Hemoglobin–albumin cross-linking with disuccinimidyl suberate (DSS) and/or glutaraldehyde for blood substitutes

Abstract

Hemoglobin (Hb) derivatization for blood substitute purposes often involves multi-step processes including redox reagents such as borohydride and periodate, with possible subsequent side effects. Disuccinimidyl suberate (DSS) allows protein cross-linking without toxic side-products, forming one-step peptide bonds with the lysine residues. Here, we report that Hb polymers were obtained using DSS, making this the first report of a single-step polymerization for blood substitutes. The increase in autooxidation rate incurred by this polymerization is completely reversed when BSA is copolymerized with Hb. Copolymerization of Hb with BSA appears to be beneficial for alleviating pro-oxidant effects, regardless of the polymerizing agent employed.

Protein-Based Blood Substitutes: Recent Attempts at Controlling Pro-Oxidant Reactivity with and Beyond Hemoglobin

“…They allow protein crosslinking without toxic side-products and forming peptide bond with lysine residue in one single step [29]. …”

“…The copolymers show very little change in autooxidation and oxygen affinity compared to native Hb, and low intensity of free radicals – less than in native Hb [29]. …”

Glutaraldehyde-polymerized hemoglobin and tempol (PolyHb-tempol) has superoxide dismutase activity that can attenuate oxidative stress on endothelial cells induced by superoxide anion

“…However, this attachment method required a "linker arm" modification of Hb. The multiple modification steps in this method are laborious and have the potential to cause Hb instability or precipitation [14].…”

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

“…Designing low oxygen affinity blood substitutes with enhanced molecular size (dimensions) has been initially attempted by one-step oligomerization platform that introduces intra and intermolecular crosslinking using glutaraldehyde. Chang introduced the oligomerization concept (Chang 1964(Chang , 1971, and since then many new bifunctional crosslinkers have been advanced for the oligomerization of Hb (Bunn et al 1969;Payne 1973;Marini et al 1989;D'Agnillo and Chang 1998;Hai et al 1999;MacDonald and Pepper 1994;Hai et al 1994;Bakker et al 1992;Dellacherie and Vigneron 1991;Zhang and Palmer 2010;Faggiano et al 2010;Tarasov et al 2007;Buehler et al 2006;Scurtu et al 2013;Berbers et al 1991;Adachi et al 1991).…”