Recombinant hemoglobin betaG83C-F41Y.
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.
Vasseur-Godbillon
Corinne
C
INSERM Unité 473, Le Kremlin-Bicêtre, France.
Sahu
Sarata C
SC
Domingues
Elisa
E
Fablet
Christophe
C
Giovannelli
Janel L
JL
Tam
Tsuey Chyi
TC
Ho
Nancy T
NT
Ho
Chien
C
Marden
Michael C
MC
Baudin-Creuza
Véronique
V
D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006242:Haptoglobins / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006455:Hemoglobins, Abnormal / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D013997:Time Factors
D006242:Haptoglobins; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D008024:Ligands; D011994:Recombinant Proteins; C105876:hemoglobin rHb1.1; D010100:Oxygen