Evaluation of the Suitability of pH Buffers in Biological, Biochemical, and Environmental Studies

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Maintaining pH stability is crucial in biological, biochemical, and environmental research. pH fluctuations can affect protein structure, cell metabolism, and various biochemical analysis results. To address this issue, Good et al. proposed seven criteria for selecting appropriate buffers, one of which is that the buffer should not form insoluble complexes with cations. However, increasing research indicates that the interaction between buffers and cations may not fully align with Good's assumptions. This article provides information on the complexation between different buffers and metal ions, and discusses their suitability.

Interactions Between Buffers and Metal Ions

This article categorizes buffers into seven families and introduces the complexation of each family with metal ions

• • Morpholine Family：The Morpholine family includes buffers such as MES (2-(N-Morpholino)ethanesulfonic acid). These buffers do not form complexes with metal ions, making them suitable for studies that require the avoidance of metal ion interference, such as certain enzyme activity tests and cell culture studies.
  • Piperazine Family：The Piperazine family includes buffers like PIPES (Piperazine-N,N′-bis(2-ethanesulfonic acid)) and EPPS (N-(2-Hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)). These buffers also do not form complexes with metal ions. They are commonly used in biochemical and molecular biology experiments, especially those involving metal ion-sensitive systems.
  • Cyclohexylamino Family：The Cyclohexylamino family includes buffers like CHES (2-(Cyclohexylamino)ethanesulfonic acid). These buffers do not form complexes with metal ions, making them suitable for experiments requiring precise pH control and avoiding metal ion effects, such as protein crystallography studies.
  • Bis(2-hydroxyethyl)amine Family：The Bis(2-hydroxyethyl)amine family includes buffers like Bicine (N,N-Bis(2-hydroxyethyl)glycine). These buffers form complexes with metal ions, so their impact on metal ions should be considered, especially in experiments involving metalloenzymes or other metal-dependent processes.
  • Tris Family：The Tris family includes buffers like Tris (Tris(hydroxymethyl)aminomethane) and TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid). Tris buffers can penetrate cells and are toxic to them, and they also inhibit certain enzymes. They form complexes with metal ions, so they should be used cautiously in experiments involving metal ions.
  • Acetamido Family：The Acetamido family includes buffers like ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid) and HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid). These buffers form complexes with metal ions, so their potential interference should be noted in studies involving metal ions.
  • Propanol Family：The Propanol family includes buffers like TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid). These buffers form complexes with metal ions, and their interaction with metal ions should be considered when used in experiments.

Applications of Buffers

Buffers have a wide range of applications. Table 2 lists the use of different buffers in various research fields such as biomolecular, cell biology, toxicology, and environmental studies. Choosing the appropriate buffer based on the specific research needs can improve the accuracy and reliability of experiments.

HEPES in Chilled Bovine Embryo Survival

Studies have shown that chilled bovine embryos have a significantly higher survival rate in HEPES-containing culture media compared to other buffers like TES, PIPES, MOPS, and EPPS. This indicates the superior performance of HEPES in certain biological systems, making it suitable for high-survival-rate embryo preservation experiments.

Advantages of MES in Yeast Cultures

MES is not metabolized by bacteria or eukaryotic cells, allowing yeast cells to maintain integrity in culture media with pH 6 and 10 mM MES. This makes MES an ideal buffer for cell cultures, especially yeast cultures.

Buffers in Electrophoresis

Electrophoresis requires precise pH control to separate DNA and proteins, typically using Tris-containing TAE or TBE buffers. However, for RNA separation, denaturing agents may cause the buffer to become alkaline and change pH, so MOPS is added to stabilize the pH.

Effects of Buffers on Spectrophotometric Measurements

Spectrophotometric measurements related to DNA, RNA, and proteins typically occur at wavelengths above 230nm. Buffers with absorption peaks at 230nm can interfere with measurements. For example, Tris interferes with Bradford protein assays; HEPES, PIPES, Bicine, and MOPS interfere with Lowry protein assays; HEPES and MOPS do not interfere with Bradford protein assays and bicinchoninic acid assays.

Cytotoxicity of Tris

Tris can penetrate cells, making it toxic to them, and it can inhibit certain enzymes. Therefore, caution should be exercised when using Tris in cell experiments to avoid unnecessary effects on the results.

Conclusion

Choosing the right buffer is crucial for the success of biological, biochemical, and environmental research. Different buffers have distinct properties and application ranges. Researchers should select the most suitable buffer based on specific experimental needs, considering the buffer's interaction with metal ions, performance in specific applications, and impact on measurement methods to ensure the accuracy and reliability of their experiments.

Further reading: HEPES handling and storage tips that you must know
Further reading：Optimizing pH Control: The Synergistic Blend of Tris and Tris HCl in Buffer Systems
Further reading：The 9 best biological buffers for electrophoresis
Further reading：12 uses of HEPES Buffer you didn't know