Abstract
Artificial horseradish peroxidases (HRP) were reconstituted from the apoenzyme and the following unnatural hemins; hematohemin, mesohemin, deuterohemin, 2, 4-diacetyldeuterohemin, and protohemin dimethyl ester. The HRP preparations used were HRP-A, a mixture of isozymes of A group, and HRP-(B+C), a mixture of isozymes B and C.
The peroxide compounds such as Compounds I and II and the oxy-form (or Compound III) were obtained in these artificial enzymes except for protohemin dimethyl ester-HRP. Their spectral patterns were similar to those of natural HRP but absorption maxima were shifted to longer wavelengths in the order of deutero-, meso-, hemato-, proto-, and 2, 4-diacetyldeuterohemin.
The pKa of the transition between the acid and alkaline forms and the enzymatic activity were measured for the natural and artificial HRP. Although the pKa values of HRP-A and -(B+C) were very different they had similar correlation with 2, 4-sub-stituents. For the pKa of two HRP samples containing the same hemin, the relation could be represented by: pKa (B+C)=pKa (A)+1.6
It was concluded that with the exception of hemato-HRP the pKa decreases in the increasing order of electron-withdrawing capacities of 2, 4-substituents of deuterohemin IX. The enzymatic activity also appeared to be related to some extent with such capacities of 2, 4-substituents.
