Diethylpyrocarbonate inhibition of estradiol receptor binding to oligonucleotides

Exposure of calf uterine estradiol-receptor complexes to diethylpyrocarbonate (ethoxyformic anhydride) at pH 6.3–6.5 results in a decrease in the ability of the receptor to bind to oligodeoxyribonucleotides. The inhibition of binding to oligodeoxypyrimidines is greater than the inhibition of binding to oligodeoxyguanylate. The inhibition by 6.6 mM diethylpyrocarbonate is complete within 10 min at 4°C. Addition of equimolar quantities of histidine or imidazole prior to exposure to diethylpyrocarbonate prevents subsequent inhibition of oligodeoxyribonucleotide binding. In comparison to histidine, other amino acids tested were deficient in this ability. Diethylpyrocarbonate modification of the receptor causes complete loss of oligodeoxyribonucleotide binding activity at times when there is a loss of less than 20% of bound steroid. Pyridoxal 5'-phosphate treatment of receptor does not prevent subsequent modification by diethylpyrocarbonate, suggesting that the site of reaction is not an essential lysine of the DNA-binding domain. Treatment of the ethoxyformylated receptor with 0.45 M hydroxylamine results in recovery of 70% of the receptor's oligonucleotide-binding ability. The time course of the reaction of diethylpyrocarbonate with the estradiol receptor and the demonstration of hydroxylamine reversal of inhibition suggest that histidine is involved in the binding of estradiol receptor to oligodeoxyribonucleotides.