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Abstract
Previous studies suggested that gamma 87 Gln in hemoglobin (Hb) F is an important site for promoting inhibition of Hb S (alpha 2 beta 2(6 Glu-->Val) polymerization by Hb F. We engineered and isolated the double mutant (Hb alpha 2 beta 2(6 Glu-->Val,87 Thr-->Gln) using a yeast expression system and characterized polymerization properties of this modified tetramer in an effort to clarify the role of Gln at position 87 in inhibiting Hb S polymerization. Electrophoretic mobility and absorption spectra of this double mutant were the same as that of Hb S, while oxygen affinity was higher, and effects of organic phosphates on oxygen affinity were reduced. The deoxy form of the double mutant showed a characteristic delay time prior to polymerization in vitro. The critical concentration for polymerization of the double mutant was about 1.5 times higher than Hb S, and delay and polymerization times were much longer than Hb S at the same hemoglobin concentrations. The logarithmic plot of delay time versus hemoglobin concentration for the double mutant showed a straight line that was intermediate between lines for AS and FS mixtures. These results and those of kinetics of polymerization of Hb S/double mutant mixtures indicate that substitution of Gln for Thr at beta 87 in Hb S prolongs delay time and inhibits polymerization, although the double mutant forms polymers like Hb S.
Highlights
From the The Children’s Hospitaol f Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
We present studies usainrgecombinant those of kinetics of polymerization of H b Wdouble mu- DNA-engineered hemoglobin tetramer containing G+luVal at tant mixtures indicate that substituotifonGlnfor Thr at the p6 position as well as Thr + Gln at t h e p87 position
Double Mutant-The purified double mutant, a$; *' ""I, ('I" ( H b teristic delay time prior to polymer formation with the olfength pE6V, T87Q), migrated as a single band and had the same the delay time depending on hemoglobin concentration
Summary
PCR-based Mutagenesis-After generation of PCR products with overlapping homologous ends and transformation intoE. Hb tetramers containing the Sp6polymer formation is characterized by a delay time prior to and p87substitutionswerethenexpressedinyeast,and polymerization in vitro [19]. 6-globin chains from the double mutant were identical to that time than Hb S to complete polymerization. The line for the double mutant was intermediate between in p" corresponding toTpX-TpXI The ab- in p" inhibitsnucleiformationandpolymerization, sorption maximum and extinctioncoefficient of the double mu- the double mutant clearly forms polymers in vitro like deoxy tantinthe UV-visible range for the oxy, carbonmonoxy, and Hb S. deoxy forms were the same as the corresponding values for Kinetics ofPolymerization ofMixtures ofHSba n d the Double recombinant Hb S [14, 18].Oxygen binding properties of the Mutant-Polymerization of 1:l mixtures of Hb S and the double double mutant, differed from native and recombinant mutant was evaluated and compared with1:l Hb S/Hb F.
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