Protein–protein coupling and its application to functional red cell substitutes

Abstract

The need for an alternative to red cells for oxygen transport in transfusions has led to the creation of hemoglobin-based oxygen carriers, materials produced by chemical modification or genetic engineering of human or bovine hemoglobin. Modifications of the native proteins are necessitated by the spontaneous dissociation of the functional hemoglobin tetramers (alpha(2)beta(2)) into non-functional alphabeta dimers. Based on clinical observations of hypertension resulting from some of these materials, it was proposed that the stabilized tetramers are sufficiently small to extravasate through blood vessels and scavenge nitric oxide, depleting the endothelium of the signal for smooth muscle relaxation. In order to increase size and minimize extravasation while maintaining structure and function, methods for producing larger entities through protein-protein conjugation were developed. Approaches have included the use of nonspecific reagents that polymerize proteins (e.g., polyglutaraldehyde), conjugation to polyethylene glycol, expression of naturally occurring multimers and the use of selective reagents, which is the focus of this article.

Hemoglobin‐Based Nanoarchitectonic Assemblies as Oxygen Carriers

“…Acellular HBOCs refer to chemically modified Hb and can be separated into two generations. The first generation of HBOCs aims to prevent the dissociation of Hb tetramers into dimers, and mainly includes intramolecular crosslinked Hb, intermolecular crosslinked Hb, polymer‐conjugated Hb, and recombinant Hb . The second generation of HBOCs is on the basis of the first generation, that co‐assembly of Hb with antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) further stabilizes the assemblies and meanwhile avoids the formation of inactive methemoglobin (metHb) .…”

“…To date, some of the above‐mentioned Hb‐based assemblies have gone through different stages of clinical trials and a few have been approved for routine clinical uses in South Africa or for anemia treatment in canines. Readers can get more‐detailed information in previous reviews . Here, we mainly introduce some recent improvements on acellular HBOCs.…”

Subunit-directed click coupling via doubly cross-linked hemoglobin efficiently produces readily purified functional bis-tetrameric oxygen carriers

“…[23][24][25][26] Specific chemical modification of proteins can introduce bioorthogonal functional groups that lead to formation of derivatives with unique and predictable reactivity. 16,[27][28][29][30][31] Cu(I)catalyzed azide-alkyne cycloaddition (CuAAC) 32,33 has been employed for coupling proteins but the process has been surprisingly inefficient despite the excellent reputation of the process with smaller species. 28 In principle, introduction of an azide into a specific site in cross-linked Hb places it on a stabilized location that would allow a reaction to occur with the displayed functional group.…”

“…Since nitric oxide is the essential local signal for relaxation of blood vessels, its resulting reduced concentration leads to vasoconstriction that increases blood pressure. [14][15][16][17] In an important study, Vandegriff and coworkers 18 noted that increasing the size of the circulating Hb species by the addition of chains of polyethylene glycol (PEG) is sufficient to prevent vasoactivity in circulation. 19 Presumably, the increased size prevents these species from penetrating the endothelia.…”

Haemoglobin wrapped covalently by human serum albumin mutants containing Mn(iii) protoporphyrin IX: an O2complex stable in H2O2solution

“…Haemoglobin (Hb)-based O 2 carriers (HBOCs) of several types have been developed in the last few decades as red blood cell (RBC) substitutes and as O 2 -therapeutic reagents, [1][2][3][4] such as cross-linked Hb, 5,6 polymerized Hb 7,8 and (polyethylene glycol)-conjugated Hb. 9,10 Nevertheless, the HBOCs gradually lose their O 2 -transporting capability by autoxidation of Hb to the ferric haem form (metHb) with the release of a superoxide (O 2 À ) that is disproportionated to hydrogen peroxide (H 2 O 2 ).…”