Details

Autor(en) / Beteiligte

• Rae, Tracey Douglas

Titel

The heme prosthetic group of the mammalian antimicrobial enzyme lactoperoxidase

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1996

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• The focus of this project is the isolation and structural characterization of a heme group from the bovine milk enzyme lactoperoxidase (LPO), herein termed heme l. Through an approach that combines proteolytic hydrolysis of LPO and reverse-phase chromatographic separation of the resulting digest, a heme group is obtained in a form that allows for complete structural characterization. Digestion of LPO with both trypsin and chymotrypsin yields several heme l-peptide species upon HPLC separation, whereas digestion of LPO with Proteinase K (protease with esterase activity) yields primarily a heme l species without peptide content. Both heme l and heme l-peptide species were investigated by paramagnetic $\sp1$H NMR spectroscopy, electrospray mass spectrometry, and peptide sequence analysis. Paramagnetic $\sp1$H NMR spectra of the high-spin bis(dimethylsulfoxide)Fe(III) complexes of the isolated heme species provide information regarding peptide content. Paramagnetic $\sp1$H NMR experiments (inversion-recovery, COSY, and steady-state NOE-difference spectra) of the low-spin bis(cyano)Fe(III)heme l complex conclusively define the heme l structure as a 1,5-bis(hydoxymethyl) derivative of heme b. The electrospray mass spectrum of heme l confirms the two-site hydroxyl functionalization on this heme. Sequence analysis of peptides released from two heme l-peptide species by base hydrolysis confirms heme-protein ester linkages in LPO occur between the two hydroxyl groups of heme l and the carboxylic side chains of glutamate 275 and aspartate 125. The reactivity of heme l in LPO was investigated through a study of the thiocarbamide-induced heme-modification reaction that results in enzyme inactivation. Two thiocarbamide compounds (methimazole and propylthiouracil) which efficiently inactivate LPO through heme modification are found to be unreactive toward the heme b containing peroxidases from plant and fungal sources, implying the heme l structure is specifically vulnerable to these compounds. Attempts to study the methimazole-modified LPO heme through both paramagnetic $\sp1$H NMR spectra of LPO-CN and isolation of the heme by the proteolysis/HPLC method were precluded by the apparent reversion and/or degradation of the modified heme. Observations made in these experiments strongly suggest the formation of a sulfheme-type structure.