|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 90 No. 8 (October 15), 1997:
pp. 2916-2920
RAPID COMMUNICATION
Amino Acids Responsible for Decreased 2, 3-Biphosphosphoglycerate Binding to Fetal Hemoglobin
By
Kazuhiko Adachi,
Patrick Konitzer,
Jian Pang,
Konda S. Reddy, and
Saul Surrey
From the The Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, PA; the Department of Biophysics, University of Pennsylvania, Philadelphia, PA; the Departments of Pediatrics and Research, the duPont Hospital for Children, Wilmington, DE; and the Department of Pediatrics, Jefferson Medical College, Philadelphia, PA.
 |
ABSTRACT |
To clarify the role of  N-terminal Gly, 5 Glu, and 143 Ser in 2, 3-biphosphosphoglycerate (BPG) binding to fetal hemoglobin (Hb F ), we engineered and produced normal human Hb F and two Hb F variants (Hb F G1V, S143H, and Hb F G1V, E5P, S143H) using a yeast expression system and then compared their oxygen-binding properties with those of native human Hb F and adult Hb (Hb A). Oxygen affinity of Hb F G1V, S143H in the absence of 2, 3-BPG was slightly higher than that of normal Hb F. The decrease in oxygen affinities for Hb F G1V, S143H with increasing 2, 3-BPG concentrations was larger than that of normal Hb F, but significantly less than that of Hb A. In contrast, oxygen affinities of Hb F G1V, E5P, S143H in the absence and presence of 2, 3-BPG were much lower than those of Hb F G1V, S143H and were similar to those of Hb A. These results indicate that differences between Pro and Glu at the A2 position in the A helix in Hb A and Hb F, respectively, are critical for reduced binding of 2, 3-BPG to Hb F, even though  5 Pro does not interact directly with 2, 3-BPG in Hb A. Hb F variants such as Hb F G1V, E5P, S143H, which exhibit reduced oxygen affinity, should facilitate design of efficient antisickling fetal Hb variants for potential use in gene therapy for sickle cell disease.
| |
INTRODUCTION |
OXYGEN AFFINITY of fetal blood is higher than that of maternal blood and may help to provide the fetus with an adequate oxygen supply during pregnancy. In mammals, 2, 3-biphosphosphoglycerate (BPG) mediates intracellular hemoglobin function by lowering oxygen affinity. Fetal red blood cells have higher affinity for oxygen because of a smaller effect of 2, 3-BPG on oxygen affinity of fetal hemoglobin (Hb F ) compared with adult Hb (Hb A).1-5 Hb A is much more sensitive to 2, 3-BPG, so that fetal red blood cells that contain Hb F and normal levels of 2, 3-BPG have significantly higher oxygen affinity than adult red blood cells containing Hb A. The higher affinity is in part due to decreased binding of 2, 3-BPG to Hb F, which results in higher oxygen affinity than Hb A in red blood cells, even though the affinity of purified Hb F for oxygen in the absence of 2, 3-BPG is slightly lower than that of Hb A.1,2,4,5 The phosphate groups of 2, 3-BPG form salt bonds with the N-terminus and the 2 and 143 His imidazoles in addition to binding of the carboxyl group of 2, 3-BPG to the  -amino group of 82 Lys in Hb A.6 Differences between the and chains at amino acids 1 and 143 include 1 Val versus 1 Gly and 143 His versus 143 Ser and are thought to contribute to the reduced binding of 2, 3-BPG.1,5 In addition, the stereo-chemical basis of this decreased interaction in Hb F has been explained by x-ray analysis and may be the result of the increased distance between the phosphate groups of 2, 3-BPG and 2 His in Hb F.6 X-ray diffraction studies also showed that structural differences between the and chains do not cause any gross functional perturbations7; however, differences in primary structure of the and chains might help explain the lower binding to 2, 3-BPG of Hb F. We can now assess reasons for differences between Hb A and Hb F by preparing Hb A and Hb F variants and studying their oxygen-binding properties using recombinant DNA technology. Because Hb F inhibits polymerization of Hb S, these basic studies should define the best antisickling Hb F variants with desired oxygen affinity, stability, and thermodynamic characteristics for potential use in gene therapy for sickle cell disease. In this report, we define amino acids responsible for the higher oxygen affinity of Hb F in the presence of 2, 3-BPG compared with Hb A.
