Heat inactivation and reactivation of broccoli (Brassica oleracea var. Italica) peroxidases

Three peroxidase (POD) isoenzymes were purified from a soluble extract of broccoli stems. The acidic (A), neutral (N) and basic (B) POD were purified to homogeneity by using ion exchange and hydrophobic and gel filtration chromatography. N and B had molecular masses of approximately 43 kDa and A had molecular mass of 48 kDa by SDS-PAGE. pI was approximately 4, 5 and 8 for A, N and B, respectively. Optimum activity using guaiacol as the H donor were obtained at approximately pH 6 for both N and B and about pH 4 for A. All three of the purified isoenzymes are glycosylated. Reaction rate with various substrates, Km and amino acid composition were different among the isoenzymes. At 65°C, A was the most heat stable followed by N and B. The activation energies for denaturation were 388, 189 and 269 kJ/mol for A, N and B, respectively. Reactivation occurred rapidly, within 10 min after the heated enzyme was cooled and incubated at room temperature. The extent of reactivation varied from 0 to 50% depending on the isoenzyme and heating conditions (temperature and time). The denaturation temperatures allowing the maximum reactivation differed among the isoenzymes. In all cases, heat treatment at low temperatures for long times prevented reactivation of the heated enzymes. Effect of calcium and bovine serum albumin on reactivation was also reported. Cooling of heat-treated enzymes resulted in rapid refolding of the secondary structure to an inactive structural species similar in conformation to the native enzyme. Reassociation of heme to the refolded peroxidase as well as molecular rearrangement of the structure around the heme occurs during incubation at ∼25°C resulting in a return of the biological activity. N has a higher level of reactivation compared to that of A and horseradish POD (HRP) because the structure at the heme pocket is relatively stable, compared to its rapid loss of enzyme activity due to alteration of secondary structure. Loss of activity of A and HRP is mainly due to alteration of the structure at the heme pocket. Effects of BSA and pH on reactivation were also discussed.