Peroxidase/hydrogen peroxide—or bone marrow homogenate/hydrogen peroxide—mediated activation of phenol and binding to protein

Abstract

1. ¹⁴C-Phenol was metabolized by rat bone marrow homogenate and H₂0_2. The homogenate catalyst, however, was inactivated by preincubation with H₂0₂, presumably due to inactivation of the enzyme(s) involved in phenol metabolism.

2. The majority of the metabolized ¹⁴C-phenol was bound to bone marrow proteins. o,o'-Biphenol and p,p'-biphenol were the principal non-protein-bound products. Ascor-bate was unable to remove phenol oxidation products bound to protein, although o.o'-biphenol recovery from the reaction mixture was markedly enhanced. Prior alkylation of protein thiols with N-ethylmaleimide decreased the binding of ¹⁴C-phenol oxidation products to bone marrow proteins by only 10-20%.

3. ¹⁴C-Phenol (200μM) metabolism by horseradish peroxidase (10μ) and H₂0₂ (200M) also resulted in extensive binding to externally added bovine serum albumin. The absorption spectrum of ¹⁴C-phenol oxidation products bound to bovine serum albumin was similar to that of bound oxidation products of o,o'-biphenol but not of p,p'-biphenol.

4. Protease digestion of bovine serum albumin bound ¹⁴C-phenol oxidation products, followed by ethyl acetate extraction, extracted 75% of the ¹⁴C, indicating that most of the binding is probably non-covalent. Up to 32% of the ¹⁴C-phenol oxidation products binding to bovine serum albumin may be covalent, since derivation with dinitrofluorobenzene and extraction under acid but not alkaline, conditions extracted the ¹⁴C. The percentage of metabolites covalently bound to bovine serum albumin was increased to 59% when horseradish peroxidase concentration was decreased to 0.2μg.

5. The thiol groups of bovine serum albumin were unaffected by o,o'-biphenol oxidation products, slightly decreased by phenol oxidation products, but were completely depleted by p,p'-biphenol oxldation products.

6. These results indicate that o,o'-biphenol oxidation products are responsible for much of the ¹⁴C-phenol binding to protein.