| |
MATERIALS AND METHODS |
The G-globin cDNA (0.44 kb) was obtained from a cDNA library made from mRNA isolated from K562 cells. The shuttle vector, pGS 200-, containing full-length human G-globin cDNA, was created by overlap polymerase chain reaction (PCR) from pGS189, which contains human -globin cDNA, by exchanging the Xho I -globin cDNA fragment with the G-globin cDNA (1.4 kb) region.8 Plasmids containing Val-1, His-143 or Val-1, Pro-5, His-143 -globin cDNAs were constructed using PCR mutagenesis and subcloning, as described previously.8,9 The complete coding sequence of the wild-type and mutated G-globin cDNAs flanked by the GGAP promoter and MF 3'-UN regions was determined using site-specific primers and fluorescently tagged terminators in a cycle sequencing reaction in which extension products were analyzed on an automated laser-activated fluorescence-emission DNA sequencer.9 The entire -globin cDNA region was excised by Xho I digestion and substituted for the -globin cDNA region by subcloning into the Xho I site of the expression vector pGS389.9 The resultant vector contains full-length human cDNAs for and G-globin variants under transcriptional control of dual pGGAP promoters, as well as a partially functional yeast LEU2d gene and the URA3 gene for plasmid amplification and selection in yeast.8
Expression in yeast and isolation of the Hb F variants were as described previously.8,9 The purified Hb F variants were subjected to electrospray mass analysis (Fisons Instruments, VG Biotech, Altricham, UK) using the multiply charged ion peaks from the -globin chain (molecular weight = 15,126.4 Daltons) as an external reference for mass scale calibrations.10 Absorption spectra of the purified Hb variants were recorded using a Hitachi U-2000 spectrophotometer (Hitachi Instruments, Inc, Danbury, CT). Circular dichroism spectra of the variants were recorded using an Aviv-Model 62 DS instrument (Varian Analytical Instruments, San Fernando, CA) using a 0.1-cm light path cuvette at approximately 10 µmol/L Hb concentrations. Methods for determination of kinetics of polymerization of deoxyhemoglobins in 1.8 mol/L phosphate buffers using the temperature-jump method were as reported previously.11 Oxygen dissociation curves were determined in 50 mmol/L Bis-Tris containing 5 mmol/L EDTA, pH 7.2, at 20°C using a Hemox Analyzer (TCS Med Co, Huntington Valley, PA).12
| |
RESULTS AND DISCUSSION |
To clarify the role of N-terminal Gly, 5 Glu, and 143 Ser in 2, 3-BPG binding to Hb F, we engineered and produced normal human Hb F and two Hb F variants (Hb F G1V, S143H, and Hb F G1V, E5P, S143H) using a yeast expression system and then compared their oxygen-binding properties with those of native human Hb F and Hb A.8,9 Purified Hb F G1V, S143H showed similar migration on cellulose acetate electrophoresis to normal human Hb F made in yeast or isolated from human cord blood (Fig 1). In contrast, cellulose acetate electrophoresis showed, as expected, that surface charge of the triple variant Hb F G1V, E5P, S143H at pH 8.6 was more positive compared with Hb F and Hb F G1V, S143H (Fig 1). Small amounts of abnormal forms of recombinant hemoglobins are produced in yeast, including sulphaem-containing hemoglobin and/or misfolded hemoglobins.13,14 These will affect oxygen-binding properties of recombinant hemoglobins and, therefore, were removed by chromatographic purification. Lack of sulphaem-containing contaminants was confirmed by measuring absorption spectra (Fig 2A). Mass spectral analysis using electrospray ionization mass spectrometry (ES-MS) showed expected G-globin chain molecular masses of 15,993 Daltons for the G chain purified from yeast, 16,088 Daltons for the G1Val, 143His- and 16,056 Daltons for the G1Val, 5Pro, 143Hisglobin chains in addition to 15,126 Daltons for the -globin chain. Absorption spectra of the CO forms of the two Hb F variants were the same as those of native and recombinant Hb F (Fig 2A). In addition, circular dichroism spectra in the region from 190 to 290 nm for recombinant Hb F and the two Hb F variants were similar to that of native Hb F (Fig 2B), indicating that these substitutions do not significantly affect globin folding and/or the overall secondary structure of Hb tetramers.
View larger version (47K):
[in this window]
[in a new window]
| Fig 1.
