DEPC treated water is a highly specialized reagent used almost exclusively in molecular biology laboratories. It is purified water chemically treated with Diethyl pyrocarbonate to remove microscopic contaminants that would otherwise interfere with sensitive scientific procedures. The process ensures the resulting water is free of destructive biological agents, making it a prerequisite for advanced genetic analyses.
Defining Diethyl Pyrocarbonate
Diethyl pyrocarbonate (DEPC), also known as diethyl dicarbonate, is a colorless liquid used as a potent chemical inactivator in laboratory settings. Chemically, it is an ethyl diester of pyrocarbonic acid, classifying it as a reactive acid anhydride. It functions by reacting irreversibly with specific chemical groups found on proteins, effectively denaturing and inactivating them.
The primary function of DEPC in water treatment is to modify the active sites of enzymes. This modification is achieved through a covalent reaction, where the DEPC molecule binds to certain amino acid residues within the enzyme structure. It has a strong affinity for histidine residues, but it can also react with lysine, cysteine, and tyrosine residues, permanently disabling the enzyme’s function.
Addressing the RNase Problem in Water
The specific contamination problem DEPC-treated water solves is the presence of Ribonucleases (RNases), which are enzymes that rapidly degrade RNA molecules. RNases are notoriously stable and ubiquitous contaminants, found on human skin, in dust, and throughout the environment. Even trace amounts carried on lab equipment or in water can quickly destroy a valuable RNA sample.
For experiments involving RNA, such as gene expression studies or sequencing, the integrity of the RNA is paramount. Since RNases are robust enzymes, they often survive standard sterilization methods like simple autoclaving. This necessitates the use of a chemical agent like DEPC to ensure complete inactivation and prevent the degradation of the target RNA.
The Methodology of DEPC Treatment and Removal
The preparation of DEPC-treated water begins by adding the chemical to highly purified water, typically at a concentration of 0.1% volume-to-volume (v/v). This mixture is shaken vigorously and allowed to react with any RNases present for at least one to two hours, sometimes at an elevated temperature like 37°C.
The next important step is the removal of the remaining, unreacted DEPC, which is toxic. This is achieved by autoclaving the solution at high temperature and pressure, such as 121°C for 15 to 20 minutes per liter. The heat and pressure cause the remaining DEPC to hydrolyze into harmless byproducts.
This hydrolysis reaction converts the toxic DEPC into ethanol and carbon dioxide (CO2). The carbon dioxide is released as a gas, and the ethanol remains dissolved in the water. The final product is autoclaved water free of both active RNases and the toxic DEPC chemical.
Safety and Use Context
DEPC itself must be handled with extreme caution, as it is a powerful acylating agent and a suspected carcinogen. For this reason, the initial treatment step is always performed in a controlled environment, such as a chemical fume hood, to prevent exposure.
The final DEPC-treated water, after thorough autoclaving, is considered safe for its intended laboratory use. The breakdown into ethanol and carbon dioxide ensures that no residual toxic chemical can interfere with subsequent biochemical reactions. This specialized water is solely intended for use as a laboratory reagent, such as for dissolving RNA pellets or preparing other RNase-free solutions.