摘要
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 H202, 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 3H-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 1991VGYLDEEGVLDQNR214 with two bound genistein molecules or a genistein dimer （2 259 Da）, 486TPDNIDIWlGGNAEPMVER504 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 2 792 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.
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 H202, 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 3H-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 1991VGYLDEEGVLDQNR214 with two bound genistein molecules or a genistein dimer （2 259 Da）, 486TPDNIDIWlGGNAEPMVER504 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 2 792 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.
基金
supported by the National Science Council, Taiwan #NSC 95‐2320‐B‐408001
the Interagency Agreement between NCTR/FDA and the National Institute for Environmental Health Sciences/National Toxicology Program, USA
