboratory period, to fully explore enzyme kinetics/ inhibition and obtain accurate values for Km, Vmax, and KI.
In this laboratory experiment we have used LDH as our enzyme as we have heard of the build-up of pyruvate in muscle cells during heavy exercising (as part of the anaerobic fate). We also did have the opportunity of studying LDH multiple times again during lectures both in this year and last year. Finally, for us who are interested in medical research, LDH is used as a diagnostic enzyme for a number of diseases, including heart attack.
It is also important to state that we have used LDH because it is present in nearly all human living cell and has a role of catalyzing the conversion of pyruvate to lactic acid as it converts NADH to NAD+. It can also catalyze the reverse reactions as part of the cori cycle in liver gluconeogenesis.
One of the main reasons we are using LDH is because its catalyzing reaction is relatively easy to monitor. We just have to take advantage of the absorption differences of coenzymes NADH and NAD+ using a simple technique to determine the concentration of protein sample (this technique is only applicable when the identity and the amino acid content of the protein(s) are known):
Using a spectrophotometer, we can measure the absorbance of the coenzymes at 340 nm, which is sufficient to provide a reliable measurement of the protein concentration. Then, using the Beer-Lambert Law, we calculate the concentration of the appropriate enzymes and proteins.
Methods
Before even starting the experiment, we obviously have to know the necessary background information to proceed with the experiment. That includes all the information mentioned in the introductory segment as well as the information provided in our lectures related to enzymes (enzymatic kinetics: Michaelis-Menten equation, Km, Vmax, and KI, the Lineweaver-Burk plot & enzyme inhibition: types and subtypes of inhibition, effects on enzymes).
Materials
The experiment requires the following materials, for each student:
12 Disposable cuvettes
1 marker
3ml of 6mM NAD+
10.3ml of 150mM CAPS at pH=10 (buffering agent, keeps pH at a relatively constant level)
5.1ml of 150mM lactate
1.2ml of 100mM oxalic acid
1.2ml of H2O
1.2ml of LDH (15U/ml)
Test tubes for all liquid substances mentioned above.
Plastic pipette
Spectrophotometer (to set at an absorption wavelength of 340nm and its timer to 120 seconds).
Protocol
Initially, we obtained the 12 disposable cuvettes and label them on the one side from 1 to 12 and on the other time we wrote our name. Then we prepared the samples in each cuvette by adding the following substances with the following volume:
Following, we carefully mixed the contents of each cuvette by using a plastic pipette. At the meantime, our professor showed us how to set the spectrophotometer to “time measurements” and specifically how to set the absorbance wavelength at 340nm and time to 120 seconds.
After being told by our professor how to blank the previous data on the spectrophotometer, we placed cuvette number 1 in the spectrophotometer and added 100μl of the LDH solution to the cuvette. As soon as we put the solution, we clicked “Start” and start measuring. We were careful yet fast because the reaction begins immediately after we put the enzyme inside the cuvette.
We proceed on doing exactly the same steps for the rest of the samples.
Results
In order to examine the results in a clear manner we plot the A340 vs time (seconds) for all 12 experiments:
Then we determine the slope for each of the 12 curves. The slope of each curve corresponds to the V0. Note that samples 1 and 7 are negative controls so their slope is expected to be zero.
The we analyze those results as follows:
Finally, we are able to plot the Lineweaver-Burk plot of the data in the absence and presence oxalic acid.
In order to calculate the Km, we have to set y=0:
For the inhibitory plot, Km is x=1/Km => Km=17.241
For the non-inhibitory plot, Km is Km= 3.1407 mM
In order to calculate the Vmax, we have to set x=0:
For both the plots the Vmax is equal to y=1/Vmax => Vmax= 0.0086 s-1
The inhibition type is competitive reversible because Vmax is the same for both inhibitory and non-inhibitory plots, whereas Km increases in the inhibitory plot.
Finally, to calculate the Ki of inhibitor we do as follows:
References
Peifeng tang, Jianlin Xu, Christopher L, Oliveira, Zheng Jian Li and Shile Liu (2017). “A mechanistic kinetic description of lactate dehydrogenase elucidating cancer diagnosis and inhibitor evaluation”, Taylor and Francis Online, 24th of January, 7 pages. Available at: https://www.tandfonline.com/doi/full/10.1080/14756366.2016.1275606
Larson, C, Reid, TR & Oronsky, BT 2018, Immunomodulatory fusion proteins, US20180134766, viewed 23 May 2018, retrieved from Scopus.
Yang Wang, Lian Wei, Dengbang Wei, Xiao Li, Lina Xu and Linna Wei (2016): “Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)”, MDPI Open Access Journal, 7th of January, 14 pages. Available at: https://www.mdpi.com/journal/ijms
Jennifer L. Powers,Natalie E. Kiesman,Connie M. Tran,John H. Brown and Vicky L. H. Bevilacqua (2007): “Lactate dehydrogenase kinetics and inhibition using a microplate reader”, IUBMB Journals, 11th of July, 5 pages. Available at: https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bmb.74
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