Protein Protocols Protein Protocols

p-Hydroxyphenylglyoxal-Modified Arginine 475
66
Modification of Arginine Side Chains
with p-Hydroxyphenylglyoxal
Dan S. Tawfik
1. Introduction
A variety of dicarbonyl compounds including phenylglyoxal, 2,3-butanendione, and
1,2-cyclohexanedione selectively modify the guanidine group of arginine (1–3). The
main advantage of p-hydroxyphenylglyoxal is in the ability to determine the number of
modified arginines spectrophotometrically. This reagent is also reactive at mildly alkaline
pH (usually 8.0–9.0) and yields a single product that is relatively stable (1).
2. Materials
1. p-Hydroxyphenylglyoxal.
2. 1 M NaOH.
3. Protein for modification (approx 10 µM) diluted in 0.1 M sodium pyrophosphate buffer,
pH 9.0.
4. Sephadex G-25 column.
3. Method
1. Prepare a 100 mM solution of p-hydroxyphenylglyoxal in water and adjust the pH of the
solution with 1 M NaOH to 9.0.
2. Prepare a series of dilutions (5–50 mM; see Note 2) of the solution in Step 1 in 0.1 M
sodium pyrophosphate buffer, pH 9.0 (see Note 1).
3. Add 10-µL aliquots of the p-hydroxyphenylglyoxal solutions to 90-µL aliquots of the
protein solution. Check the pH and if necessary adjust it back to pH 9.0.
4. Incubate for 60–180 min in the dark.
5. Pass the sample through a Sephadex G–25 column. Elute with deionized water or with an
appropriate buffer (see Note 4).
6. Determine the activity of the modified protein.
7. Determine the number of modified arginines by measuring the absorbance of the purified
protein (see Notes 3 and 4) at 340 nm (at pH 9.0, ε = 18,300 M –1 cm –1 ).
4. Notes
1. An optimal rate and selectivity of modification is generally obtained at pH 8.0–9.0; however,
in some proteins a higher pH might be required to modify a particular arginine residue.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
475