Cellulose acetate electrophoresis of Hb F variants. Mobilities after cellulose acetate electrophoresis of Hb F purified from cord blood (lane 3) and yeast (lane 4) and the two Hb F variants (Hb F G1V, S143H [lane 5] Hb F G1V, E5P, S143H [lane 6]) were compared with native Hb A (lane 2) and Hb S (lane 1).
|
|
View larger version (22K):
[in this window]
[in a new window]
| Fig 2.
Absorption and circular dichroism spectra of Hb F and the two Hb F variants. Absorption (A) and CD (B) spectra were recorded for the CO forms of human and recombinant Hb F and the two Hb F variants in 0.1 mol/L phosphate buffer, pH 7.0, at 5°C. Results were corrected for small differences in protein concentration. a, b, c, and d represent Hb F purified from cord blood, yeast, and the two purified Hb F variants (Hb F G1V, S143H and Hb F G1V, E5P, S143H), respectively.
|
|
Oxygen affinities of Hb F and the Hb F variants were measured in the absence and presence of 2, 3-BPG in 50 mmol/L Bis-Tris buffer, pH 7.4, containing 0.1 mol/L NaCl at 20°C, and P50 values (the partial oxygen pressure required to give 50% saturation of hemoglobin) are shown in Fig 3. Oxygen affinities of Hb F made in yeast or purified from human cord blood were identical in the absence of 2, 3-BPG and were slightly lower than that of normal human Hb A, as indicated by their slightly higher P50 values (4.1 v 3.4 for Hb A). P50 values of Hb A and Hb F increased with increasing 2, 3-BPG; however, effects of 2, 3-BPG on Hb F were much less than that of Hb A. The P50 value of Hb F G1V, S143H in the absence of 2, 3-BPG was slightly lower than that of normal Hb F (3.0 v 4.0 for Hb F ). The increases in P50 for Hb F G1V, S143H with increasing 2, 3-BPG concentrations were larger at greater than 0.5 mmol/L 2, 3-BPG than that of normal Hb F (Fig 3). Effects of 2, 3-BPG on increases in P50 for this variant were still significantly less than that of Hb A. These results clearly indicate that amino acids other than the N-terminal and 143 positions in the - and -globin chains are responsible for differences in 2, 3-BPG effects on oxygen affinity of Hb A and Hb F. Therefore, we engineered an additional substitution of Pro for Glu at the 5 (A2) position and expressed Hb F G1V, E5P, S143H in yeast to examine the role of 5 Glu in decreased 2, 3-BPG binding to Hb F.
View larger version (14K):
[in this window]
[in a new window]
| Fig 3.
Effects of 2, 3-BPG on oxygen affinity of the Hb F variants. Oxygen affinities of recombinant Hb F ( ), Hb F G1V, S143H ( ), and Hb F G1V, E5P, S143H ( ) were measured in 50 mmol/L Bis-Tris, pH 7.2, containing 5 mmol/L EDTA and 0.1 mol/L NaCl at 20°C as a function of various 2, 3-BPG concentrations, and results were compared with those of Hb A ( ) and Hb F ( ) isolated from human adult and cord red blood cells, respectively.
|
|
P50 values of the triple variant Hb F G1V, E5P, S143H in the absence and presence of 2, 3-BPG were much higher than those of Hb F G1V, S143H and were similar to those of Hb A (Fig 3). These results clearly show that the difference between Pro and Glu at the A2 position in the A helix in Hb A and Hb F, respectively, is critical for reduced binding of 2, 3-BPG to Hb F, even though 5 Pro does not interact directly with 2, 3-BPG in Hb A. Negatively charged Glu (A2) at this position may affect the size, charge, and/or shape of the cavity as well as the position of 2 His, in comparison with Pro (A2) of Hb A. These differences might then result in decreased 2, 3-BPG binding to Hb F, which results in higher oxygen affinity for Hb F in the presence of 2, 3-BPG compared with Hb A.
