Modication of Arginine
The chemical modication of arginyl residues in proteins presents a considerable challenge. The average pKa for the guanidino group of arginine is 12.48 (Table 1.1), making it the most basic group in a protein and, as such, a poor nucleophile. As with other amino acids in proteins with functional groups, arginyl residues are subject to posttranslational modication. The methylation of arginine resulting in three different derivative forms (Figure 15.1)33,34 is of considerable importance in the control or protein-protein interactions35 and protein-nucleic acid interactions.36 Arginyl residues are subject to glycation (Figure 15.1) with metabolites such as glyoxal37 and methylglyoxal.38 Chetyrkin and coworkers39 observed that the nonoxidative addition of glucose to proteins (lysozyme and the RDG-α3NCI domain of collagen IV; see Chapter 13 for the discussion of the reaction of glucose with amino groups in proteins) did not affect activity, while reaction with glucose under oxidative conditions, which produces glyoxal and methylglyoxal, did result in inactivation. Arginine undergoes reversible phosphorylation (Figure 15.1) in process considered to be an important regulatory event.40,41 There is early work on the nitration of arginine residues in proteins with nitric acid in fuming sulfuric acid,42 and nitro group was used to protect the guanidine group during peptide synthesis,43 but other derivatives have been the subject of recent study.44 Nitroarginine is an inhibitor of nitric oxide synthesis45 and, as the methyl esters (L-NAME), has in vivo physiological effect.46 Kethoxal is a chemical used as a footprinting reagent for RNA47-50 where it modies guanine residues.49-51 The antiviral activity of kethoxal dates at least to 1959,52 and there was some early clinical use of this chemical.53,54 Delihas and coworkers55 demonstrated the kethoxal formed a stable complex with proteins in E. coli ribosomes. These investigators also demonstrated reaction with bovine serum albumin. Subsequent work from this laboratory56 showed that kethoxal reacted with arginyl residues. Somewhat later, Iijima and coworkers57 reported the reaction of kethoxal with amino acids and pancreatic ribonuclease. The primary site of reaction was arginine; there was slow reaction with primary amines including the ε-amino group of lysine. Reaction with Nα-arginine resulted in multiple products, which were stable at pH 7 and reactive with periodate; 15% of the modied arginine reverts to arginine on acid hydrolysis (6 N HCl/110°C/22 h). The extent of modication depended on solvent with the greatest extent of modication in water with decreasing modication in cacodylate and acetate with the lowest degree of modication in phosphate buffer. The degree of loss of activity also varied with solvent and pH of assay. Aggregation of RNase was also observed on reaction with kethoxal. In the most recent work, Akinsiku and coworkers58 used MS to determine the structure of the adduct formed between kethoxal and arginine and the borate complex (Figure 15.2). I could nd no current use of kethoxal for the modication of proteins.