Coexpression of Human α- and Circularly Permuted β-Globins Yields a Hemoglobin with Normal R State but Modified T State Properties

Abstract

For the first time, a circularly permuted human β-globin (cpβ) has been coexpressed with human α-globin in bacterial cells and shown to associate to form α-cpβ hemoglobin in solution. Flash photolysis studies of α-cpβ 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 α-cpβ was observed by gel chromatography. The rate constants for O 2 and CO binding to the R state forms of α-cpβ are almost identical to those of native HbA (k′ R(CO) ≈ 5.0 μM −1 s −1 ; k′ R(O 2 ) ≈ 50 μM −1 s −1 ), and the rate of O 2 dissociation from fully oxygenated α-cpβ is also very similar to that observed for HbA (k R(O 2 ) ≈ 21-28 s −1 ). When the equilibrium deoxyHb form of α-cpβ is reacted with CO in rapid mixing experiments, the observed time courses are monophasic and the observed bimolecular association rate constant is ∼1.0 μM −1 s −1 , which is intermediate between the R state rate measured in partial photolysis experiments (∼5 μM −1 s −1 ) and that observed for T state deoxyHbA (k′T(CO) ≈ 0.1 to 0.2 μM −1 s −1 ). Thus the deoxygenated permutated β subunits generate an intermediate, higher affinity, deoxyHb quaternary state. This conclusion is supported by equilibrium oxygen binding measurements in which α-cpβ exhibits a P 50 of ∼1.5 mmHg and a low n-value (∼1.3) at pH 7, 20 °C , compared to 8.5 mmHg and n ≈ 2.8 for native HbA under identical, dilute conditions.In the 2005 Nationwide Blood Collection and Utilization Survey Report, the American Association of Blood Banks reported on the shrinking margin between the number of available units of blood approved for administration and the number of transfusions (1). Between 1989 and2004 this margin decreased by 53%, and the 2004 margin was the smallest ever reported by this survey (1). In response to this trend, there is increased interest in developing a safe and effective red blood cell (RBC) substitute. Given the unique properties of RBCs, these endeavors face many design challenges. RBCs contain a high concentration of hemoglobin (Hb) inside a protective membrane, which provides Hb a circulatory half-life of several months and protects tissues from oxidative damage (2). Additionally, RBCs contain methemoglobin reductase, † Supported by NIH Grants HL081068 (S.J.A.-C.) and GM035649 and HL047020 (J.S.O.), Grant C0612 from the Robert A. Welch Supporting Information Available: Experimental methods for cloning of the genes for tandem repeat of β-globin, cpβ-globin, and the coexpression vector pALA2, amino acid sequence of cpβ-globin, and SDS-PAGE of purified proteins. This material is available free of charge via the Internet at http://pubs.acs.org. Three main classes of RBC substitutes have been investigated in response to these design challenges (2,3): perfluorocarbon emulsions, liposome encapsulated hemoglobin solutions, and extracellular hemoglobin-based oxygen carriers (HBOCs). The ad...

Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers

“…Data were analyzed as described elsewhere [25] and the values of P 50 and n max reported are calculated from curve fitting. The stoichiometry of ligand binding was determined by titrating deoxy dihemoglobin with CO-saturated buffer at 20 °C.…”

“…The oxygen affinity and cooperativity of the purified protein were determined at 37 °C in 50 mM HEPES (pH 7.4) 100 mM NaCl using a Hemox analyzer (TCS Medical Products). Data were analyzed as described elsewhere [ 25 ] and the values of P 50 and n max reported are calculated from curve fitting. The stoichiometry of ligand binding was determined by titrating deoxy dihemoglobin with CO-saturated buffer at 20 °C.…”