title: Reactivation of broccoli peroxidases: structural changes of partially denatured isoenzymes.
abstract: Structural changes involved in the reactivation of peroxidases (PODs) from broccoli and horseradish (HRP) following heat denaturation were investigated by using circular dichroism and absorption spectroscopy. Cooling heat-treated enzymes resulted in rapid refolding of the secondary structure into 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 approximately 25 degrees C and results in the return of biological activity. The secondary structure of neutral broccoli POD (N) is relatively heat labile, resulting in a rapid loss of activity, but the level of reactivation is high because the structure at the heme pocket is relatively stable. Acidic broccoli POD and HRP are more heat stable than N, but have a low degree of reactivation. Loss of activity is due primarily to alteration of the structure at the heme pocket. Effects of bovine serum albumin and pH on reactivation of PODs are also discussed.
  
  
  BACKGROUND
  Peroxidase; reactivation; horseradish; broccoli; circular dichroism; absorption spectroscopy.
authors.affiliation: Department of Agro-Industry, Faculty of Agriculture Natural Resources and Environment, Naresuan University, Phitsanulok 65000, Thailand.
authors.forename: Tipawan
authors.initials: T
authors.lastname: Thongsook
authors.forename: John R
authors.initials: JR
authors.lastname: Whitaker
authors.forename: Gary M
authors.initials: GM
authors.lastname: Smith
authors.forename: Diane M
authors.initials: DM
authors.lastname: Barrett
mesh_terms: D001937:Brassica / Q000201:enzymology*; D002942:Circular Dichroism; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D013053:Spectrophotometry
chemical_list: D004793:Enzyme Reactivators; D007527:Isoenzymes; D009195:Peroxidase