Tris(hydroxymethyl)aminomethane, commonly known as Tris, is a frequently used buffer substance in biological and chemical laboratories worldwide. A buffer is a solution that resists changes in acidity or alkalinity, helping keep the pH stable even when small amounts of acid or base are introduced. Tris ensures that the environmental conditions necessary for biological processes, such as those involving enzymes and DNA, remain constant. Its high solubility and effectiveness in the slightly alkaline range make Tris an indispensable tool for researchers across biochemistry and molecular biology.
The Core Function: Maintaining pH Stability
The primary role of Tris buffer is to prevent fluctuations in the concentration of hydrogen ions, measured as pH. Biological molecules, such as proteins and nucleic acids, are sensitive to their environment; small pH changes can cause them to lose structure and function. Enzymes, for instance, operate within a narrow, optimal pH range, and moving outside this range can stop their activity.
Tris achieves stability because it is a weak base in chemical equilibrium with its conjugate acid, TrisH\(^+\). When an acid is added, the Tris base absorbs excess hydrogen ions, forming TrisH\(^+\) and neutralizing the acid. Conversely, if a base is added, TrisH\(^+\) releases hydrogen ions to neutralize the base, resisting a pH increase.
This proton-accepting and proton-donating action allows the solution to absorb moderate amounts of added acid or base without a significant pH shift. The capacity to absorb these changes is the buffering capacity, which is greatest when the concentrations of the weak base and its conjugate acid are nearly equal. This balance ensures that the molecular machinery remains functional and experimental results are reliable.
Unique Chemical Properties of Tris
Scientists select Tris because its chemical properties align well with the needs of many biological systems. The effectiveness of a buffer is defined by its pKa value, the pH at which the buffer is half-dissociated and has its maximum buffering capacity. Tris has a pKa of approximately 8.1 at 25°C, making it highly effective in the pH range of 7.0 to 9.0.
This slightly alkaline range closely matches the physiological pH of many living systems and is ideal for molecular biology techniques. The Tris molecule contains a primary amine group and three hydroxymethyl groups, contributing to its high solubility and buffering action.
A defining property of Tris is its considerable temperature dependence. For every 1°C increase in temperature, the pKa decreases by about 0.031 units, causing the pH of the solution to drop significantly. Due to this sensitivity, researchers must always adjust the Tris buffer’s pH at the same temperature at which the experiment will be run to ensure accuracy.
Another advantage is that Tris does not strongly interact with many common metal ions, unlike phosphate-based buffers which can form precipitates. This non-interference is beneficial in experiments where metal ions are required cofactors for enzyme activity.
Essential Applications in Biological Research
The stable environment provided by Tris buffer makes it a foundational component in numerous laboratory procedures, spanning molecular biology and biochemistry. One widespread use is in gel electrophoresis, a technique used to separate DNA, RNA, or proteins based on size and charge. Tris is a primary ingredient in running buffers like Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE) for nucleic acids, and Tris-Glycine-SDS for proteins (SDS-PAGE). In these electrophoresis systems, the buffer ensures molecules maintain a consistent charge and that the electric current flows uniformly for accurate separation.
Tris is also a component of TE buffer (Tris-EDTA), used for the long-term storage of DNA and RNA. Here, pH stability prevents the degradation of nucleic acids over time.
In biochemistry, Tris buffer is used in enzyme assays, ensuring activity is measured accurately under standardized conditions. It is also incorporated into solutions used for breaking open cells (cell lysis) and for purifying proteins. Tris-based saline solutions are commonly used as wash buffers in immunological techniques like Western blotting and ELISA, helping maintain the integrity of proteins and antibodies.