4-(Oxoacetyl)phenoxyacetic acid (OAPA) forms a stable, covalent bond between its glyoxal group and the guanidino group of arginine and arginine derivatives [Duerksen, P. J., & Wilkinson, K. D. (1987) Anal. Biochem. 160, 444-454]. Studies were carried out to determine the chemical nature of this linkage, and the structure of the stable adduct between OAPA and methylguanidine was elucidated. The stable product results from an internal oxidation-reduction of the Schiff base adduct to form a cyclic alpha-aminoamide, 4-[4-(carboxymethoxy)phenyl]-2-(methylimino)-5-oxoimidazolidine. OAPA coupled to polyacrylamide beads was used to immobilize ubiquitin via its arginine residues, and the resulting affinity support was shown to specifically and reversibly bind a previously described enzyme, ubiquitin carboxyl-terminal hydrolase [Pickart, C. M., & Rose, I. A. (1985) J. Biol. Chem. 260, 7903-7910]. The resin was then used to isolate three newly identified ubiquitin carboxyl-terminal hydrolytic activities, which did not bind to ubiquitin immobilized via lysine residues. Significant purification was achieved in each case, and one isozyme was further purified to homogeneity.