Humana Press Humana Press Hemoglobin Disorders Molecular Methods and Protocols Edited by Ronald L. Nagel, MD M E T H O D S I N M O L E C U L A R M E D I C I N E TM Hemoglobin Disorders Molecular Methods and Protocols Edited by Ronald L. Nagel, MD X-ray Crystallography of Hemoglobins 1 1 1 From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L. Nagel © Humana Press Inc., Totowa, NJ X-ray Crystallography of Hemoglobins Martin K. Safo and Donald J. Abraham 1. Introduction X-ray crystallography has played a key role in understanding the relation- ship between protein structure and physiological function. In particular, X-ray analysis of hemoglobin (Hb) crystals has been pivotal in the formulation of basic theories concerning the behavior of allosteric proteins. Methemoglobin (MetHb) from horse was the first three-dimensional (3D) structure of liganded Hb to be solved (1–4). It was followed by crystallographic determination of the unliganded (deoxygenated) form nearly a decade later (5). The X-ray analyses provided 3D atomic resolution structures and confirmed that Hb was tetrameric, containing two subunit types (α and β), and one oxygen-binding heme group per subunit. John Kendrew (myoglobin) and Max Perutz (Hb) received the Nobel Prize for their pioneering work, being the first to determine the 3D struc- tures of proteins, using X-ray crystallography. Since the crystallographic deter- mination of these structures, there has been an almost exponential increase in the use of X-ray crystallography to determine the 3D structures of proteins, i.e., as evidenced by the history of structures deposited in the protein data bank. Comparison of the quaternary structures of liganded and deoxygenated horse Hb clearly showed significantly different conformational states. The Hb X-ray structures were the first to confirm the two-state allosteric theory put forward by Monod et al. (6), which is referred to as the MWC model. The liganded Hb conformation conformed to the MWC relaxed (R) state, while unliganded Hb conformation conformed to the MWC tense (T) state. The source of the tension in the T state was attributed to crosslinking salt bridges and hydrogen bonds between the subunits. The relaxed (R) state has only a few intersubunit hydro- gen bonds and salt bridges. 2 Safo and Abraham Muirhead and Greer (7) published the first structure of human adult deoxy- genated hemoglobin (deoxyHbA). Several years later, Baldwin and Chothia (8,9) and Baldwin (9) published the structure of human adult carbonmonoxy- hemoglobin (HbCOA), and Shaanan (10) published the structure of human adult oxyhemoglobin (oxyHbA). Interestingly, the structure of oxyHbA was delayed because of complications resulting from heme iron autoxidation. Sub- sequently, a new quaternary ligand-bound Hb structure known as R2 (11) or Y (12,13) provided another relaxed structure. R2 was proposed to be a low-energy intermediate in the T-to-R allosteric transition. However, further analysis has revealed that R2 is not an intermediate but, rather, another relaxed end-state structure (14). Quite recently, our laboratory discovered two more novel HbCO A relaxed structures (R3 and RR2); RR2 has a structural conformation between that of R and R2 (unpublished results). The quaternary structural difference between T and R3 is as large as that of T and R2. However, R2 and R3 have