Abstract
1, 2-Cyclohexanedione reacts specifically with the guanidino group of arginine or arginine residues at pH 8 to 9 in sodium borate buffer in the temperature range of 25-40 degrees. The single product, N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine (DHCH-arginine) is stable in acidic solutions and in borate buffers (pH 8 to 9). DHCH-Arginine is converted to N-7-adipyl-L-arginine by periodate oxidation. The structures of the two compounds were elucidated by chemical and physicochemical means. Arginine or arginyl residues can be regenerated quantitatively from DHCH-arginine by incubation at 37 degrees in hydroxylamine buffer at pH 7.0 FOR 7 TO 8 hours. Analysis of native egg white lysozyme and native as well as oxidized bovine pancreatic RNase, which were treated with cyclohexanedione, showed that only arginine residues were modified. The utility of the method in sequence studies was shown on oxidized bovine pancreatic ribonuclease A. Arginine modification was complete in 2 hours at 35 degrees in borate buffer at pH 9.0 with a 15-fold molar excess of the reagent. The derived peptides showed that tryptic hydrolysis was entirely limited to peptide bonds involving lysine residues, as shown both by two-dimensional peptide patterns and by isolation of the resulting peptides. The stability of DHCH-arginyl residues permits isolation of labeled peptides.
Highlights
1,2-Cyclohexanedione reacts with the guanidino group of arginine or arginine residues at pH 8 to 9 in sodium borate buffer in the temperature range of 25-40”
In this paper we describe the reaction of 1,2-cyclohexancdione with arginine and arginyl residues of proteins at pH 8 to 9 in borate
(1.05 g, 5 mmol) was dissolved in 100 ml of freshly prepared 0.2 M sodium borate buffer at pII9.0 and 0.67 g (6 mmol) of 1,2-cyclohexanedione were added; the reaction mixture was kept under Nz at room temperature
Summary
1,2-Cyclohexanedione reacts with the guanidino group of arginine or arginine residues at pH 8 to 9 in sodium borate buffer in the temperature range of 25-40”. Modification with 2,3-butanedione, glyoxal, phenylglyoxal, and cyclohexanedione can be performed under milder conditions [5, 7,8,9,10,11] All of these procedures may result in significant side reactions with a- or t-amino groups. In most cases arginine modification is irreversible, a major disadvantage in protein-sequencing studies In other instances, such as the reaction of phenylglyoxal with arginine [11] , rcgeneration to arginine occurs spontaneously in neutral and alkaline solutions. This instability of the product places limitations on its use in studies of sequences and for specific labeling.
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