In addition to 2, 3-BPG, inorganic ions, protons, CO2 , and organic phosphates have marked effects on oxygenation of hemoglobin.1,4,15,16 For example, oxygen affinity is lowered in the presence of increasing chloride ion concentrations (Fig 4).1,4 Binding sites for chloride in deoxyhemoglobin have been identified by x-ray analysis and include the N-terminal amino group of the chain as well as specific sites in the chain.17 Previous studies showed that chloride effects on oxygen affinities of purified Hb F and Hb A are slightly different (see Bunn and Forget1 and Bunn and Briel4 and see Fig 4). At low chloride ion concentrations, Hb F has a significantly lower oxygen affinity (higher log P50 ) than Hb A, as shown in Fig 4. It is likely that, at low chloride concentrations, the deoxy or T quaternary structure of Hb F is more stable than that of Hb A, because Hb F may have fewer repelling positive charges in its central cavity.18,19 In contrast, at near physiologic concentrations of chloride, Hb F and Hb A have similar oxygen affinities. Effects of chloride ion on oxygen affinity of Hb F were less than that of Hb A as previously reported (see Bunn and Forget1 and Bunn and Briel4 and see Fig 4). Chloride ion binding to Hb A occurs at the 2, 3-BPG binding site, and therefore may have less effect on oxygenation of Hb F than on that of Hb A.16,18 The alkaline Bohr effect of Hb F ( log P50 / pH between pH 6.5 and 8.5), like that of Hb A, is markedly dependent on chloride ion concentration. At low chloride concentrations (eg, 0.005 mol/L), both Hb A and Hb F have identical Bohr effects; whereas at physiologic chloride ion concentrations, the Bohr effect of Hb F is about 20% higher than that of Hb A.1,18 It is likely that the smaller Bohr effect of Hb A is due to the presence of 143 His instead of 143 Ser.
View larger version (15K):
[in this window]
[in a new window]
| Fig 4.
Effects of chloride ion on oxygen affinity of the Hb F variants. Oxygen affinities of recombinant Hb F (), Hb F G1V, S143H (), and Hb F G1V, E5P, S143H () were measured in 50 mmol/L Bis-Tris, pH 7.2, containing 5 mmol/L EDTA at 20°C as a function of different chloride ion concentrations and were compared with those of Hb A () and Hb F () isolated from human adult and cord red blood cells, respectively.
|
|
Oxygen affinities of Hb F G1V, S143H in the absence and presence of chloride ion were higher than those of Hb F (Fig 4). Effects of chloride on oxygen affinity of Hb F G1V, S143H were similar to those of Hb F. Substitution of Pro for Glu at the 5 (A2) position in Hb F G1V, S143H increased P50 values in the presence or absence of chloride, but P50 values were lower than those of normal Hb F. Effects of chloride on oxygen affinity of Hb F G1V, E5P, S143H were also similar to those of Hb F G1V, S143H and normal Hb F. These results indicate that the N-terminal Gly and/or 143 Ser instead of Val and His, respectively, in Hb F increases oxygen affinity, whereas the Glu to Pro change at the A2 position in Hb F decreases oxygen affinity and increases 2, 3-BPG binding. There may be no differences between the two Hb F variants when comparing effects of chloride ion on oxygen affinity because of the smaller size and, therefore, ease of entry of chloride into the cavity in contrast to the larger size of 2, 3-BPG.
Proline plays an important role in the native structure of proteins and usually does not participate in direct formation of helices. Introduction of proline in the middle of a helical segment usually leads to subunit destabilization, as evidenced by several naturally occurring Hb variants such as Hb Saki (14 Leu  Pro), Hb Crete (129 Ala Pro), Hb Bicetre (63 His Pro), and Hb Atlanta (75 Leu Pro).20-22 In contrast, x-ray analysis indicated that Pro at the second position of the A helix in the chain is not conserved, suggesting that Pro at this position is not critical for structure. In fact, Hb Warwickshire (5 Pro Arg) showed no difference in oxygen-binding properties in comparison with Hb A at physiologic chloride ion concentrations.20 In addition, this substitution caused mild instability and a slight decrease rather than increase in oxygen affinity in the presence of 2, 3-BPG in comparison with Hb A (11.0 v 9.96 mm Hg of P50 for Hb A). These results indicate that there is an increase in 2, 3-BPG binding to hemoglobin as a result of the 5 Pro Arg change.23 However, the recombinant Hb A variant (5 Pro Ala) showed increased oxygen affinity (0.68 for the P50 ratio of Hb A P5A/Hb A) without any affects on heat stability.24 Effects of 2, 3-BPG on oxygen affinity of this variant were less than those of Hb A in the presence of 0.1 mol/L NaCl. In addition, it is interesting to note that the decreased senstivity to 2, 3-BPG of horse Hb compared with human Hb A was suggested to be due to perturbations induced by amino acid differences in the A-helix at positions -A1 (4 Thr v Ser) and -A2 (5 Pro v Gly) in addition to the amino acid replacement at position -NA2 (2 His v Gln).25
X-ray diffraction analysis of crystals of Hb F showed that the only detectable difference in tertiary structure of the and chains in Hb F and Hb A, respectively, is in the N-terminal regions.6,7 Differences in primary structure of and chains near the N-terminal region in the A helix (eg, Gly [NA1], Phe [NA3] Glu [A2], Asp [A4] Ala [A6], and Ser [A10] in v Val, Leu, Pro, Glu, Ser, and Ala in ) may decrease interaction of Hb F with 2, 3-BPG. Our results also show that Glu instead of Pro at the A2 position in the chain slightly increased oxygen affinity. These findings suggest that amino acids at the A2 position may influence the first turn of the A helix, which may affect interactions between the A and EF helices in the absence of 2, 3-BPG. Differences in flexibility of the first turn of the A helix in Hb A versus Hb F may be affected by Pro in Hb A and the negative charge of Glu in Hb F at the A2 position, and these differences could also modify 2 His binding to 2, 3-BPG.
