Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens

Abstract

Recent work in our laboratory showed that products formed by the antibody-catalyzed water-oxidation pathway can kill bacteria. Dihydrogen peroxide, the end product of this pathway, was found to be necessary, but not sufficient, for the observed efficiency of bacterial killing. The search for further bactericidal agents that might be formed along the pathway led to the recognition of an oxidant that, in its interaction with chemical probes, showed the chemical signature of ozone. Here we report that the antibody-catalyzed water-oxidation process is capable of regioselectively converting antibody-bound benzoic acid into para-hydroxy benzoic acid as well as regioselectively hydroxylating the 4-position of the phenyl ring of a single tryptophan residue located in the antibody molecule. We view the occurrence of these highly selective chemical reactions as evidence for the formation of a short-lived hydroxylating radical species within the antibody molecule. In line with our previously presented hypothesis according to which the singlet-oxygen ((1)O*(2)) induced antibody-catalyzed water-oxidation pathways proceeds via the formation of dihydrogen trioxide (H(2)O(3)), we now consider the possibility that the hydroxylating species might be the hydrotrioxy radical HO(3)*, and we point to the remarkable potential of this either H(2)O(3)- or O(3)-derivable species to act as a masked hydroxyl radical HO* in a biological environment.

Figure 1

(A) Rendering of the combining site of 4C6 Fab bound to the benzoic-acid ligand. (B) Molecular surface representation showing a putative channel that links the interfacial variable and constant domains with the antigen-binding site of murine 4C6 Fab and the benzoic-acid ligand bound directly above this channel. The images were generated by using APPLICATION VISUALIZATION SYSTEM (AVS) software (23), and the molecular surfaces were computed by using msms software (24).

Figure 2

HPLC analysis of sodium benzoate hydroxylation. R_T benzoic acid, 6.4 min; R_T 4-hydroxybenzoic acid, 3.8 min. (A) H₂O₂ (1 mM) + UV irradiation + benzoic acid (2 mM). (B) 4C6 (20 μM) + UV irradiation + benzoic acid (2 mM). The peak area of para-hydroxy benzoic acid is equivalent to ≈200 μM. (C) 4C6 + UV irradiation + benzoic acid (2 mM) + catalase (13 milliunits/ml). The peak area of para-hydroxy benzoic acid is equivalent to ≈220 μM. (D) 6G6 (20 μM) + UV irradiation + benzoic acid (2 mM).

Figure 3

(A) Standard view of the 4C6 Fab with the light (L) and heavy (H) chains colored in pink and blue, respectively. The locations of all tryptophans (highlighted in yellow) within the Fab are also shown. The hydroxylated tryptophan, L163, is highlighted in green. (B) Electron-density view of the hydroxylated tryptophan L163 and a proximate water molecule. A 2Fo-Fc electron-density map was calculated to 2.5 Å from a crystal structure with the tryptophan refined as alanine and contoured at 0.8 σ. The figure was generated in BOBSCRIPT (25) and rendered in RASTER3D (26).

Figure 4

Chemical formulae illustrating the central position of the hydrotrioxide radical between H₂O₃ and O₃ and its presumed conversion into ³O₂ and the hydroxyl radical.