The effects of pH and aniline trapping on the partitioning of the A-21 cyclic anhydride intermediate of human insulin into deamidated insulin and covalent dimer were investigated at low pH and 35 degrees C. Characterization of the covalent dimer was achieved by proteolytic cleavage and electrospray mass spectrometry and indicated that the deamidated A-21 asparagine of one insulin molecule and the B-1 phenylalanine residue of another are involved. Anhydride trapping with aniline at pH 4.0 provided evidence that the rate-limiting generation of a cyclic anhydride intermediate is involved in the formation of both deamidated and dimeric insulin. In the presence of aniline at pH 4.0 insulin formed two anilide products, A-21 N delta 2-phenylasparagine and N delta 2-phenylasparagine and N gamma 2-phenylaspartic acid human insulin at the expense of both desamido A-21 and covalent dimer formation, consistent with the formation of a common intermediate. At 35 degrees C and under conditions where the insulin monomer predominates, the fraction of insulin reacting to form [desamidoA-21] insulin decreased with a concurrent increase in formation of [desamidoA-21-PheB-1] dimer with an increase in pH from 2.0 to 5.0. The pH dependence of insulin product distribution could not be quantitatively rationalized solely in terms of the fraction of the PheB-1 amine group in un-ionized form. Rather, consideration of the charge states of ionizable residues near the reacting groups was necessary to fully account for the observed pH effects on product formation.