Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the beta 6 position.
We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-->Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)). rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis. The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C. The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb. However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K. Crystallization of native Hb C and both rHbs was inhibited by Hb F. These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C.
Adachi
K
K
Division of Hematology, Children's Hospital of Philadelphia, PA 19104.
Lai
C H
CH
Konitzer
P
P
Donahee
M
M
Campbell
A
A
Surrey
S
S
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D003460:Crystallization; D004247:DNA / Q000737:chemistry; D004588:Electrophoresis, Cellulose Acetate; D006444:Hemoglobin C / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D006454:Hemoglobins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
D006454:Hemoglobins; D011994:Recombinant Proteins; D004247:DNA; D006444:Hemoglobin C; D001120:Arginine; D008239:Lysine