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In an attempt to alter the catalytic properties of horseradish peroxidase (HRP, EC 1.11.1.7), aspartic, glutamic and arginine residues were modified using ethanedithiol and diacetyl. Modification of Asp and Glu led to a marked increase in Vmax along with denaturation of the protein. The thiol groups introduced were thought to be responsible, despite being situated on the periphery of the molecule as shown by the modification of the apoenzyme. The role of Arg 38 in the activation of hydrogen peroxide was indicated by the modifications of both enzyme and apoenzyme. An amino acid residue close to Arg 38 was thought to take over its function after blocking the group.

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.