Experiments with peroxidase. III. Recombination of hemin and protein to the active enzyme

Abstract

The recombination of equimolecular amounts of protohemin and apoprotein to horse-radish peroxidase (HRP) in aqueous solution was studied using spectrophotometric techniques. In the Soret region (360–450 mμ) two intermediate compounds, E and F, were observed. The formation of E is very fast, whereas the two reactions E → F and F → HRP are slower and of different velocities. They could not be measured accurately since it is difficult to separate them spectrophotometrically. Kinetic observations showed that the first recombination reaction (C + D) → E was mainly due to a rapid change in the hemin molecule with pH, but another process, presumably a disaggregation of hemin, takes place simultaneously. The step E → F is attributed to the establishment of one or two linkages of the same type between hemin and protein. During this reaction the ability of the enzyme to form H 2O 2 complexes is re-established. The apparent free energy of activation was measured and a value of E′ a = 11,900 ± 300 cal./mole was found. The apoprotein loses its ability to combine with hemin when heated at low pH. The reaction F → HRP is the slowest step and is superimposed on the step E → F. During the final reaction F → HRP the intrinsic activity of the enzyme-substrate complex HRP-H 2O 2II increases to its maximum value, which can lie somewhat above the value of the initial untreated HRP.