very different conformations. The quaternary difference is determined by superimposing the α1β1 subunit interfaces and calculating the rotation angle between the nonsuperimposed α2β2 dimers (8,9). The first 3D structures of horse Hb were solved using isomorphous replace- ment techniques (1–3,5). A number of published Hb structures also crystallize isomorphously, thus making it possible to use phases from the known isomor- phous Hb structure for further structural analysis. The development of molecu- lar replacement methods (15,16) for the solution of protein structures enabled routine structure solutions for nonisomorphous Hb crystals. When the structure horse Hb was determined, no computer refinement pro- grams existed. Therefore, the atomic positions were refined visually against the electron density map. With isomorphous mutant crystals (17) or isomorphous crystals with bound allosteric effectors (18), simple electron density difference map calculations have been shown to be powerful tools in analyzing structural differences. Currently, all new protein structures are refined using modern, faster computing methods, such as CNS (19) and REFMAC (20). The crystal structures of more than 250 Hbs have been solved and published, including mutants and Hb cocrystalized with allosteric effector molecules. Selected examples of native and mutant Hbs including quaternary states, crys- tallization conditions, and unit cell descriptions are given in Tables 1–3. The structures of mutant Hbs provided the first concrete correlation between struc- tural changes and disease states, while Hb cocrystallized with small effector molecules has advanced our understanding of the fundamental atomic-level interactions that regulate allosteric function of an important protein. The general methodologies for isolating, purifying, crystallizing and crystal mounting for data collection follow. The X-ray structure solution of Hb and variants is routine and employs the techniques discussed above: isomorphous X-ray Crystallography of Hemoglobins 3 3 Table 1 Crystallization Conditions and Structural Properties of Selected Human Hbs Quater- Chemical Resolution Name nary state form Crystallization condition Unit cell characteristics a (Å) Reference DeoxyHbA T Normal 2.2–2.8 M NH 4 phosph/sulfate, a = 63.2, b = 83.5, c = 53.8 Å, 1.7 25 pH 6.5 β = 99.3°, SG = P2 1 , AU = 1 tetramer DeoxyHbA T Normal 10–10.5% PEG 6000, 100 mM a = 97.1, b = 99.3, c = 66.1 Å, 2.15 26 KCl, 10 mM K phosph, pH 7.0 SG = P2 1 2 1 2, AU = 1 tetramer RSR13-deoxy T Normal 2.5–2.9 M NH 4 phosph/sulfate, pH 6.5 a = 63.2, b = 83.6, c = 53.9 Å, 1.85 27 HbA complex b β = 99.2°, SG = P2 1 , AU = 1 tetramer DeoxyHbF c T Fetal 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 SG = P2 1 , AU = 1 tetramer 2.5 28 OxyHbA R Normal 2.25–2.75 M Na/K phosph, pH 6.7 a = 53.7, b = 53.7, c = 193.0 Å, 2.1 10 SG = P4 1 2 1 2, AU = 1 dimer HbCO A R Normal 2.25–2.75 M Na/K phosph, pH 6.7 a = 53.7, b = 53.7, c = 193.8 Å, 2.7 8 SG = P4 1 2 1 2, AU = 1 dimer HbCO A R2 Normal 16% PEG 6000, 100 mM,a = 97.5, b = 101.7, c = 61.1 Å, 1.7 11 Na cacodylate, pH 5.8 SG = P2 1 2 1 2 1 , AU = 1 tetramer CO Gower II R2 Embryonic 21% MME PEG 5000, a = 62.8, b = 62.8, c = 320.9 Å, 2.9 29 (α2ε2) d 0.2 M TAPS-KOH, pH 8.5 SG = P4 3 2 1 2, AU = 1 tetramer HbCO A e R3 Normal 2.34–2.66 M Na/K phosph, pH 6.4–6.7 a = 61.5, b = 61.5, c = 176.3 Å, 2.65 Unpublished SG = P4 1 22, AU = 1 dimer data HbCO A e RR2 Normal 2.34–2.66 M Na/K phosph, pH 6.4–6.7 a = 65.5, b = 154.6, c = 55.3 Å, 2.18 Unpublished SG = P2 1 2 1 2, AU = 1 tetramer data CNMetHbA f Y Normal 16–17% PEG 8000, 0.1 M Tris, a = 106.1, b = 86.2, c = 64.3 Å, 2.09 13 0.12% BOG SG = P2 1 2 1 2 1 , AU = 1 tetramer a SG , space group; AU, and asymmetric unit. b RSR13 is an allosteric effector. c The authors of deoxyHbF did not provide the cell constants, however, the crystal is isomorphous to the high-salt deoxyHbA cryst al (25). d The quaternary structure of carbonmonoxy embryonic Gower II Hb lies between that of R and R2 states, though closer to the R2 st ate. e Relaxed end-state structures (see text). f The quaternary structures of Y and R2 state Hbs are similar. 4 Safo and Abraham 4 Table 2 Crystallization Conditions and Structural Properties of Selected Natural Mutant Human Hbs Quater- Chemical Resolution Name nary state form Crystallization condition Unit cell characteristics a (Å) Reference DeoxyHbA Sickle cell T Glu6βVal 33% PEG 8000, 5.5 mM citrate, pH 4.0–5.0 a = 52.9, b = 185.7, c = 63.3 Å, 2.05 24 β = 92.6°, SG = P2 1 , AU = 2 tetramers Catonsville T Pro37α-Glu- 2.2–2.8 M NH 4 Phosph/sulfate pH 6.5 a = 63.2, b = 83.6, c = 53.8 Å, 1.7 30 Thr38αβ = 99.4°, SG = P2 1 , AU = 1 tetramer Rothschild T Trp37βArg 10–10.5% PEG 6000, 100 mM KCl, a = 97.1, b = 99.3, c = 66.1 Å 2.0 26 10 mM K phosph, pH 7.0 SG = P2 1 2 1 2, AU = 1 tetramer Thionville T Val1αGlu 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 63.2, b = 83.6, c = 53.8 Å, 1.5 31 AcetMet(-1)1αβ = 99.4°, SG = P2 1 , AU = 1 tetramer COYpsilanti Y Asp99βTyr 2.25–2.30 M Na/K phosph, pH 6.7 a = 93.1, b = 93.1, c = 144.6 Å 3.0 12 SG = P3 2 21, AU = 1 tetramer Cowtown R His146βLeu 2.25–2.75 M Na/K phosph, pH 6.7 a = 54.38, b = 54.38, c = 195.53 Å, 2.3 32 SG = P4 1 2 1 2, AU = 1 dimer Knossos b T Ala27βSer 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 SG = P2 1 , AU = 1 tetramer 2.5 33 Grange- T Ala27βVal 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 SG = P2 1 , AU = 1 tetramer 2.5 33 Blanche b Brockton b T Ala138βPro 2.2–2.8 M NH 4 phosph/sulfate, pH 6.8 SG = P2 1 , AU = 1 tetramer 3.0 34 Suresnes b T Arg141αHis 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 SG = P2 1 , AU = 1 tetramer 3.5 35 Kansas T Asn102βThr 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 63.4, b = 83.6, c = 53.9 Å, 3.4 36 β = 99.3 o , SG = P2 1 , AU = 1 tetramer a SG, space group and; AU, asymmetric unit. b The authors of Hb Knossos, Grange-Blanche, Brockton, and Suresnes did not provide the cell constants, however, the crystals are isomorphous to the high-salt deoxyHbA crystal (25). X-ray Crystallography of Hemoglobins 5 5 Table 3 Crystallization Conditions and Structural Properties of Selected Artificial Mutant Human Hbs Quater- Chemical Resolution Name nary state form Crystallization condition Unit cell characteristics a (Å) Reference Yα42H T Tyr42αHis 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 62.4, b = 81.2, c = 53.3 Å, 1.8 38 β = 99.65°, SG = P2 1 , AU = 1 tetramer rHb(α96Val→Trp) T Val96αTrp 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 63.3, b = 83.4, c = 53.8 Å, 1.9 39 β = 99.5°, SG = P2 1 , AU = 1 tetramer rHb(α96Val→Trp) R Val96αTrp 2.25–2.75 M Na/K phosph, pH 6.7 a = 54.3, b = 54.3, c = 