[16 Heme Oxygenase 1 in Regulation of Inflammation and Oxidative Damage

By Shaw-Fang Yet, Luis G. Melo, Matthew D. Layne, and Mark A. Perrella

Introduction

Heme oxygenase (HO) enzymes catalyze the initial reaction in heme catabolism.1 Heme is a diverse metalloporphyrin molecule that is incorporated into important hemoproteins, such as hemoglobin and cytochromes, in the form of iron protoporphyrin IX.2 Distinct forms of heme oxygenase have been identified2-4: (1) HO-1, a 32-kDa protein induced by diverse stimuli, including inflammatory cytokines and factors that promote oxidative stress; (2) HO-2, a 36-kDa protein that is predominantly a constitutive enzyme; and (3) HO-3, a 33-kDa protein highly homologous to HO-2 (~90%) at the amino acid level.4 HO-1 and HO-2 are clearly products of distinct genes as they differ in chromosomal localization, gene

1 R. Tenhunen, H. S. Marver, and R. Schmid, Proc. Natl. Acad. Sci. U.S.A. 61, 748 (1968).

2 M. D. Maines, Annu. Rev. Pharmacol. Toxicol. 37, 517 (1997).

4 W. K. McCoubrey, Jr., T. J. Huang, and M. D. Maines, Eur. J. Biochem. 247, 725 (1997).

for parent ions, while holding the third quadrupole at a constant m/z value (315, daughter ion) after subjecting all ions to CAD in the second quadrupole. Using this scan mode, the conjugate between GSH- and biotin-BMCC with an m/z value of 841 is readily identified as the parent ion (Fig. ID). We have also performed experiments to detect the presence of the BMCC-GSH modification in complex peptide mixtures. Using these scanning techniques, at least 100 fmol of BMCC-GSH conjugate parent ion (m/z — 841) can be detected in the presence of a 10-pmol mixture of tryptic peptides derived from an in-gel digest, using a nanoelectrospray ionization source (data not shown). Precursor ion scanning for m/z = 315 is most specific for the presence of the BMCC-GSH conjugate in complex peptide mixtures. In principle, this approach could be used to identify the position of biotin-BMCC-tagged sulfhydryls in any protein, including integrin.

Acknowledgment

This work was supported by Grants HL-58925, CA-69306, and CA-30199 from the NIH.

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