Protein Protocols Protein Protocols

474 Tawfik
4. Notes
1. The concentration of commercial DEP is often lower than indicated owing to hydrolysis.
The concentration of the sample after dilution with an organic solvent can be readily
determined by adding an aliquot of an imidazole solution (1–10 mM in phosphate, pH 7.0)
and measuring the increase in absorbance at 230 nm after 5 min (ε = 3000 M –1 cm –1 ).
2. The final acetonitrile concentration in the protein reaction mixture should be 5< %, see
Chapter 64, Subheading 3.1. for typical dilutions and reaction volumes.
3. A molar excess of 10–300 of DEP is usually sufficient; however, with some proteins
higher concentrations of the reagent might be needed. Likewise, if no modification is
observed at pH 5.0, the reaction can be performed at higher pH. In such cases it is
recommended that one ensure that loss of activity is indeed due to the ethxyformylation of
histidine (see Note 4).
4. Measuring the differential UV spectra during modification with DEP is useful not only to
determine the number of modified histidines but also to eliminate the possibility that
residues other than histidines were modified. The ethoxyformylation of histidine side
chains by DEP should result in an increase in the absorbance of the protein at 242 nm
(ε = 3200 M –1 cm –1 ). Likewise, restoration of the activity of the modified protein after
treatment with hydroxylamine should be accompanied by a parallel decrease in the
absorbance at 242 nm. Ethoxyformylation of the hydroxyl of tyrosine would increase the
absorbance at approx 280 nm whereas similar modification of serine or threonine does not
cause a significant changes of absorbance at this range.
5. The ethoxyformyl product is quite unstable. Because the reagent is rapidly hydrolyzed
(to give ethanol and carbonate) there is hardly a need to purify the protein after modification.
In any case, dialysis even in neutral buffers may result in a significant removal of the
ethoxyformyl group. Hence, purification by exclusion chromatography (e.g., on Sephadex
G-25) is preferred.
References
1. Dominicini, P., Tancini, B., and Voltattorni, C. B. (1985) Chemical modification of pig
kidney 3, 4-dihydroxy-phenylalanine decarboxylase with diethyl pyrocarbonate. J. Biol.
Chem. 260, 10,583–10,589.
2. Melchior, W. B., Jr. and Fahrney, D. (1970) Ethoxyformylation of proteins. Reaction of
ethoxyformic anhydride with a-chymotrypsin, pepsin and pancreatic ribonuclease at pH 4.
Biochemistry 9, 251–258.
3. Sheflyan, G. Y., Duewel, H. S., Chen, G., and Woodard, R. W. (1999) Identification of
essential histidine residues in 3-deoxy-D- manno-octulosonic acid 8-phosphate synthase:
analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis.
Biochemistry 38, 14,320–14,329.