title: The covalent binding of genistein to the non-prosthetic-heme-moiety of bovine lactoperoxidase leads to enzymatic inactivation.
abstract: OBJECTIVE
  Genistein, a major soy isoflavone metabolite (SIF), inactivates oxidation activity of bovine lactoperoxidase (LPO). Modification of the heme moiety of LPO by nitrogen-containing compounds has been shown to inactivate LPO. In contrast, SIF mediated inactivation of LPO does not involve a heme modification and the mechanism of SIF inhibition is poorly understood.
  
  
  METHODS
  After inactivation of LPO by genistein in the presence of H(2)O(2), trypsin-digested LPO peptide fragments were collected and analyzed by MALDI-TOF-MS to characterize the chemical binding of genistein(s) to LPO.
  
  
  RESULTS
  The heme moiety of LPO was not modified by genistein. A covalent binding study showed that (3)H-genistein bound to LPO with a ratio of ∼12 to 1. After HPLC analysis and peak collection, trypsin-digested peptide fragments were analyzed by MALDI-TOF-MS. The 3H-genistein co-eluted peptide fragments (RT=24 min) were putatively identified as 199IVGYLDEEGVLDQNR214 with two bound genistein molecules or a genistein dimer (2 259 Da), 486TPDNIDIWIGGNAEPMVER504 with two bound genistein molecules or a genistein dimer (2 663 Da), and 161ARWLPAEYEDGLALPFGWTQR182 with three bound genistein molecules or a genistein trimer (3 060 Da). The fragment with a mass of 1 792 Da (RT=36 min) was identified as 132CDENSPYR139 with three genistein molecules or a genistein trimer.
  
  
  CONCLUSIONS
  The results suggest that LPO was inactivated by irreversible covalent binding of genistein or genistein polymers to particular peptide fragments constituting regions of the outward domain. No genistein interaction with the prosthetic heme moiety of LPO was observed.
authors.affiliation: Department of Biotechnology, Asia University, Taichung, Taiwan, China. hebronchang@asia.edu.tw
authors.forename: Hebron C
authors.initials: HC
authors.lastname: Chang
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Doerge
authors.forename: Chenghong
authors.initials: C
authors.lastname: Hsieh
authors.forename: Yingju
authors.initials: Y
authors.lastname: Lin
authors.forename: Fuujen
authors.initials: F
authors.lastname: Tsai
mesh_terms: D000818:Animals; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects*; D019833:Genistein / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D007784:Lactoperoxidase / Q000378:metabolism*; D010928:Placental Lactogen; D011485:Protein Binding
chemical_list: D007529:Isoflavones; D010928:Placental Lactogen; D006861:Hydrogen Peroxide; D019833:Genistein; D007784:Lactoperoxidase