| Heme oxygenase-2 (HO-2) is the constitutive isoform of heme oxygenase which catalyzes the NADPH, O2 and cytochrome P450 reductase dependent oxidation of heme to carbon monoxide, iron and biliverdin that is immediately reduced to bilirubin. These products of the HO reaction have important physiological effects: carbon monoxide is a potent vasodilator, biliverdin and its product bilirubin are potent antioxidants, ifreei 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. To date, three heme oxygenase isoforms HO-1, HO-2 and HO-3 have been identified. HO-1 or HSP32 , a major heat shock/stress response protein is ubiquitous and its mRNA as well as its activity can be increased several-fold by heme, other metalloporphyrins, transition metals and stimuli that induce cellular stress. In contrast to HO-1, HO-2 is highly concentrated in neurons and testes (1). Multiple HO-2 transcripts which differ in size and use 3 different 5i untranslated regions (referred to as rHO-2, rHO-2-1 and rHO-2-2) and 2 poly(A) signals have been identified and a functional glucocorticoid response element (GRE) in the promoter region of rHO-2 has been characterized. In the adult rat testis, there is developmentally regulated expression of two transcripts for HO-2 of ~2.1kb and ~1.45kb which are not present in the brain, kidney, thymus, heart, spleen, liver or in prepubertal 14 day old rat testis. Both of these transcripts exclusively use rHO-2 and they contain all of the coding region exons present in the ~1.3kb and ~1.9kb transcripts which are common to all other organs including the adult and prepubertal rat testis. This data suggests that HO-2 levels in the testis are controlled by glucorticoids and that developmental and tissue-specific factor(s) determine generation of transcripts unique to the organ. The apparent exclusive use of rHO-2 by the mature testis suggests that HO-2 may play a role in male reproduction (2). The recombinant protein is produced in E. coli in a soluble form as it does not contain the C-Terminal membrane spanning region. This recombinant human HO-2 (M.W.~34,400) preparation consists of amino acid residues 1-291 of human HO-2 followed by V-E-H-H-H-H-H-H. The recombinant product consists of only the full length HO-2 protein due to an elimination of the second start codon. Applications: Suitable for use in Western Blot and Immunohistochemistry (1:2,000). Other applications not tested. Recommended Dilution: Western Blot: We recommend that the material be diluted in 1X sample buffer (e.g. Laemmli, U.K., (1970) Nature 277: 680-685). On a 15-well minigel system, 50ng of protein per lane should be sufficient when used in a Western Blot. This protein mixture yields a band of 34.4kD. An ~70kD band is also observed, which may be aggregates of HO-2. Storage and Stability: May be stored at 4C for short-term only. For long-term storage, aliquot to avoid repeated freezing and thawing and freeze at -70C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for at least 12 months. |
