Tris(hydroxymethyl)aminomethane, commonly known as Tris buffer, is a frequently used buffering agent in modern biological and chemical laboratories. A buffer is a solution designed to resist changes in \(\text{pH}\) when small amounts of acid or base are added. This resistance is necessary because biological molecules, such as \(\text{DNA}\), \(\text{RNA}\), and proteins, are sensitive to fluctuations in acidity or alkalinity. Tris maintains the stable \(\text{pH}\) environment required for sensitive biological processes.
The Chemical Makeup of Tris
The chemical name for Tris is Tris(hydroxymethyl)aminomethane, and its formula is \(\text{C}_4\text{H}_{11}\text{NO}_3\) or \(\text{(HOCH}_2)_3\text{CNH}_2\). This molecule is classified as a primary amine, containing a nitrogen atom bonded to one carbon atom, which provides its buffering capacity. The central carbon atom is also bonded to three hydroxymethyl groups (\(\text{-CH}_2\text{OH}\)), which increases its solubility in water.
The \(\text{pKa}\) value defines the \(\text{pH}\) where a solution contains equal amounts of the acid and base forms. For Tris, this value is approximately 8.1 at \(25^\circ \text{C}\). A buffer is most effective one \(\text{pH}\) unit above and below its \(\text{pKa}\). The functional buffering range for Tris is generally between \(\text{pH}\) 7.2 and 9.0, aligning with the slightly alkaline conditions needed for many molecular biology experiments.
The Mechanism of Buffering
Tris maintains a stable \(\text{pH}\) through the equilibrium between its two forms: the unprotonated Tris base and the protonated Tris acid (Tris- \(\text{H}^+\)). In solution, the amine group on the Tris molecule readily accepts a proton (\(\text{H}^+\)), transforming it into its conjugate acid form. This process allows the solution to neutralize introduced acids or bases, minimizing \(\text{pH}\) change.
When an acid is introduced, the free Tris base molecules absorb the excess hydrogen ions (\(\text{H}^+\)), preventing them from lowering the overall \(\text{pH}\) of the solution. Conversely, when a base is added, the Tris- \(\text{H}^+\) (the acidic form) releases a proton to neutralize the introduced hydroxide ions (\(\text{OH}^-\)). This acid-base pair action creates a “reservoir” of protons that buffers the solution against external \(\text{pH}\) shifts.
Primary Uses in Biochemistry and Molecular Biology
Tris buffer is a standard reagent across many laboratory disciplines, finding wide application due to its effective \(\text{pH}\) range and compatibility with biological systems. One recognized application is in nucleic acid work, where it forms the basis of two common electrophoresis buffers: \(\text{TAE}\) (Tris-Acetate-EDTA) and \(\text{TBE}\) (Tris-Borate-EDTA). These buffers are used to separate \(\text{DNA}\) and \(\text{RNA}\) fragments by size in an electric field, requiring a stable \(\text{pH}\) for predictable molecular movement.
In protein research, Tris is used extensively for purifying, storing, and analyzing proteins and enzymes. The stable \(\text{pH}\) environment prevents proteins from denaturing or aggregating, which would destroy their function. It is a common component in buffers used for enzyme assays, where a precise and constant \(\text{pH}\) is required to measure enzyme activity accurately.
Tris is frequently incorporated into cell culture media, often in combination with other buffering systems, to maintain the extracellular \(\text{pH}\) that supports healthy cell growth and metabolism. It is also a component in buffers for \(\text{DNA}\) and \(\text{RNA}\) isolation and purification processes, protecting nucleic acid molecules from degradation. The slightly alkaline \(\text{pH}\) range of Tris helps to keep \(\text{DNA}\) soluble and stable.
Handling and Practical Limitations
While Tris is versatile, its use requires careful consideration of its significant temperature dependence. The \(\text{pKa}\) of Tris is highly sensitive to temperature changes, meaning the \(\text{pH}\) of the solution will decrease substantially as the temperature increases. The \(\text{pH}\) drops by approximately \(0.02\) to \(0.03\) units for every one \(\text{degree}\) Celsius increase. For example, a Tris buffer prepared to \(\text{pH}\) 7.8 at \(25^\circ \text{C}\) could shift to \(\text{pH}\) 8.4 if cooled to \(4^\circ \text{C}\), or drop to \(\text{pH}\) 7.4 if heated to \(37^\circ \text{C}\). Researchers must measure and adjust the \(\text{pH}\) of the buffer at the specific temperature at which the experiment will be run.
Another limitation is Tris’s ability to chelate, or bind, certain metal ions like copper, which can be problematic in assays where these ions are necessary cofactors for enzyme activity. Tris can also interfere with certain assays, especially those involving metal-dependent enzymes. Furthermore, it is incompatible with some \(\text{pH}\) electrodes that use silver, as it can form a precipitate that clogs the electrode junction. These factors sometimes lead scientists to choose alternative buffers, such as phosphate or \(\text{HEPES}\).