Polymerization of recombinant Hb S-Kempsey (deoxy-R state) and Hb S-Kansas (oxy-T state).
In order to investigate the role of the R (relaxed) to T (tense) structural transition in facilitating polymerization of deoxy-Hb S, we have engineered and expressed two Hb S variants which destabilize either T state (Hb S-Kempsey, alpha 2 beta 2 Val-6,Asn-99) or R state structures (Hb S-Kansas, alpha 2 beta 2 Val-6, Thr-102). Polymerization of deoxy-Hb S-Kempsey, which shows high oxygen affinity and increased dimer dissociation, required about 2- and 6-fold higher hemoglobin concentrations than deoxy-Hb S for polymerization in low and high phosphate concentrations, and its kinetic pattern of polymerization was biphasic. In contrast, oxy- or CO Hb S-Kansas, which shows low oxygen affinity and increased dimer dissociation, polymerized at a slightly higher critical concentration than that required for polymerization of deoxy-Hb S in both low and high phosphate buffers. Polymerization of oxy- and CO Hb S-Kansas was linear and showed no delay time, which is similar to oversaturated oxy- or CO Hb S. These results suggest that nuclei formation, which occurs during the delay time prior to deoxy-Hb S polymerization, does not occur in T state oxy-Hb S-Kansas, even though the critical concentration for polymerization of T state oxy-Hb S-Kansas is similar to that of T state deoxy-Hb S.
Adachi
K
K
Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Sabnekar
P
P
Adachi
M
M
Reddy
L R
LR
Pang
J
J
Reddy
K S
KS
Surrey
S
S
D002263:Carboxyhemoglobin / Q000737:chemistry / Q000235:genetics; D014644:Genetic Variation; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics; D006455:Hemoglobins, Abnormal / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010108:Oxyhemoglobins / Q000737:chemistry / Q000235:genetics; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D010108:Oxyhemoglobins; D011108:Polymers; D011994:Recombinant Proteins; C032577:hemoglobin Kempsey; C100003:deoxyhemoglobin; C017360:hemoglobin Kansas; D002263:Carboxyhemoglobin