It is well known that Hb F and Hb A2 inhibit polymerization of Hb S in vitro and that high levels of Hb F in red blood cells of patients with sickle cell disease have been frequently associated with reduced disease severity.1,26 Therefore, there is considerable interest in finding ways to increase synthesis of Hb F in adults for the purpose of ameliorating sickle cell disease.26 The net effect of Hb F or Hb A2 on Hb S polymerization is to dilute Hb S by exclusion of FS or A2S hybrids (2s or 2s ) from deoxy Hb S polymerization that results in inhibition of polymerization.1,8,27,28 We compared polymerization of Hb S/Hb F G1V, E5P, S143H versus Hb S/Hb F and Hb S/Hb A2 mixtures to assess inhibitory effects of Hb F G1V, E5P, S143H on Hb S polymerization. Polymerization of 1:1 mixtures of Hb S/Hb F G1V, E5P, S143H was much delayed compared with Hb S alone, just like Hb S/Hb F and Hb S/Hb A2 mixtures, and also showed a delay time before polymerization. Polymerization rates for Hb S/Hb F G1V, E5P, S143H mixtures were similar to those of Hb S/Hb A2 mixtures and slightly faster than those for Hb S/Hb F mixtures at the same Hb concentrations.28 Logarithmic plots of delay time versus concentration for Hb S/Hb F G1V, E5P, S143H showed a straight line like Hb S/Hb A2 mixtures; this line was shifted right 0.44 U from the line for Hb S and slightly left from the line for Hb S/Hb F mixtures (Fig 5). In addition, the critical concentration required for polymerization of Hb S/Hb F G1V, E5P, S143H mixtures was the same as that of Hb S/Hb A2 mixtures. These results indicate that Hb F G1V, E5P, S143H inhibits Hb S polymerization like Hb A2 and that Hb F G1V, E5P, S143H/S hybrids are excluded like Hb A2/S hybrids from the initiation of polymerization with Hb S.28
View larger version (12K):
[in this window]
[in a new window]
| Fig 5.
Relationship between log of reciprocal delay time and Hb concentration for polymerization of Hb F G1V, E5P, S143H/Hb S mixtures. Kinetics of polymerization of the deoxy-forms of 1:1 mixtures of Hb F G1V, E5P, S143H/Hb S compared with Hb F/Hb S or Hb A2 /Hb S mixtures were performed in 1.8 mol/L phosphate buffer, pH 7.4, at 30°C using the temperature-jump method. , , and X represent Hb F G1V, E5P, S143H/S, F/S, and A2 /S mixtures, respectively, whereas represents HbS.
