Recombinant hemoglobin βG83C‐F41Y

Abstract

We have engineered a stable octameric hemoglobin (Hb) of molecular mass 129 kDa, a dimer of recombinant hemoglobin (rHb betaG83C-F41Y) tetramers joined by disulfide bonds at the beta83 position. One of the major problems with oxygen carriers based on acellular hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb betaG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb betaG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 M(-1) x h(-1) (observed time coefficient of 1 h at 1 mM glutathione) at 25 degrees C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 microM Hb and 25 degrees C. In addition, the octameric rHb betaG83C-F41Y was resistant to potential reducing agents present in fresh plasma.

Differential Thermal Stability and Oxidative Vulnerability of the Hemoglobin Variants, HbA2 and HbE

“…From uv-visible absorption, and synchronous fluorescence spectroscopic measurements, it was revealed that HbE starts disintegrating or melting at a lower temperature (~ 38°C) compared to that of HbA (~ 42°C) and HbA 2 (~ 45°C) at physiological pH range. These melting temperatures are comparable with the same at high alkaline pH, though a recent study indicated that the melting of HbA starts at much higher temperature (72°C) compared to the value reported here [ 40 ]. Though direct in vitro measurement of thermal stability of HbE was not performed before, earlier studies on other unstable Hb variants along with HbA and HbA 2 have shown that thermal stability of HbA 2 to be the highest among others studied [ 14 , 15 ].…”

Interaction of haptoglobin with hemoglobin octamers based on the mutation <i>α</i>Asn78Cys or <i>β</i>Gly83Cys

“…Thus, after a 15 min incubation of HbA with Hp at room temperature, the SEC profile shows the presence of a single peak (Ve = 9.85 ml) corresponding to the elution volume of the Hp( αβ ) 2 complex (results not shown) [17]. When the same experiment was performed with the β octamer, the SEC profile of this mixture shows the presence of two species corresponding to free Hp1-1(Ve = 10.3 ml) and free β octamer (Ve = 11.6 ml), indicating that the β octamers do not react with Hp after 15 min of incubation (Figure 3(a), red profile) [17]. For the α octamer (Figure 3(b), pink profile) there was about 6% of a Hp/ α octamer complex (Ve = 8.5 ml), in addition to the main species corresponding to free Hp (Ve = 10.3 ml) and free α octamer (Ve = 11.6 ml).…”

“…As previously described, the experiments were run at 29°C as a function of pH in 0.1 M sodium phosphate buffer and contained 0.1 mM Hb (on a heme basis) [13, 14,17]. The P 50 values (in mmHg) and the n 50 values are given with an accuracy of ± 10%.…”

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

“…This is reminiscent of the genetically engineered species consisting of two fused tetramers that is presented as a functional octamer. 73,75,76 Dendrimeric assemblies Reaction of hemoglobin with trimesoyl tris(3,5-dibromosalicylate) (TTDS, Fig. 9) forms a bb cross-linked protein with the residual ester available to function as an acylation site for conjugation reactions with nucleophiles.…”