INSIGHTS INTO STOICHIOMETRY OF ARGININE MODIFICATION BY PHENYLGLYOXAL AND 1,2-CYCLOHEXANEDIONE PROBED BY LC-ESI-MS

Several arginine modification studies on enzymes have mostly been done by phenylglyoxal (PG) or 1,2-cyclohexanedione (CHD) as the modifying reagents. It has been found that one molecule or two molecules of PG or one CHD molecule covalently modify sidechain guanidine of arginine of the enzymes. To seek clearer insights into stoichiometry of this reaction, herein, we decided to investigate some model amino acids that include L-arginine (L-Arg) and two model peptides (an octapeptide and a 30 amino acids long peptide). Reactions were conducted at room temperature (RT: ~25 ° C), involving ‘equimolar concentrations’ of reactants in seven different mediums: five buffers (all at basic pH, 7.4 - 8.4) and two solvents. The two solvents are: (1) water (H 2 O) and (2) mixture of acetonitrile and water (ACN:H 2 O, 1:1, v/v). Progress of every reaction was monitored as a function of time by liquid chromatography - electrospray ionization mass spectrometry (LC-ESI-MS). With PG, L-Arg forms 1:1 adduct ([L-Arg+PG]; m/z 309), 1:2 adduct ([L-Arg+2PG]; m/z 443) and water condensed products of respective 1:1 and 1:2 adducts ( m/z 291 & m/z 425) in all six mediums, except in borate, where only uncondensed 1:1 adduct ([L-Arg+PG]; m/z 309) was observed. However, with CHD, L-Arg yielded 1:1 adduct ( m/z 287), 1:2 adduct ( m/z 399) and corresponding water condensed products (m/z 269 & m/z 381) in borate. Interestingly, in H 2 O and in ACN:H 2 O (1:1) too, L-Arg undergoes modification. This is the first LC-ESI-MS study illustrating modification of LArg by phenylglyoxal and 1,2-cyclohexanedione. Molecular structures are proposed for every observed modified product, depicting stoichiometry and mode of binding by PG or CHD onto guanidine moiety of L-Arg. Keywords : Arginine Modification; Electrospray Ionization-Mass Spectrometry (ESI-MS); Liquid Chromatography- MS (LC-MS); L-Arginine; Peptides and Proteins; Arginyl Enzymes