In an effort to clarify the role of amino acid hydrophobicity at the beta 6 position in sickling we have made recombinant hemoglobin tetramers containing beta 6 Val (Hb S) and beta 6 Leu (Hb Leu). Recombinant Hb S and Hb Leu had the same electrophoretic mobility, chromatographic behavior, and absorption spectrum. The deoxy form of both tetramers polymerized in high phosphate buffer (1.8 M) and exhibited distinct delay times prior to polymerization. The kinetics of polymerization for recombinant and native Hb S were similar, while recombinant Hb Leu polymerized more readily. The solubility of deoxy Hb Leu was less than deoxy Hb S, indicating that rapid polymerization and decreased solubility of deoxyhemoglobin is accelerated with increasing hydrophobicity at the beta 6 position.

The relationship between different amino acids at the/l6 position of hemoglobin and tetramer stability was addressed by a site-directed mutagenesis approach. Precipitation rates during mechanical agitation of oxyhemoglobins with Gln, Ala, Val, Leu and Trp at the 86 position increased 2, 5, 13, 21 and 53 times, respectively, compared with that for Hb A. There was a linear relationship between the log of the precipitation rate constant and amino acid hydrophobicity at the 86 position, suggesting that enhanced precipitation of oxy Hb S during mechanical agitation results in part from increased hydrophobic&y of/?6 Val. Deoxyhemoglobin solubility increased in the order of/36 Be, Leu, Val, Trp, Gln, Ala and Glu suggesting that hydrophobic interactions between 86 Val and the acceptor site of another hemoglobin molecule during deoxy-Hb S polymerization not only depend on hydrophobicity but also on stereospecificity of the amino acid side chain at the 86 position. Furthermore, our results indicate that hydrophobic amino acids at the 86 position which promote tetramer instability in the oxy form do not necessarily promote polymerization in the deoxy form.

Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)