|
|
One approach to treatment of sickle cell disease is gene therapy that might involve viral transfer of the human -globin or variant gene into self-renewing hematopoietic stem cells in the marrow, hopefully resulting in long-term expression at high levels in adult blood cells after transplantation of the virally transduced bone mallow cells. Oxygen affinities of mixtures of Hb S and Hb F show higher oxygen affinity than that of Hb S or Hb A alone, which is caused by different affinities for 2, 3-BPG. This results in oxygen being first removed from Hb S in FS mixtures under low oxygen pressure. Therefore, reduction in oxygen affinity of Hb F in red blood cells containing Hb S and Hb F would be beneficial to patients by allowing more Hb S in these mixtures to be oxygenated under low oxygen tension and, therefore, significantly reduce sickling of red blood cells. The presence of Hb F variants with lower oxygen affinity than native Hb F in Hb S-containing red blood cells would then delay polymerization of Hb S. Therefore, studies aimed at production of more efficient Hb F variants for use as antisickling agents for gene therapy are critical. This is also important because there are limitations on expression levels of Hb F with viral vectors in cells; therefore, design of the most efficient Hb variant could be rate-limiting. Construction of Hb F variants like Hb F G1V, E5P, S143H with reduced oxygen affinity should facilitate optimal selection of Hbs for potential use in gene therapy for sickle cell disease and thalassemia in the future. Furthermore, the growing knowledge of hematopoetic stem cell biology will also help efforts to enhance gene transfer efficiency and expression of vectors containing Hb F variants.
| |
FOOTNOTES |
Submitted March 7, 1997;
accepted July 28, 1997.
Supported in part by grants from the National Institutes of Health (No. P60 HL38632 and DK 16691), the American Heart Association, and the March of Dimes Birth Defects Foundation and by the Nemours Foundation.
Address reprint requests to Kazuhiko Adachi, PhD, Division of Hematology, The Children's Hospital of Philadelphia, 34th St and Civic Center Blvd, Philadelphia, PA 19104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hearly marked
``advertisment'' in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Eric Rappaport and members of the Nucleic Acid/Protein Core at The Children's Hospital of Philadelphia for automated DNA sequence analysis. We are grateful to Dr H.E. Witkowska for mass spectral analysis of the -chain variants performed at the Children's Hospital Mass Spectrometry Facility (Oakland, CA; Dr C. Shackleton, Director).
| |
REFERENCES |
1. Bunn HF, Forget BG: Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia, PA, Saunders, 1986, p 37
2.
Allen DW,
Wyman J,
Smith CA:
The oxygen equilibrium of foetal and adult human hemoglobin.
J Biol Chem
203:81,
1953
3.
Bauer C,
Ludwig I,
Ludwig M:
Different effects of 2, 3 diphosphoglycerate and adenosine triphosphate on the oxygen affinity of adult and foetal human haemoglobin.
Life Sci
7:1336,
1968
4.
Bunn HF,
Briel R:
The interaction of 2, 3-diphosphoglycerate with various human hemoglobins.
J Clin Invest
49:1088,
1970
5.
Tomita SJ:
Modulation of the oxygen equilibria of human fetal and adult hemoglobins by 2, 3-diphosphoglycerate acid.
J Biol Chem
256:9495,
1981[Free Full Text]
6.
Arnone A:
X-ray diffraction study of binding of 2, 3-diphosphoglycerate to human deoxyhaemoglobin.
Nature
237:146,
1972[Medline]
[Order article via Infotrieve]
7.
Frier JA,
Perutz MFJ:
Structure of human foetal deoxyhemoglobin.
J Mol Biol
112:97,
1977[Medline]
[Order article via Infotrieve]
8.
Adachi K,
Pang J,
Konitzer P,
Surrey S:
Polymerization of recombinant Hb F E6V and Hb F E6V, Q87T alone, and in mixtures with Hb S.
Blood
87:1617,
1996[Abstract/Free Full Text]
9.
Adachi K,
Konitzer P,
Surrey S:
Role of 87 Gln in the inhibition of Hb S polymerization by Hb F.
J Biol Chem
269:9562,
1993[Abstract/Free Full Text]
10. Shackleton CH, Witkowska HE: Mass spectrometry in the characterization of variant hemoglobins, in Desiderio DM (ed): Mass Spectrometry: Clinical and Biomedical Applications. New York, NY, Plenum, 1994, p 135
11.
Adachi K,
Asakura T:
Nucleation-controlled aggregation of deoxyhemoglobin S: Possible difference in the size of nuclei in different phosphate concentrations.
J Biol Chem
254:7765,
1979[Free Full Text]
12.
Festa RS,
Asakura T:
The use of oxygen dissociation curve analyzer in transfusion therapy.
Crit Care Med
7:391,
1979[Medline]
[Order article via Infotrieve]
13.
Adachi K,
Konitzer P,
Lai CH,
Kim J,
Surrey S:
Oxygen-binding properties and mechanical stability of human hemoglobin made in yeast.
Protein Eng
5:807,
1992[Abstract/Free Full Text]
14.
Hofmann OM,
Mould RM,
Brittain T:
The incorporation of sulphaem into recombinant adult human haemoglobin produced in a yeast expression system.
