Good’s buffers
Good’s buffers
Maintaining a stable extracellular pH minimizes nonspecific fluctuations in metabolism and signaling. Good’s buffers are characterized by low toxicity, weak interactions with metals, small salt effects, and minimal interference with assays, and are often used as a complement or alternative to bicarbonate systems during culture at 37 °C with CO₂ and during open‐lid handling.
I. Background and concept
Good’s buffers are a set of 20 buffers screened and described by Norman Good and colleagues between 1966 and 1980 for biochemical and biological research. Most of them were novel zwitterionic compounds first prepared and tested by Good’s group, while a few (MES, ADA, BES, Bicine) were known chemicals previously overlooked by biologists. Before Good’s work, there were very few hydrogen-ion buffers available to biologists for pH 6–8, and many in use were unsuitable—toxic, highly reactive, and inefficient. Many Good’s buffers have become, and remain, key tools in modern biological laboratories.
II. Screening and design criteria
To ensure usability and reproducibility in biological systems, Good’s team set the following criteria:
1.pKa and effective buffering range: Target pH 6–8, with pKa near this range to maximize usable buffering capacity.
2.Solubility: High water solubility; low solubility in nonpolar solvents to reduce accumulation in hydrophobic compartments such as membranes.
3.Low membrane permeability: To minimize transmembrane diffusion, intracellular accumulation, and disturbance of cellular homeostasis.
4.Minimal salt effects: Avoid strong ionic strength–related side effects.
5.Stability of dissociation constant: Buffer pKa should change little with concentration, temperature, or medium ionic composition.
6.“Benign” interactions with metal ions: If complexes form, they should remain soluble; ideally, no strong chelation of common mono-/divalent cations.
7.Chemical and enzymatic stability: Resist spontaneous or enzymatic degradation and minimize side reactions.
8.Biochemical inertness: Do not participate in or significantly perturb the reaction network under study.
9.Spectroscopic compatibility: No appreciable absorbance at ≥230 nm to avoid interference with routine UV/Vis measurements.
10.Availability and preparability: Straightforward synthesis/preparation, controlled cost, and easy purification.
III. Common Good’s buffers
Buffer | pKa (20 °C) | Effective pH range | Year added |
6.15 | 5.5–6.7 | 1966 | |
6.62 | 6.0–7.2 | 1966 | |
6.82 | 6.1–7.5 | 1966 | |
6.88 | 6.1–7.5 | 1966 | |
6.95 | 6.2–7.6 | 1980 | |
7.10 | — | 1966 | |
7.15 | 6.5–7.9 | 1972 | |
7.17 | 6.4–7.8 | 1966 | |
7.50 | 6.8–8.2 | 1966 | |
7.55 | 6.8–8.2 | 1966 | |
7.60 | 7.0–8.2 | 1980 | |
7.60 | 7.0–8.2 | 1980 | |
7.70 | — | 1966 | |
7.85 | — | 1980 | |
7.90 | — | 1980 | |
8.10 | 7.6–8.6 | 1972 | |
8.15 | 7.4–8.8 | 1966 | |
8.20 | — | 1966 | |
8.20 | 7.5–8.9 | 1966 | |
8.35 | 7.6–9.0 | 1966 | |
8.55 | 7.7–9.1 | 1972 |
IV. Functions and mechanisms
1.Buffering mechanism: As weak acid/ conjugate base pairs, Good’s buffers provide maximal capacity near their pKa, dampening pH shifts caused by metabolic acid production, CO₂ exchange, or open-lid handling during culture.
2.Positive effects on cellular homeostasis: Stable pH narrows drift in ion channel/transporter activity, reduces stress signaling and metabolic flux (glycolysis/oxidative phosphorylation) variability, and favors adhesion and morphology of adherent cells.
3.Analytical impacts: Most Good’s buffers have low absorbance ≥230 nm and low fluorescence background, making them compatible with nucleic acid/protein quantification and common probes. However, under strong light (blue) with riboflavin present, HEPES can generate ROS; for long-term imaging consider MOPS/PIPES or reduce light/ add ROS scavengers.
4.Metal interactions: Tricine/Bicine chelate some divalent metals (e.g., Cu²⁺) relatively strongly and may affect metal-dependent enzymes or fluorescent probes; for metal-sensitive experiments, prefer HEPES/MOPS/PIPES.
5.Relation to bicarbonate systems: Used together, they add buffering capacity and improve pH stability during open-lid handling/transport. If completely replacing bicarbonate, recalibrate pH (typically 7.2–7.4) and osmolality at 37 °C under the target gas atmosphere, and verify cell viability over 48–72 h.
V. Practical notes
1.Do not use in redox studies
Piperazine-containing buffers—PIPES, HEPES, POPSO, HEPES—may generate reactive radicals; avoid them for redox/free-radical related experiments.
2.Use Tricine with caution in strong light/flavin systems
Tricine is prone to flavin photo-oxidation and can reduce flavin enzyme activity under daylight; for strong light or flavin-related work, switch to MOPS/PIPES.
3.Handling solubility
The free acids of ADA, POPSO, and PIPES have poor water solubility—prefer their monosodium salts (easier to dissolve, more stable formulations).
4.Avoid UV absorption bands
ADA shows noticeable absorbance <260 nm; ACES absorbs at ≤230 nm—avoid those bands in UV/Vis measurements or select alternative buffers.
In summary, Good’s buffers provide a stable, low-interference pH microenvironment under complex culture and measurement conditions. Select by target pH, light/metal sensitivity, and detection window; after preparation, recheck at 37 °C under the target gas and perform a 48–72 h cell-viability check to obtain reproducible results in most cell and biochemical experiments.
References
【1】Good, Norman E.; Winget, G. Douglas; Winter, Wilhelmina; Connolly, Thomas N.; Izawa, Seikichi; Singh, Raizada M. M. (1966). "Hydrogen Ion Buffers for Biological Research". Biochemistry. 5 (2): 467–477.
【2】Good, Norman E.; Izawa, Seikichi (1972). "Hydrogen ion buffers". Photosynthesis and Nitrogen Fixation Part B. Methods Enzymol. Vol. 24. pp. 53–68.
【3】Ferguson, W. J.; Braunschweiger, K. I.; Braunschweiger, W. R.; Smith, J. R.; McCormick, J. J.; Wasmann, C. C.; Jarvis, N. P.; Bell, D. H.; Good, N. E. (1980). "Hydrogen Ion Buffers for Biological Research". Anal. Biochem. 104 (2): 300–310.
【4】Grady, J. K.; Chasteen, N. D.; Harris, D. C. (1988). "Radicals from "Good's" buffers". Anal. Biochem. 173 (1): 111–115.
【5】Kirsch, M.; Lomonosova, E. E.; Korth, H.-G.; Sustmann, R.; de Groot, H. (1998). "Hydrogen peroxide formation by reaction of peroxynitrite with HEPES and related tertiary amines. Implications for a general mechanism". J. Biol. Chem. 273 (21): 12716–12724.
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