Author(s): Kaca W, Roth RI, Levin J
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Abstract Cell-free hemoglobin (Hb) is a purified preparation of human hemoglobin that is being developed as a resuscitation fluid. In vivo administration of hemoglobin has resulted in significant toxicity, due in part to contamination with bacterial endotoxin (lipopolysaccharide (LPS)). To better understand this toxicity, we have studied the interaction between Hb and LPS. Mixtures of each of three different Hb preparations (cross-linked alpha alpha Hb, cross-linked carbon monoxy-alpha alpha HbCO, and non-cross-linked (native) HbAo) and LPS (Escherichia coli O26:B6 or Proteus mirabilis S1959) were examined by several independent methods for evidence of Hb.LPS complex formation. Binding assays in microtiter plates demonstrated saturable binding of LPS to immobilized Hb, with a kD of 3.1 x 10(-8) M. Binding of LPS to Hb also was demonstrated wiht a radiolabeled LPS photoaffinity probe. Ultrafiltration of Hb/LPS mixtures by 300- and 100-kDa cut-off membranes showed that the majority of LPS in these mixtures (87-97 and 64-72\%, respectively) was detected in the filtrates, in contrast to the lack of filterability of LPS in the absence of Hb. Density centrifugation demonstrated that LPS co-migrated with each of the three Hbs, whereas unbound LPS had a distinctly greater sedimentation velocity than Hb or Hb.LPS complexes. Nondenaturing polyacrylamide gel electrophoresis demonstrated that in the presence of Hb, LPS migrated into the gel and co-electrophoresed with Hb, whereas LPS alone did not appreciably enter the gel. Finally, precipitation by ethanol of each of the three Hb preparations was increased in the presence of LPS compared with precipitation in the absence of LPS. Interaction of LPS with each of the three Hb preparations was also associated with altered biological activity of LPS, as shown by enhancement of LPS activation of Limulus amebocyte lysate. Therefore, our data provide several lines of independent evidence for Hb-LPS complex formation and indicated that LPS exhibited altered physical characteristics and enhanced biological activity in the presence of Hb.
This article was published in J Biol Chem
and referenced in Journal of Microbial & Biochemical Technology