Ursula Muller-Eberhard

Synthesis of hemopexin and cysteine protease inhibitor is coordinately regulated by HSF-II and interferon-β2 in rat hepatoma cells

Rat hepatoma (H-35) cells respond to hepatocyte-stimulating factors by increased expression of major acute phase plasma proteins. The synthesis of hemopexin is stimulated 10-fold by either hepatocyte-stimulating factor-II of human squamous carcinoma cells or hepatocyte-stimulating factor/interferon-beta 2 of activated human blood monocytes. The hormone specificity, time course and dose-dependence of hemopexin regulation is closely correlated with that of cysteine protease inhibitor. The coordinate expression of hemopexin and other type II acute phase proteins suggests the existence of common molecular regulatory mechanisms.

Porphyrin-induced photodynamic cross-linking of hepatic heme-binding proteins

Three types of hepatic proteins, a heme-binding Z protein, a mixture of the glutathione S-transferases and a cytochrome P450 isozyme, were shown to be susceptible to photodynamic cross-linking and loss in antigenicity by naturally occurring porphyrins. At 50 microM, uroporphyrin caused the most and protoporphyrin the least photodecomposition. Hemopexin, a specific serum heme carrier, was photodecomposed but no cross-linking was detected. Heme and scavengers of singlet oxygen partially prevented protein photodecomposition.

Long-term intercalation of residual hemin in erythrocyte membranes distorts the cell

The effect of long-term incubation of residual globin-free hemin on whole red blood cell and isolated cytoskeletal proteins was studied. Hemin at concentrations found in pathological red cells was inserted to fresh erythrocytes. Increased hemolysis developed in the hemin-containing cells after a few days at 37 degrees C and after about four weeks at 4 degrees C. Since lipid and hemoglobin peroxidation did not depend on the presence of hemin, time-dependent effects on the cytoskeleton proteins were studied. Observations were: (1) spectrin and protein 4.1 exhibited a time-dependent increasing tendency to undergo hemin-induced peroxidative crosslinking. (2) The ability of the serum proteins, albumin and hemopexin, to draw hemin from spectrin, actin and protein 4.1 decreased with time of incubation with hemin. These results were attributed to time-dependent hemin-induced denaturation of the cytoskeletal proteins. Albumin taken as a control for physiological hemin trap was unaffected by hemin. Small amounts of hemo-spectrin (2-5%) were analyzed in circulating normal cells, and this in vivo hemo-spectrin also failed to release hemin. It was concluded that slow accumulation of hemin, a phenomenon increased in pathological cells, is a toxic event causing erythrocyte destruction.

A turbidometric assay for measuring proteins in culture media

An immunoturbidometric assay was developed for the measurement of proteins in culture fluids of hepatocytes. The assay is simple to perform and avoids the biohazards associated with radioimmunoassays. The limit of detection of this assay exemplified by hemopexin and transferrin is 5 ng/ml protein. This degree of sensitivity is attained by incorporating into the procedure the addition of polyethylene glycol to enhance formation of primary antigen-antibody complexes and of a second antibody to further increase the immune complex size, which favors the ratio of specific to background light scattering.

The reactions of hemopexin and liver fatty acid-binding protein (L-FABP) with aurothiomalate

Abstract We investigated whether gold(I) binds to hemopexin, Hx, a serum heme carrier with a molecular weight similar to that of serum albumin, or to L-FABP, a liver cytosolic heme- and fatty acid-binding protein whose molecular weight is similar to that of metallothionein. These proteins and, for comparison, similar concentrations of bovine albumin were incubated with sodium aurothiomalate and then fractionated by gel exclusion chromatography. At 16 μM of Hx and gold the ratio of gold bound to Hx was 0.30 ± 0.04. The corresponding ratio for albumin was 0.98 ± 0.04 per mercaptalbumin. Considering the much lower serum levels of Hx compared to albumin it is unlikely that Hx plays a role in serum gold binding and transport. L-FABP also binds gold: at 52 μM L-FABP and 108 μM gold, the gold to protein ratio was 0.55 ± 0.05. The corresponding ratio for albumin under identical conditions was 1.18 ± 0.08 per mercaptalbumin. L-FABP failed to bind zinc or cadmium, two other metals bound by metallothionein.

Studies on species cross-reactivity of hemopexin by use of monoclonal and polyclonal antibodies.

The extent of immunological cross-reactivity between hemopexins of four species (rat, human, rabbit and chicken) was assessed with four affinity purified polyclonal antibodies and three monoclonal antibodies using RIA, Western blotting and rocket immunoelectrophoresis. Neither the two monoclonal antibodies to rabbit hemopexin (Rb3D11 and Rb3H9), the monoclonal antibody (R4B3) to rat hemopexin nor any of the polyclonal antibodies showed shared antigenic determinants between avian and mammalian hemopexins as judged by RIA or rocket immunoelectrophoresis. Western blotting with polyclonal antibodies revealed some reactivity raising the possibility of a few shared, though distantly related, epitopes. Polyclonal antibodies, raised to the mammalian hemopexins cross-reacted to variable extents with the respective antigens by RIA, results paralleled by data obtained by Western blotting. Anti-rat monoclonal antibodies reacted only with rat hemopexin in Western blots and minimally with rabbit hemopexin in RIA. The anti-rabbit monoclonal antibodies recognized two distinct epitopes one of which is shared with human hemopexin and presumably highly conserved.