Introduction of a new regulatory mechanism into human hemoglobin.
Previous studies on bovine hemoglobin (HbBv) have suggested amino acid substitutions, which might introduce into human hemoglobin (HbA) functional characteristics of HbBv, namely a low intrinsic oxygen affinity regulated by Cl(-). Accordingly, we have constructed and characterized a multiple mutant, PB5, [beta(V1M + H2 Delta + T4I + P5A + A76K)] replacing four amino acid residues of HbA with those present at structurally analogous positions in HbBv, plus an additional substitution, beta T4I, which does not occur in either HbBv or HbA. This 'pseudobovine' hemoglobin has oxygen binding properties very similar to those of HbBv: the P(50) of HbA, PB5 and HbBv in the absence of Cl(-) are 1.6, 4.6 and 4.8 torr, respectively, and in 100 mM Cl(-) are 3.7, 10.5 and 12 torr, respectively. Moreover, PB5 has 3-fold slower autoxidation rate compared to HbA and HbBv. These are desirable characteristics for a human hemoglobin to be considered for use as a clinical artificial oxygen carrier. Although the functional properties of PB5 and HbBv are similar, van't Hoff plots indicate that the two hemoglobins interact differently with water, suggesting that factors regulating the R to T equilibrium are not the same in the two proteins. A further indication that PB5 is not a functional mimic of HbBv derives from PB5(control), a human hemoglobin with the same substitutions as PB5, except the beta T4I replacement. PB5(control) has a high oxygen affinity (P(50)=2.3 torr) in the absence of Cl(-), but retains the Cl(-) effect of PB5. The Cl(-) regulation of oxygen affinity in PB5 involves lysine residues at beta 8 and beta 76. PB4, which has the same substitutions as PB5 except beta A76K, and PB6, which has all the substitutions of PB5 plus beta K8Q, both have a low intrinsic oxygen affinity, like HbBv and PB5, but exhibit a decreased sensitivity to Cl(-). Since HbBv has lysine residues at both beta 8 and beta 76, these results imply that Cl(-) regulation in HbBv likewise involves these two residues. The mechanism responsible for the low intrinsic oxygen affinity of HbBv remains unclear. It is suggested that residues peculiar to HbBv at the alpha(1)beta(1) interface may play a role.
Department of Anesthesiology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. cfrontic@jhmi.edu
Clara
C
Fronticelli
Kevin M
KM
Bobofchak
Michael
M
Karavitis
Maria Teresa
MT
Sanna
William S
WS
Brinigar
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016296:Mutagenesis; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001059:Apoproteins; D002712:Chlorides; D006454:Hemoglobins; D009211:Myoglobin; D011994:Recombinant Proteins; C010857:apomyoglobin; D010100:Oxygen