Hemopexin liver

Hemopexin was first identified as a heme binding P-globin in elec-trophoretograms of plasma of patients with hemolysis (17-19). The protein is synthesized and secreted by the liver (20-22), and during secretion the signal peptide is removed and the protein is glycosylated (23). Tissue forms of hemopexin are expected due to the presence of mRNA in brain (24), peripheral neurons (25), and neural retina (26), pointing to a function of hemopexin in barrier tissues. 

The human hemopexin gene has been cloned (38) and is located near the P-globin gene cluster on human chromosome 11 (39). The promoters of the human, rat, and mouse hemopexin genes have been cloned, and the human gene contains a liver specific element (40) and an interleukin-6 responsive element (41), consistent with the positive acute phase response ofhemopexin (33,34). 

In patients clearance of intravenous heme is rapid until hemopexin levels are depleted (148), and lack of interaction with hemopexin may explain the higher clinical efficacy of heme-arginate compared with hemin itself (149, 150). In intact animals, i.v. heme causes rapid association of hemopexin but not albumin with the liver (47, 63, 68), and heme uptake from heme-albumin complexes into isolated rat hepato-cytes occurs via diffusion of heme released from its loose complex with BSA (137). Moreover, unlike uptake from heme-hemopexin, free heme uptake by cells occurred even at 4°C, as expected for nonspecific membrane association and in total disagreement with a membrane-receptor-mediated or active transport uptake process. 

Heme that is released into the blood stream by lysis of red cells, catabolism of haptoglobin-hemoglobin complexes, or other mechanisms binds to albumin (Ka = 10 M) and hemopexin (Ka 10 M) (48). The hemopexin-heme complex is taken up in the liver by a receptor-mediated process. A candidate for the hemopexin-heme receptor has been identified (49). Other heme-binding proteins are a-l-microglobulin and the glutathione transferases. 

The use of isotope-labelled synthetic peptides as IS was proposed for the absolute quantification of proteins in protein expression studies [113]. If necessary, these synthetic peptides can be covalently modified to be applied as IS in for instance phosphopeptide quantification. This approach was applied in the quantitative analysis of two glutathione 5 -transferase isoforms in human liver cytosol by LC-MS in SRM mode [114]. A series of pilot experiments were performed to select the most suitable IS peptides for four human plasma proteins (hemopexin, ocl antichymotrypsin, interleiddn-6, and tumor necrosis factor-oc) [115]. Rabbit polyclonal antibodies were raised against these selected peptides and immobilized on POROS supports. These lAC columns were applied to achieve a 120-fold emichment of the antigen peptide from digested human plasma proteins. The peptides and its IS were measured by LC-MS in SIM or SRM mode. The methods appears to be a tailor method for targeted protein analysis. 

Newborn Period Hp is absent or present in very low concentration in most newborn infants as a result of hepatic immaturity. In addition, hemopexin levels in newborns ai e on average one fifth of adult levels. The absence of Hp and hemopexin in the normal fetus may prevent in utero stimulation of the hepatic excretory mechanisms for bilirubia (fetal bilirubin is normally excreted by the placenta and the maternal liver). 

The chemistry and biochemistry of Hpx has been reviewed and a crystal structure is available. Hemopexin is present in serum at about 10 pM and its primary function is to transport released heme to its degradation site in the parenchymal cells of the liver via receptor-mediated endocytosis. Encapsulation of a single heme by Hpx occurs via bis-histidyl protein side-chain coordination of the Fe. Spectroelectrochemical investigation of the heme-Hpx assembly gives insight into the role of Hpx in controlling the reduction potential of the heme Fe, the efficiency of electron transfer at the metal centre, the influence of bis-histidyl coordination at the Fe centre, and the possible role of Fe redox in the Hpx-mediated transport and recycling of heme. 

Figure 5-2 Expression of hemopexin mRNA levels in barrier tissues. The data show the results of a dot blot analysis of various amounts of mouse liver (0.2, 0.5,0.8, 1.0 and 2.0 pg), brain (10 and 20 pg), ovary (5 and lOpg), testis (10 and 20pg) RNA samples (Ambion, Texas, USA) as indicated and the levels of mRNA were determined by hybridization with the same hemopexin probe as described in the legend to Figure 5-1.

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