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1.  Hb Baden: Structural and functional characterization 
American journal of hematology  2010;85(11):848-852.
Hb Baden (β18Val→Met) is a rare variant hemoglobin that has never been functionally or clinically characterized. We describe a Hb Baden heterozygote who exhibits normal growth and development, as well as age- and gender-appropriate hematological parameters. Surprisingly, in vitro analyses demonstrate that Hb Baden is relatively unstable and exhibits an abnormally high affinity for O2. These properties are likely to affect the physiologies of individuals who inherit the βBaden mutation in trans to a determinant for either a functionally relevant hemoglobin-opathy or a mild thalassemia. The data also provide insights into the function of the AB-segment/A-helix of the β-globin, supporting a structural model in which this poorly understood region serves as a scaffold that fixes the positions of other helices that directly impact β-globin function.
PMCID: PMC3618950  PMID: 20872549
2.  Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin. Corrigendum 
A correction to the paper by Abdulmalik et al. [(2011), Acta Cryst. D67, 920–928].
The affiliation of one of the authors of Abdulmalik et al. (2011) [Acta Cryst. D67, 920–928] is corrected.
PMCID: PMC3337008
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state; corrigendum
3.  Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin 
Pyridyl derivatives of vanillin increase the fraction of the more soluble oxygenated sickle hemoglobin and/or directly increase the solubility of deoxygenated sickle hemoglobin. Crystallographic analysis reveals the structural basis of the potent and dual antisickling activity of these derivatives.
Vanillin has previously been studied clinically as an antisickling agent to treat sickle-cell disease. In vitro investigations with pyridyl derivatives of vanillin, including INN-312 and INN-298, showed as much as a 90-fold increase in antisickling activity compared with vanillin. The compounds preferentially bind to and modify sickle hemoglobin (Hb S) to increase the affinity of Hb for oxygen. INN-312 also led to a considerable increase in the solubility of deoxygenated Hb S under completely deoxygenated conditions. Crystallographic studies of normal human Hb with INN-312 and INN-298 showed that the compounds form Schiff-base adducts with the N-terminus of the α-subunits to constrain the liganded (or relaxed-state) Hb conformation relative to the unliganded (or tense-state) Hb conformation. Interestingly, while INN-298 binds and directs its meta-positioned pyridine-methoxy moiety (relative to the aldehyde moiety) further down the central water cavity of the protein, that of INN-312, which is ortho to the aldehyde, extends towards the surface of the protein. These studies suggest that these compounds may act to prevent sickling of SS cells by increasing the fraction of the soluble high-affinity Hb S and/or by stereospecific inhibition of deoxygenated Hb S polymerization.
PMCID: PMC3211971  PMID: 22101818
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state
4.  Relationship between β4 Hydrogen Bond and β6 Hydrophobic Interactions during Aggregate, Fiber or Crystal Formation in Oversaturated Solutions of Hemoglobin A and S 
Oversaturated deoxy-α2β2T4V aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-α2β2E6V, D73H. Solubility of deoxy-α2β2T4V was ~10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-α2β2E6V,T4V. These results indicate that β4Val in HbA in the oxy and deoxy forms with or without β6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a β4/β73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(α2β2E6V,D73N), while α2β2T4V and α2β2D73H as well as HbS, α2β2E6V,D73H and α2β2E6V,T4V in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of β6 amino acid hydrophobicity that the synergism between the β4Thr hydrogen bond and β6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.
PMCID: PMC2636675  PMID: 19022217
Hemoglobin; Polymerization; Fiber formation; Crystallization; Hydrogen bond; Hydrophobic interaction; Sickle hemoglobin; Phase transition
5.  A Colorimetric Microplate Assay Method for High Throughput Analysis of Arginase Activity in Vitro 
Analytical biochemistry  2008;383(2):332-334.
Several analytical methods have been developed for the determination of arginase activity in physiological samples. These methods are limited by the considerable effort and time required to obtain reliable and reproducible measurements. Here we describe a simple high throughput colorimetric assay for the determination of arginase activity based on the ornithine-ninhydrin reaction. This method is an improvement over the original single cuvette assay developed by Chinard in that no boiling step is required. The turn-around time has been reduced, with improved precision and reproducibility. The method was extended to the determination of arginase activity in human leukemic (K562) cells and sickle erythrocytes. We believe that the method will find applications for routine analysis as well as for characterizing the action of novel and potent inhibitors on arginase activity.
PMCID: PMC2605851  PMID: 18789882

Results 1-5 (5)