Mutational analysis of phenylalanine beta 85 in the valine beta 6 acceptor pocket during hemoglobin S polymerization.
Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at beta 85 were made and expressed in yeast in an effort to evaluate the role of Phe-beta 85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these beta 85 substitutions do not significantly affect heme-globin interactions and tetramer helix content. Hb S containing Trp-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-beta 85 had increased oxygen affinity. All four supersaturated beta 85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe-beta 88, Ala-beta 88, Glu-beta 88, or Glu-beta 85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477-17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563-31566). Leu substitution at beta 85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these beta 85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb SF veta 85L, Hb SF beta 85A, Hb SF beta 85T, and Hb SF beta 85W are 0.65-, 2.2-, 2.5- and 3-fold higher, respectively, than Hb S.(ABSTRACT TRUNCATED AT 250 WORDS)
Adachi
K
K
Division of Hematology, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine 19104, USA.
Reddy
L R
LR
Reddy
K S
KS
Surrey
S
S
D001665:Binding Sites; D004588:Electrophoresis, Cellulose Acetate; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014633:Valine
D006451:Hemoglobin, Sickle; D046911:Macromolecular Substances; D011108:Polymers; D010649:Phenylalanine; D014633:Valine