This project analyzed how non-covalent bonding affects the structure and activity of lactate dehydrogenase (LDH) isoenzymes. Spectrophotometric assays were used to measure reaction rates of rabbit muscle LDH and pig heart LDH exposed to heat or vortexing, with and without cofactors NADH and oxamate. Results showed that non-covalent bonding did not affect LDH's active site but increased stability, with pig heart LDH more stable than rabbit muscle LDH. The study provides insight into LDH isoenzyme functioning.

1. LDH STABILITY AND NON-
COVALENT BONDS
PhoebeYang
Mentor: Robert A. Edwards

2. Introduction
Lactate dehydrogenase is an enzyme that plays an important role in the conversion
between pyruvate and lactate in the process of cellular respiration. It catalyzes the
conversion of pyruvate to lactate as it converts NADH to NAD+. NAD+ and oxamate are
molecules that commonly and naturally bond with LDH. NAD+ is a cofactor, while
oxamate is a noncompetitive inhibitor.The presence of these additional bonds change the
bonding structures within the LDH. Different isoenzymes of LDH are found in different
parts of the human body.These isoenzymes also vary structurally.
Spectrophotometric assays are a method used to measure the rate of enzyme reactions.
Changes in intensity of the intensity of light absorbed or reflected are measured to
determine the rate of reaction.

3. Objective
The objective of this project is to observe how structures and bonding of different LDH
isoenzymes affect their activity and stability.This would allow for a better understanding
of the functioning of LDH.

4. PROCEDURE AND
METHOD

5. General Method:
■ Use protein database to calculate non-covalent bond strengths
■ Use spectrophotometric assays to measure rate of reactions before and after
vortexing or heating using LDH both with and without NADH and oxamate bonds
Materials:
■ Buffer solution (15mM potassium buffer containing 50mM KCl at pH 7.2)
■ Rabbit muscle LDH (diluted 1000 times)
■ Pig Heart LDH (diluted 100 times)
■ Pyruvate (20mM)
■ NADH (2mM and 600um)
■ Oxamate (20mM)

6. Set up:
Enzyme Solution: Enzyme
only
Assay mix: 2.5mL Pyruvate
(20mM), 2.5mL NADH
(600um), 4mL Buffer
Each Cuvette: 900uL assay
mix, 100uL enzyme
solution (enzyme solution
is added immediately
before assay)
Assay:
Assay each cuvette at
340um and record in 10s
intervals for 1 minute
Heat enzymes at 56
degrees celsius for 10
minutes
Or
Vortex enzymes for 1
minute
Assay:
Assay each cuvette at
340um and record in 10s
intervals for 1 minute
Experiments on LDH containing no
NADH and oxamate bonds
Experiments on LDH containing
NADH and oxamate bonds
Set up:
Enzyme Solution: 200uL
Oxamate (20mM), 200uL
NADH (2mM), 1600ul
Enzyme
Assay mix: 2.5mL Pyruvate
(20mM), 2.5mL NADH
(600um), 3mL Buffer
Each Cuvette: 800uL assay
mix, 200uL enzyme
solution (enzyme solution
is added immediately
before assay)
Assay:
Assay each cuvette at
340um and record in 10s
intervals for 1 minute
Heat enzymes at 56
degrees celsius for 10
minutes
Or
Vortex enzymes for 1
minute
Assay:
Assay each cuvette at
340um and record in 10s
intervals for 1 minute

7. NON-COVALENT
BONDING

11. DATA COLLECTION
AND ANALYSIS

12. Pig Heart LDH (no NADH and oxamate)Vortex
Control: Manipulated:

13. Rabbit Muscle LDH (no NADH and oxamate)Vortex
Control: Manipulated:

14. Pig Heart LDH (no NADH and oxamate) Heat
Control: Manipulated:

15. Rabbit Muscle LDH (no NADH and oxamate) Heat
Control: Manipulated:

16. Pig Heart LDH (with NADH and oxamate)Vortex
Control: Manipulated:

17. Rabbit Muscle LDH (with NADH and oxamate)Vortex
Control: Manipulated:

18. Pig Heart LDH (with NADH and oxamate) Heat
Control: Manipulated:

19. Rabbit Muscle LDH (with NADH and oxamate) Heat
Control: Manipulated:

20. T-test and Statistical Significance
■ 3 t-tests were conducted in each set of experiments:
– T-test between rabbit muscle LDH control and manipulated
– T-test between pig heart LDH control and manipulated
– T-test between change in activity of rabbit muscle LDH and pig heart LDH

21. Results:
■ Vortex (no NADH and oxamate)
– Pig heart LDH: Statistically significant
– Rabbit muscle LDH: Statistically significant
– Change in pig heart LDH vs Change in rabbit muscle LDH: Statistically significant
■ Heat (no NADH and oxamate)
– Pig heart LDH: Statistically significant
– Rabbit muscle LDH: Statistically significant
– Change in pig heart LDH vs Change in rabbit muscle LDH: Statistically significant
■ Vortex (with NADH and oxamate)
– Pig heart LDH: Statistically significant
– Rabbit muscle LDH: Not statistically significant
– Change in pig heart LDH vs Change in rabbit muscle LDH: Statistically significant
■ Heat (with NADH and oxamate)
– Pig heart LDH: Statistically significant
– Rabbit muscle LDH: Statistically significant
– Change in pig heart LDH vs Change in rabbit muscle LDH: Statistically significant
T-test and Statistical Significance

22. RESULTS AND
CONCLUSIONS

23. Overall Findings
■ Non-covalent bonding within and between molecules of LDH do not affect bonding at
the active site of the enzyme
Conclusions
■ Analysis of non-covalent bonds showed stronger non-covalent bonding to be found in
muscle LDH
■ Experiments showed heart LDH to be more stable

24. ACKNOWLEDGEMENTS
Many thanks to Dr. Robert A. Edwards for mentoring
me throughout this projecy

25. References
Durdenko, E.V., Kuznetsova, S. M.,Tikhonenko, S. A., Emelyanenko,V. I., & Saburova, E.
A. (2010).Temperature stability of lactate dehydrogenase in complex with
anionic polyelectrolyte poly(styrenesulfonate). Biophysics, 55(4), 535–543. doi:
10.1134/s0006350910040032
Karl,W. F., & Peters,T. (1967).Thermal Stability of Lactic Dehydrogenase from Human
Tissues. AmericanJournal of Clinical Pathology, 47(2), 171–174. doi:
10.1093/ajcp/47.2.171
Vesell, E. S., &Yielding, K. L. (1966). Effects of pH, ionic strength, and metabolic
intermediates on the rates of heat inactivation of lactate dehydrogenase
isozymes. Proceedings of the National Academy of Sciences, 56(4), 1317–
1324. doi: 10.1073/pnas.56.4.1317