Effect of cross-linker length on the stability of hemoglobin.

A series of cross-linking reagents with 4 to 7 carbons have been synthesized and used to modify human hemoglobin. The product yields and biochemical properties of these cross-linked hemoglobins are compared to those made with both longer and shorter cross-linkers. Several trends become apparent. The yields decrease as the cross-linker becomes longer, which correlates well with molecular dynamics studies of reagent binding pathways presented here. The autooxidation rates increase while thermal stability decreases with longer reagents. Cross-linking under deoxy conditions also increases autooxidation rates, but the effect is less than that of increased cross-linker length. The results suggest that shorter reagents may provide better-stabilized tetramers for the construction of more complex hemoglobin-based oxygen carriers.

Synthesis of a hemoglobin polymer containing antioxidant enzymes using complementary chemistry of maleimides and sulfhydryls.

To increase the overall size of hemoglobin (Hb), we developed a novel system of polymerization based on the complementary chemistry between sulfhydryls and maleimides. The maleimides were introduced onto the protein through N-(-maleimidobutyryloxy) succinimide, while the sulfhydryls were added using 2-iminothiolane hydrochloride (Trauts reagent). Resulting polymers showed SDS-PAGE bands with molecular weights as high as 96 kDa. Size exclusion chromatography has demonstrated species with molecular weight > 700 kDa. The flexibility of the sulfhydryl-maleimide chemistry has also allowed insertion of two antioxidant enzymes, catalase (Cat) and superoxide dismutase (SOD), into the Hb polymer. Cat was incorporated into the heavier fractions of the polymer, while SOD was found throughout the molecular weight range.