Coexpression of human alpha- and circularly permuted beta-globins yields a hemoglobin with normal R state but modified T state properties

Abstract

For the first time, a circularly permuted human beta-globin (cpbeta) has been coexpressed with human alpha-globin in bacterial cells and shown to associate to form alpha-cpbeta hemoglobin in solution. Flash photolysis studies of alpha-cpbeta show markedly biphasic CO and O(2) kinetics with the amplitudes for the fast association phases being dominant due the presence of large amounts of high-affinity liganded hemoglobin dimers. Extensive dimerization of liganded but not deoxygenated alpha-cpbeta was observed by gel chromatography. The rate constants for O(2) and CO binding to the R state forms of alpha-cpbeta are almost identical to those of native HbA (k'(R(CO)) approximately 5.0 microM(-1) s(-1); k'(R(O(2))) approximately 50 microM(-1) s(-1)), and the rate of O(2) dissociation from fully oxygenated alpha-cpbeta is also very similar to that observed for HbA (k(R(O(2))) approximately 21-28 s(-1)). When the equilibrium deoxyHb form of alpha-cpbeta is reacted with CO in rapid mixing experiments, the observed time courses are monophasic and the observed bimolecular association rate constant is approximately 1.0 microM(-1) s(-1), which is intermediate between the R state rate measured in partial photolysis experiments (approximately 5 microM(-1) s(-1)) and that observed for T state deoxyHbA (k'(T(CO)) approximately 0.1 to 0.2 microM(-1) s(-1)). Thus the deoxygenated permutated beta subunits generate an intermediate, higher affinity, deoxyHb quaternary state. This conclusion is supported by equilibrium oxygen binding measurements in which alpha-cpbeta exhibits a P(50) of approximately 1.5 mmHg and a low n-value (approximately 1.3) at pH 7, 20 degrees C, compared to 8.5 mmHg and n approximately 2.8 for native HbA under identical, dilute conditions.

Figure 1

UV–visible spectra of HPSEC-purified α-cpβ (black line) and rHb0.0 (gray line) recorded in 10 mM sodium phosphate and 150 mM NaCl, pH 7.0.

Figure 2

HPSEC of oxy and deoxy α-cpβ. Samples of oxy α-cpβ (160 μM in heme)were subjected to analytical HPSEC using mobile phases that were either equilibrated with air (gray trace) or deoxygenated by sparging with N₂ and addition of 5 mM sodium dithionite (black trace). Inset: Apparent molecular masses (kDa) calculated from calibration standards.

Figure 3

ESIMS mass spectra of HPSEC-purified α-cpβ (7.5 nmol; top) and rHb0.0 (11.6 nmol; bottom).Inboth spectra the peaks for the β-globin are labeled with boxes. The spectra were taken in 50% (v/v) acetonitrile in water containing 0.1% (v/v) formic acid.

Figure 4

Time courses for full photolysis of CO from HbA and α-cpβ (panel A) and partial photolysis of CO (panel B) from HbA and α-cpβ (4% photolysis for HbA and 12% photolysis for α-cpβ). Experiments were conducted in CO-saturated 0.1 M potassium phosphate and 1 mM EDTA, pH 7.0 at 20 °C, in the presence of sodium dithionite. The concentrations of heme in the HbA and α-cpβ samples were 58 and 48 μM, respectively. Time courses for full photolysis (panel C) and partial photolysis (11% photolysis for both; panel D) of O₂ from HbA and α-cpβ. Experiments were conducted in air-equilibrated 0.1 M potassium phosphate and 1 mM EDTA, pH 7.0 at 20 °C. The concentrations of heme in the HbA and α-cpβ samples were 73 and 56 μM, respectively.

Figure 5

Normalized time courses for stopped-flow measurement of O₂ displacement by CO (panel A) and CO binding to deoxyHbA and deoxy α-cpβ (panel B). Samples were prepared in 0.1 M potassium phosphate buffer and 1 mM EDTA, pH 7.0, and data were collected at 20 °C. Air-equilibrated samples were rapidly mixed with buffer equilibrated with 100% CO and containing sodium dithionite to a final concentration of 464 μM CO, 131 μM O₂, and 3.5 μM heme for the HbA sample and 2.5 μM heme for the α-cpβ sample. Deoxy samples containing sodium dithionite were mixed with 5% (v/v) CO in N₂ to a final concentration of 23.2 μM CO and 3.5 μM heme for HbA and 2.5 μM heme for α-cpβ.

Figure 6

Fractional saturation with oxygen vs log of oxygen partial pressure (mmHg) for HbA (circles) and α-cpβ (triangles). Samples were prepared in 0.1 M potassium phosphate buffer and 1 mM EDTA, pH 7.0, and data were collected at 20 °C. Samples were either 10 μM (solid symbols) or 30 μM (open symbols) in heme.

Scheme 1

Proposed Construction of the Single-Chain Hemoglobin^{a a} (1) Generationofa circularly permuted β-globin with new terminiin the G-H loop, (2) insertion of cpβ-globininto the G-H loopof α-globin to create a fused α₁(cpβ)₁ dimer, and (3) fusion of two α₁(cpβ)₁ dimers by glycine insertion (thick line) to create a single-chain hemoglobin.