Diethylpyrocarbonate
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| Diethylpyrocarbonate | |
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| IUPAC name | diethyl dicarbonate |
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| CAS number | [1609-47-8] |
| PubChem | |
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| Properties | |
| Molecular formula | C6H10O5 |
| Molar mass | 162.141 g/mol |
| Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa) Infobox disclaimer and references |
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Diethylpyrocarbonate (DEPC) also called 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 cannot be used with Tris buffer or HEPES since they inactivate DEPC by reacting with it. 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.
Water is usually treated with 0.1% v/v diethylpyrocarbonate for at least 1 hour at 37°C and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC on 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.
In a study on rat and pigeon heart mitochondria free radical leakage: "p-Chloromercuribenzoate and ethoxyformic anhydride Diethylpyrocarbonate strongly inhibited the H2O2 production induced by rotenone with pyruvate/malate in both species."[1]
[edit] References
- ^ Herrero A, Barja G. (1997). Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon. MECHANISMS OF AGING AND DEVELOPMENT. 98 (2): 95-111. PMID 9379714
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