Hemoglobin (Hb, Mw: 64 500) and albumin (Mw: 66 500) are major protein components in our circulatory system. On the basis of bioconjugate chemistry of these proteins, we have synthesized artificial O(2) carriers of two types, which will be useful as transfusion alternatives in clinical situations. Along with sufficient O(2) transporting capability, they show no pathogen, no blood type antigen, biocompatibility, stability, capability for long-term storage, and prompt degradation in vivo. Herein, we present the latest results from our research on these artificial O(2) carriers, Hb-vesicles (HbV) and albumin-hemes. (i) HbV is a cellular type Hb-based O(2) carrier. Phospholipid vesicles (liposomes, 250 nm diameter) encapsulate highly purified and concentrated human Hb (35 g/dL) to mimic the red blood cell (RBC) structure and eliminate side effects of molecular Hb such as vasoconstriction. The particle surface is modified with PEG-conjugated phospholipids, thereby improving blood compatibility and dispersion stability. Manipulation of physicochemical parameters of HbV, such as O(2) binding affinity and suspension rheology, supports the use of HbV for versatile medical applications. (ii) Human serum albumin (HSA) incorporates synthetic Fe(2+)porphyrin (FeP) to yield unique albumin-based O(2) carriers. Changing the chemical structure of incorporated FeP controls O(2) binding parameters. In fact, PEG-modified HSA-FeP showed good blood compatibility and O(2) transport in vivo. Furthermore, the genetically engineered heme pocket in HSA can confer O(2) binding ability to the incorporated natural Fe(2+)protoporphyrin IX (heme). The O(2) binding affinity of the recombinant HSA (rHSA)-heme is adjusted to a similar value to that of RBC through optimization of the amino acid residues around the coordinated O(2).

Small molecules that increase the oxygen affinity of human hemoglobin may reduce sickling of red blood cells in patients with sickle cell disease. We screened 38 700 compounds using small molecule microarrays and identified 427 molecules that bind to hemoglobin. We developed a high-throughput assay for evaluating the ability of the 427 small molecules to modulate the oxygen affinity of hemoglobin. We identified a novel allosteric effector of hemoglobin, di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide (TD-1). TD-1 induced a greater increase in oxygen affinity of human hemoglobin in solution and in red blood cells than did 5-hydroxymethyl-2-furfural (5-HMF), N-ethylmaleimide (NEM), or diformamidine disulfide. The three-dimensional structure of hemoglobin complexed with TD-1 revealed that monomeric units of TD-1 bound covalently to β-Cys93 and β-Cys112, as well as noncovalently to the central water cavity of the hemoglobin tetramer. The binding of TD-1 to hemoglobin stabilized the relaxed state (R3-state) of hemoglobin. TD-1 increased the oxygen affinity of sickle hemoglobin and inhibited in vitro hypoxia-induced sickling of red blood cells in patients with sickle cell disease without causing hemolysis. Our study indicates that TD-1 represents a novel lead molecule for the treatment of patients with sickle cell disease.