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Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Influence of salts on Michaelis-constant values for NADH
Clinical Chemistry
|December 1, 1977
Summary
Buffer and salt concentrations significantly impact enzyme assays, affecting the Michaelis constant for lactate dehydrogenase. Understanding these inhibition effects is crucial for reducing variability in clinical diagnostics.
Area of Science:
- Biochemistry
- Enzyme Kinetics
- Clinical Chemistry
Background:
- Lactate dehydrogenase (LDH) assays previously showed enzyme concentration-dependent Michaelis constants.
- This dependency was attributed to inhibitory effects of buffer and salt components.
Purpose of the Study:
- To identify and quantify the inhibitory influence of common buffers and salts on LDH enzyme kinetics.
- To determine inhibition constants for various substances competing with substrates or cofactors.
Main Methods:
- Enzyme kinetic assays measuring pyruvate to lactate conversion by LDH.
- Analysis of Michaelis constant dependence on enzyme concentration in the presence of different buffers and salts.
- Calculation of inhibition constants for buffer/salt-enzyme interactions.
Main Results:
- Buffers (NH4HCO2, tris, phosphate) and salts ( (NH4)2SO4, NaCl) inhibit lactate dehydrogenase.
- Inhibition constants vary, with values around 0.3-1.0 mol/liter.
- Despite high inhibition constants, the high buffer-to-substrate ratio in assays causes significant Michaelis constant alteration.
Conclusions:
- Buffer and salt concentrations critically influence the Michaelis constant in enzymatic assays.
- Controlling these concentrations is essential for improving the reliability and reducing variability in clinical LDH assays.
- This principle may extend to other enzyme-based diagnostic tests.
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