Characterization of polyethylene glycolated horseradish peroxidase in organic solvents: Generation and stabilization of transient catalytic intermediates at low temperature

Polyethylene glycolated horseradish peroxidase (PEG-HRP) can catalyze one- and two-electron oxidation reactions in organic solvents as well as in aqueous buffer. Even though the oxidation of guaiacol in benzene and chlorobenzene is 5 orders of magnitude slower than in phosphate buffer, compounds I and II are involved in the catalytic cycle in organic media. Factor analysis and global fittings of rapid scan data set reveal that the formation of compound I of PEG-HRP in organic media consists of two steps (the first fast and the second slow) and suggest the involvement of a H2O2-HRP complex in the catalytic cycle. The labile precursor of compound I is stabilized when PEG-HRP reacts with hydrogen peroxide in chlorobenzene at -20 degrees C. The absorption spectrum of the precursor does not exhibit the features of hyperporphyrin spectrum but has a normal Soret as previously observed in R38L HRP. More importantly, compound I of PEG-HRP can be maintained for more than an hour at -20 degrees C in chlorobenzene.