Heme oxygenase is the rate-limiting microsomal enzyme in the heme degradative pathway. Heme oxygenase catalyzes the NADPH, O2 and cytochrome P450 reductase dependent oxidation of heme to form equimolar biliverdin, carbon monoxide, a putative neurotransmitter, and iron. Biliverdin is subsequently converted to bilirubin by biliverdin reductase. These products of the HO reaction have important physiological effects: carbon monoxide is a potent vasodilator; biliverdin and its product bilirubin are potent antioxidants; "free' iron increases oxidative stress and regulates the expression of many mRNAs (e.g., DCT-1, ferritin and transferrin receptor) by affecting the conformation of iron regulatory protein (IRP)-1 and its binding to iron regulatory elements (IREs) in the 5'- or 3'-UTRs of the mRNAs. Increased heme oxygenase activity leads to accumulation of the antioxidant bilirubin, and degradation of the preoxidant heme. Moderate overexpression of the inducible form, HO-1, is associated with protection against oxidative injury. Growth retardation, anemia, iron deposition, and vulnerability to stressful injury are all characteristics observed in mice in which the heme oxygenase-1 gene has been knocked out. The isozymes have a hydrophobic region of 23 amino acids at the C-terminus that serves as membrane anchor. A conserved sequence of hydrophobic of 23 amino acids in the middle is thought to be the substrate (heme) binding site.

To date, 3 forms of heme oxygenases (HO1-3) have been identified. HO-1 or Hsp-32 (EC 1.14.99.3; mouse/rat 289 aa; human 288 aa, chromosome 22; ~88% homology between the species) is an inducible enzyme. Ho-1 is expressed in most tissues with highest levels in spleen. HO-1 gene expression is inducible by heme, suggesting an important role of HO-1 in heme metabolism. Many other agents or conditions related to oxidant damage such as longer wavelength UV radiation, hyperoxia, hypoxia, hydrogen peroxide, glutathione depletion, endotoxin, and, more recently, nitric oxide (NO) have also been found to stimulate HO-1 expression. The 5'-untranslated region (UTR) of HO-1 has several consensus regulatory elements, which include sites for

activator protein 1 (AP-1), metal responsive element (MRE), oncogene c-myc/max heterodimer binding site (Myc/Max), antioxidant response element (ARE) and GC box binding (Sp1). HO-1 expression has been shown to increase in benign prostatic hyperplasia (BPH) and malignant prostate tissue suggesting a role for this stress protein in the pathogenesis of BPH and prostate cancer. There is recent data, which indicates the ability of peroxynitrite (ONOO-) to modulate the expression of HO-1 and suggest that the heme oxygenase pathway contributes to protection against the cytotoxic action of ONOO-, which is a potent oxidizing agent, generated by the interaction of nitric oxide (NO) and the superoxide anion. ONOO- rapidly decomposes to a highly reactive hydroxyl radical and nitrogen dioxide, both of which cause oxidative damage.

HO-2 (mouse/rat 315 aa; human 316 aa; chromosome 16) is non-inducible form. Except for corticosterone, no other inducer of HO-2 has been identified to date. HO-2 has been localized to endothelial cells and adventitial nerves of blood vessels. It is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Low levels of Ho-2 are found in most tissues except spleen. At the amino acid level, HO-1 and HO-2 are 42% homologous. However, each isozyme is evolutionary highly conserved. The molecular weight of mammalian HO-1 is ~31-33kDa and HO-2 is ~36kDa.

A third enzyme, HO-3 (rat 290 aa ~ 33 kDa) has been cloned and characterized from rat brain. The HO-3 mRNA is found in the spleen, liver, thymus, prostate, heart, kidney, brain and testis. The predicted amino acid structure of HO-3 differs from both HO-1 and HO-2 but is closely related to HO-2 (~ 90% similarity). HO-3 sequence has two HRM known to be involved in heme binding. It is a poor heme catalyst. Therefore, HO-3 has a potential regulatory role in heme-dependent cellular processes.

 

M= Mouse; R=Rat; H=Human; Ha=Hamster; Rb=Rabbit; B=Bovine; CT= near C-terminus; NT=near N-terminus; Internal=Middle of protein.

m=mouse; r=rat; h=human; b=bovine; d=dog; ~CT or ~NT=near C or N-terminus. EC=Extracellular; CP=Cytoplasmic domain; Control peptides (unconjugated, free, antigenic peptides), because of their small size, are not recommended for Western. They should be used in ELISA/antibody blocking studies.

* Expected antibody crossreactivity information is mostly based upon high (>70%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera or antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.
Western blot +ve protein controls, where available, are semi-pure, pure or recombinant proteins that are formulated in SDS-PAGE sample buffer. They are recommended to be used for Western (load 10 ul/lane) for visulaization with antibodies.