Magnetic resonance spectroscopy, calcium content, and anion coordination studies of bovine and goat lactoperoxidase.
Bovine lactoperoxidase from two purebred strains and a commercial source as well as lactoperoxidase isolated from Alpine goat milk were examined by proton NMR spectroscopy for structural comparison of the heme site. Hyperfine shifted proton NMR spectra for both the native enzymes and cyanide complexes were equivalent for the protein obtained from the four separate sources. Activity assays (guaiacol and iodide ion oxidations) were also employed to compare the enzyme from various sources. Bovine lactoperoxidase was shown to contain 1.5 +/- 0.1 calcium ions per heme unit. Lactoperoxidase complexes with nitrite ion and thiocyanate ion were characterized for comparison with the cyanide complex. The nitrite complex exhibits a proton NMR hyperfine shift pattern at ambient temperature consistent with a low-spin ferric formulation. Interaction of lactoperoxidase with thiocyanate ion was monitored by NMR and EPR spectroscopy. Proton NMR spectra of lactoperoxidase in the presence of excess thiocyanate ion illustrated the retention of a high-spin ferric configuration consistent with predominant binding of the physiological thiocyanate substrate at a non-heme site at room temperature. However, EPR spectroscopy at cryogenic temperatures revealed the existence of a low-spin lactoperoxidase thiocyanate complex. This result may be explained by low-affinity ambient temperature thiocyanate heme binding that is greatly enhanced at liquid helium temperature.
Lukat
G S
GS
Department of Chemistry, University of Iowa, Iowa City 52242.
Doran
M B
MB
Utschig
L M
LM
Goff
H M
HM
D000818:Animals; D000838:Anions; D002118:Calcium / Q000032:analysis*; D002417:Cattle; D003486:Cyanides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D006041:Goats; D006418:Heme / Q000032:analysis; D007784:Lactoperoxidase / Q000032:analysis* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008892:Milk / Q000201:enzymology; D009573:Nitrites / Q000378:metabolism; D013861:Thiocyanates / Q000378:metabolism
D000838:Anions; D003486:Cyanides; D009573:Nitrites; D013861:Thiocyanates; D006418:Heme; D007784:Lactoperoxidase; C031760:thiocyanate; D002118:Calcium