Diethylpyrocarbonate
Encyclopedia
Diethylpyrocarbonate also called diethyl dicarbonate (IUPAC name), diethyl oxydiformate, ethoxyformic anhydride, or pyrocarbonic acid diethyl ester, is used in the laboratory
to inactivate the RNase enzymes from water and other laboratory utensils. It inactivates the RNases by the covalent modifications of the histidine
residues. DEPC is unstable in water and susceptible to hydrolysis to carbon dioxide and ethanol, especially in the presence of a nucleophile. For this reason DEPC cannot be used with Tris
buffer or HEPES
. In contrast it can be used with PBS or MOPS
. A handy rule is that enzymes or chemicals which have active -O:, -N: or -S: cannot be treated with DEPC to become RNase-free as DEPC reacts with these species. Furthermore DEPC degradation products can inhibit in vitro transcription.
Water is usually treated with 0.1% v/v diethylpyrocarbonate for at least 1hour at 37°C and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC in this manner yields CO2, H2O and EtOH. Higher concentrations of DEPC are competent of deactivating larger amounts of RNase but remaining traces or byproducts may inhibit further biochemical reactions such as in vitro translation. Further on, chemical modification of RNA such as carboxymethylation is possible when traces of DEPC or its byproducts are present, resulting to reduced usage of RNA even after buffer exchange (after precipitation).
DEPC treated (and therefore RNase-free) water is used in handling of RNA in the laboratory, to reduce the risk of RNA being degraded by RNases.
DEPC derivatization of histidines is also used to study the importance of histidyl residues in enzymes. Modification of histidine
by DEPC results in carbethoxylate derivates at the N-omega-2 nitrogen of the imidazole
ring. DEPC modification of histidines can be reversed by treatment with 0.5M hydroxylamine
at neutral pH.
Laboratory
A laboratory is a facility that provides controlled conditions in which scientific research, experiments, and measurement may be performed. The title of laboratory is also used for certain other facilities where the processes or equipment used are similar to those in scientific laboratories...
to inactivate the RNase enzymes from water and other laboratory utensils. It inactivates the RNases by the covalent modifications of the histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
residues. DEPC is unstable in water and susceptible to hydrolysis to carbon dioxide and ethanol, especially in the presence of a nucleophile. For this reason DEPC cannot be used with Tris
Tris
Tris is an abbreviation of the organic compound known as trisaminomethane, with the formula 3CNH2. Tris is extensively used in biochemistry and molecular biology. In biochemistry, tris is widely used as a component of buffer solutions, such as in TAE and TBE buffer, especially for solutions of...
buffer or HEPES
HEPES
HEPES is a zwitterionic organic chemical buffering agent; one of the twelve Good's buffers...
. In contrast it can be used with PBS or MOPS
MOPS
MOPS is the common name for the compound 3-propanesulfonic acid, a buffer introduced by Good et al. in the 1960s. It is a structural analog to MES. Its chemical structure contains a morpholine ring. HEPES is a similar pH buffering compound that contains a piperazine ring...
. A handy rule is that enzymes or chemicals which have active -O:, -N: or -S: cannot be treated with DEPC to become RNase-free as DEPC reacts with these species. Furthermore DEPC degradation products can inhibit in vitro transcription.
Water is usually treated with 0.1% v/v diethylpyrocarbonate for at least 1hour at 37°C and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC in this manner yields CO2, H2O and EtOH. Higher concentrations of DEPC are competent of deactivating larger amounts of RNase but remaining traces or byproducts may inhibit further biochemical reactions such as in vitro translation. Further on, chemical modification of RNA such as carboxymethylation is possible when traces of DEPC or its byproducts are present, resulting to reduced usage of RNA even after buffer exchange (after precipitation).
DEPC treated (and therefore RNase-free) water is used in handling of RNA in the laboratory, to reduce the risk of RNA being degraded by RNases.
DEPC derivatization of histidines is also used to study the importance of histidyl residues in enzymes. Modification of histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
by DEPC results in carbethoxylate derivates at the N-omega-2 nitrogen of the imidazole
Imidazole
Imidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...
ring. DEPC modification of histidines can be reversed by treatment with 0.5M hydroxylamine
Hydroxylamine
Hydroxylamine is an inorganic compound with the formula NH2OH. The pure material is a white, unstable crystalline, hygroscopic compound. However, hydroxylamine is almost always provided and used as an aqueous solution. It is used to prepare oximes, an important functional group. It is also an...
at neutral pH.