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Fig. 6 | BMC Biotechnology

Fig. 6

From: Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics

Fig. 6

rHp prevents heme release and lipid peroxidation. a Heme transfer from metHb to hemopexin was measured by spectrophotometry in a reaction mixture of 10 μM metHb(Fe3+) and 10 μM hemopexin. Plasma derived (pd) and recombinant wild-type Hp at 10 μM concentration completely blocked the reaction while only partial inhibition was observed with the cleavage site mutated rHps (green lines). b Lipoprotein peroxidation was measured by spectrophotometry as a function of oxyHb deoxygenation in a reaction mixture of 10 μM oxyHb(Fe2+) with 0.5 g/L rLP. The graph shows the time to anoxia. Peroxidation was completely blocked for > 6 h by the plasma derived and recombinant wild-type Hps, whereas lipid oxidation was not delayed compared to free Hb by the cleavage-site mutated rHp variants rHp1Smut and rHp2FSmut. c The identical lipid peroxidation reaction of rLP (0.5 g/L) and oxyHb (10 μM) as described in (b) was performed in cultures of HUVEC cells. Toxicity of lipid peroxidation products was monitored by fluorescence microscopy of β-catenin (yellow) and nuclear morphology (DAPI, blue) at 24 h after initiation of the reaction (original optical magnification 400×). Identical experiments as in (c) were performed on HUVEC monolayers monitored in an ECIS instrument. d Shows original electrical monolayer resistance data over time of biologic replicates. The colors represent the different treatments as indicated in (e). e “Time to monolayer breakdown” was calculated as a derivative from the electrical resistance data shown in (d). Data indicates mean ± SD of 6 biologic replicates

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