Mechanism of lysozyme inactivation and degradation by iron.
The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.
Sellak
H
H
INSERM U294, Faculté de Médecine Xavier Bichat, Paris, France.
Franzini
E
E
Hakim
J
J
Pasquier
C
C
D002374:Catalase / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds / Q000494:pharmacology*; D016166:Free Radical Scavengers; D005609:Free Radicals / Q000494:pharmacology; D006878:Hydroxides / Q000494:pharmacology; D017665:Hydroxyl Radical; D007501:Iron / Q000378:metabolism / Q000494:pharmacology*; D007502:Iron Chelating Agents / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009113:Muramidase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000528:radiation effects; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000494:pharmacology
D005296:Ferrous Compounds; D016166:Free Radical Scavengers; D005609:Free Radicals; D006878:Hydroxides; D007502:Iron Chelating Agents; D012710:Serum Albumin, Bovine; D017665:Hydroxyl Radical; C020748:ferrous sulfate; D007501:Iron; D002374:Catalase; D013482:Superoxide Dismutase; D009113:Muramidase