Protein Eng
7:281,
1994
15.
Rossi-Fanelli A,
Antonini E,
Caputo A:
Studies on the relations between molecular and functional properties of hemoglobin. I. The effect of salts on the molecular weight of human hemoglobin.
J Biol Chem
236:391,
1961[Free Full Text]
16.
Rossi-Fanelli A,
Antonini E,
Caputo A:
Studies on the relations between molecular and functional properties of hemoglobin. II. The effect of salts on the oxygen equilibrium of human hemoglobin.
J Biol Chem
236:397,
1961[Free Full Text]
17.
O'Donnell S,
Mandaro R,
Shuster TM,
Arnone A:
X-ray diffraction and solution studies of specifically carbamylated human hemoglobin A.
J Biol Chem
254:12204,
1979[Free Full Text]
18.
Poyart C,
Bursaux E,
Guesnon P,
Teisseire B:
Chloride binding and the Bohr effect of human fetal erythrocytes and Hb FII solutions.
Pflugers Arch
376:169,
1978[Medline]
[Order article via Infotrieve]
19.
Bonaventura J,
Bonaventura C,
Sullivan B,
Ferruzzi G,
McCurdy R,
Fox J,
Moo-Penn WF:
Hemoglobin Providence: Functional consequences of two alterations of the 2, 3-diphosphoglycerate binding site at position beta 82.
J Biol Chem
251:7563,
1976[Abstract/Free Full Text]
20.
International Hemoglobin Center:
IHIC Variants list.
Hemoglobin
19:39,
1995
21.
Morimoto H,
Lehman H,
Perutz MF:
Molecular pathology of human haemoglobin: Stereochemical interpretation of abnormal oxygen affinities.
Nature
232:408,
1971[Medline]
[Order article via Infotrieve]
22.
Brennan SO,
Shaw JG,
George PM,
Huisman THJ:
Posttranslational modification of 141 Leu associated with the 75(E19) Leu Pro mutation in Hb Atlanta.
Hemoglobin
17:1,
1993[Medline]
[Order article via Infotrieve]
23.
Wilson CID,
Cave RJ,
Lehmann H,
Close M,
Imai K:
Hemoglobin Warwickshire [5(A2) Pro Arg)], a possible fine tuning of 2, 3-BPG affinity by 5 Pro.
FEBS Lett
176:331,
1984[Medline]
[Order article via Infotrieve]
24.
Baudin V,
Kister J,
Caron G,
Jonval V,
Poyart C,
Pagnier J:
The role of proline 5(A2) in the functional properties of human adult hemoglobin.
Hemoglobin
20:55,
1996[Medline]
[Order article via Infotrieve]
25.
Giardina B,
Brix O,
Clementi ME,
Scatena R,
Nicoletti B,
Ciccehtti R,
Argentin G,
Condo G:
Differences between horse and human haemoglobins in effects of organic and inorganic anions on oxygen binding.
Biochem J
266:897,
1990[Medline]
[Order article via Infotrieve]
26. Embury SH, Hebbel PR, Mohandas N, Steinberg MH: The Sickle Hemoglobins: Science and Medicine. New York, NY, Raven, 1994
27.
Nagel RL,
Bookchin RM,
Johnson J,
Labie D,
Wajcman H,
Isaac-Sodeye W,
Honig GR,
Schiliro G,
Crookston JH,
Matsutomor K:
Structural basis of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S.
Proc Natl Acad Sci USA
76:670,
1979[Abstract/Free Full Text]
28.
Adachi K,
Pang J,
Brandley L,
Reddy LR,
Trifillis P,
Schwartz E,
Surrey S:
Polymerization of three Hb A2 variants containing Val-6 and inhibition of Hb S polymerization by Hb A2 .
J Biol Chem
271:24557,
1996[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
K. Adachi, T. Yamaguchi, J. Pang, and S. Surrey
Effects of Increased Anionic Charge in the beta -Globin Chain on Assembly of Hemoglobin In Vitro
Blood,
February 15, 1998;
91(4):
1438 - 1445.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Adachi, Y. Zhao, T. Yamaguchi, and S. Surrey
Assembly of gamma - with alpha -Globin Chains to Form Human Fetal Hemoglobin in Vitro and in Vivo
J. Biol. Chem.,
April 21, 2000;
275(17):
12424 - 12429.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract