K. Takahashi
Dec 10, 1968
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Journal
The Journal of biological chemistry
Abstract
Abstract The experiments described in this communication were carried out in order to investigate the usefulness of phenylglyoxal as a reagent for the chemical modification of arginine residues in proteins. Phenylglyoxal reacts with the guanido group of the arginine residue under mild conditions, pH 7 to 8 and 25°. The derivative, which contains two phenylglyoxal moieties per guanido group, is sufficiently stable, particularly under mildly acidic conditions (below pH 4) to permit the isolation of labeled peptides. At neutral or alkaline pH values, the derivative is decomposed slowly. About 80% of the original arginine can be regenerated by incubation of the derivative in the absence of excess reagent for 48 hours at 37° and about pH 7. Bovine pancreatic ribonuclease A (0.5% solution) is inactivated to the extent of 80 to 90% within 30 min by reaction with a 1.5% solution of phenylglyoxal at pH 8.0 and 25°. Of the 4 arginine residues in the molecule, 2 to 3 are modified. Analysis of tryptic-chymotryptic hydrolysates of the inactivated protein show that condensation occurs primarily at arginine residues 39 and 85 in the sequence. These are the 2 arginine residues which x-ray diffraction studies have shown to be closest to the active site of the enzyme. In this modified ribonuclease, the 2 histidine residues, 12 and 119, continue to be susceptible to specific alkylation by iodoacetate. In general, phenylglyoxal (14C-labeled, if desired) may be useful for modification of accessible arginine residues in proteins and may be potentially useful for reversible coverage of arginine residues so that tryptic hydrolysis can take place at lysine residues only. When proteins are treated with a large excess of the reagent for a long period, reaction may also occur with e-amino groups of lysine residues. In all cases, any available α-amino group is deaminated to give a residue of the corresponding α-keto acid. The reagent is very effective in deaminating small peptides to the corresponding α-keto acyl peptides, thus permitting the identification of the end groups by subtractive analysis.