194.1 Å 2.5 39 SG = P4 1 2 1 2, AU = 1 dimer Deoxy-Hbβ6W T Glu6βTrp 4–7 uL of 33 % PEG 8000, 5 uL a = 62.9, b = 81.3, c = 111.4 Å 2.0 40 of Na citrate, pH 4.8 SG = P2 1 2 1 2 1 , AU = 1 tetramer Deoxy-rHb1.1 T Asn108βLys 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 62.9, b = 82.0, c = 53.9 Å, 2.0 41 α1-Gly-α2 β = 99.0°, SG = P2 1 , AU = 1 tetramer CNmet-rHb1.1 B Asn108βLys 13 % PEG 3350, 10 mM KCN, a = 102.5, b = 115.2, c = 56.7 Å 2.6 41 α1-Gly-α2 150 mM NH 4 acetate, pH 5.0 SG = P2 1 2 1 2 1 , AU = 1 tetramer Deoxy-βV67T T Val67βThr 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 63.5, b = 83.2, c = 54.0 Å, 2.2 42 β = 99.15°, SG = P2 1 , AU = 1 tetramer Des-Arg141αHbA T des-Arg141α 10–10.5 % PEG 6000, 100 mM a = 96.7, b = 98.7, c = 66.0 Å, 2.1 43 KCl, 10 mM K phosph, pH 7.0 SG = P2 1 2 1 2, AU = 1 tetramer Bulltown T His146βGln 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 63.2, b = 83.4, c = 53.8 Å, 2.6 44 β = 99.4°, SG = P2 1 , AU = 1 tetramer Deoxy-βV1M T Val1βMet 2.2–2.8 M NH 4 phosph/sulfate, pH 6.5 a = 63.2, b = 83.7, c = 53.8 Å, 1.8 45 β = 99.4°, SG = P2 1 , AU = 1 tetramer a SG, space group; AU, asymmetric unit. 6 Safo and Abraham replacement, difference electron density calculations, molecular replacement, and structure refinement (for details, see references). 2. Materials 2.1. Purification of Human Hb for Crystallization 1. HbA is purified from outdated human red blood cells (RBCs) unsuitable for trans- fusion (~500 mL). Sickle cell Hb (HbS) is purified from sickle cell blood, nor- mally obtained from homozygote sickle cell patients who receive blood-exchange transfusions. To avoid clotting, blood samples are normally stored with about 1/10 vol of an anticoagulant agent, such as EDTA, heparin, or potassium citrate. 2. Buffer stock solution (5–10 L) containing 50 mM Tris buffer (pH 8.6) with EDTA: The solution is made by mixing 50 vol of 0.1 M Trizma base, 12.4 vol of 0.1 N Trizma hydrochloride, adjusting the volume to 100 mL with deionized water containing 4 g of EDTA (see Note 1). 3. Stock saline solutions (3 and 1 L) of 0.9% (9 g/L) and 1.0% NaCl (10 g/L), respectively. 4. DEAE sephacel and chromatography column equipment. 5. Cellulose dialysis tubes (Fisher Pittsburgh, PA). 6. Carbon monoxide gas cylinder (Matheson, Joliet, IL) (see Note 2). 7. NaCl, Na dithionite, and K 2 HPO 4 . 8. Three Erlenmeyers or side arm flasks (1 L). 2.2. Crystallization of Human Hb 1. Cyrstallization procedures will be described for deoxyHbA, deoxyHbS, and COHb A. These methods are also applicable to other HBs. HbA and HbS isolated and purified as described in Subheading 3.1.2. are used for all crystallization setups. 2.2.1. High-Salt Crystallization of T-State deoxyHbA 1. HbA solution (12 mL) (60 mg/mL or 6g%): Dilute the protein with deionized water if necessary to obtain the above concentration. 2. 3.6 M precipitant solution (50 mL) (pH 6.5): This is made by mixing 8 vol of 4 M (NH 4 ) 2 SO 4 , 1.5 vol of 2 M (NH 4 ) 2 HPO 4 , and 0.5 vol of 2 M (NH 4 )H 2 PO 4 . 3. Deionized water (100 mL). 4. Ten 8-mL sterile interior vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). 5. Stoppered glass jar (Aldrich, St. Louis, MO). 6. Parafilm. 7. Pipets and pipet tips (100 and 1000 mL). 8. Three 15- to 25-mL beakers or volumetric flasks. 9. Graduated cylinders (10- and 50-mL). 10. Mixture of FeSO 4 (2 g) and Na citrate (1.5 g). 11. A few grains of Na dithionite. 12. Test tube rack. X-ray Crystallography of Hemoglobins 7 2.2.2. High-Salt Crystallization of R-State HbCO A 1. HbA solution (12 mL) (40 mg/mL or 4g%) in a 50-mL round-bottomed flask equipped with a stir bar and a greased stopcock adapter. 2. 3.4 M precipitant solution (40 mL) (pH 6.4): This is made by mixing 7 vol of 3.4 M NaH 2 PO 4 and 5 vol of 3.4 M K 2 HPO 4 (see Note 3). 3. Deionized water (100 mL). 4. Toluene (50 µL). 5. Ten 8-mL sterile interior vacutainer tubes (Becton Dickinson). 6. Stoppered glass jar (Aldrich). 7. Pipets and pipet tips (100 and 1000 mL). 8. A few grains of Na dithionite. 9. Carbon monoxide gas cylinder (Matheson) (see Note 2) and nitrogen gas cylinder. 10. Test tube rack. 11. Vacuum pump and rubber tubing. 2.2.3. Low-Salt Crystallization of T-State deoxyHbS 1. HbS solution (1.2 mL) (120 mg/mL or 12g%). 2. 50% (w/v) polyethylene glycol (PEG) 6000 (12 mL). 3. 0.2 M citrate buffer (1 mL), pH 4.0–5.0 (Hampton Research, Laguna Hills, CA). 4. Deionized water (10 mL). 5. Ten 3-mL sterile interior vacutainer tubes (Becton Dickinson). 6. Parafilm. 7. Stoppered glass jar (Aldrich). 8. Pipets and pipet tips (100 and 1000 mL). 9. Two 15- to 25-mL beakers. 10. A few grains of Na dithionite. 2.3. Crystal Preparation and Mounting The methods described here are for deoxyHbA and COHb A, and are also applicable to other Hb cystals. 2.3.1. Room Temperature Data Collection 1. Vacutainer tube containing T- or R-state crystals. 2. Capillary sealant, such as epoxy or paraffin wax or any wax with a low melting point. 3. Disposable pipets and pipet rubber bulb. 4. Stainless steel blunt-end needles (Fisher). 5. Disposable syringes (3–5 mL) (Fisher Scientific). 6. Sterilized paper wicks (Hampton Research). 7. Thin-walled quartz or borosilicate capillaries (Charles Supper, Natick, MA), ranging in size from 0.1 to 1.2 mm. 8. Soldering iron. 9. Sharp tweezer. 8 Safo and Abraham 2.3.2. Cryogenic Temperature Data Collection 2.3.2.1. T-STATE DEOXYHBA CRYSTAL 1. Vacutainer tube containing T-state crystals. 2. Glycerol (100 µL). 3. Small Dewar flask with liquid nitrogen. 4. Thin fiber loop with diameter slightly larger than longest crystal dimension (Hampton Research). 5. Cryovial and cryovial tong (Hampton Research). 6. Disposable pipets and pipet rubber bulb. 7. Glass slides. 8. A few grains of Na Dithionite. 2.3.2.2. R-STATE COHB CRYSTAL 1. Vacutainer tube containing R-state crystals. 2. Cryoprotectant solution made by mixing 60 µL of mother liquor and 5–8 µL of glycerol. 3. Thin fiber loop with diameter slightly larger than longest crystal dimension. 4. Disposable pipets and pipet rubber bulb. 5. Glass slides. 3. Methods 3.1. Purification of Human Hb for Crystallization About 90% of RBC content is made up of Hb, and in healthy human adults, HbA accounts for more than 90% of the human Hb protein, while other minor components, such as fetal HbF (~1%) and hemoglobin HbA 2 (2 to 3%), make up the remainder. The method described here for isolating HbA and HbS from blood or RBCs, and further purification by ion-exchange chromatography, is a modified version of Perutz’s (21) protocol. This procedure, using appropriate buffer eluents, has also been used to separate other variant forms of human Hb and Hb from other species. 3.1.1. Purification of HbA 1. Place three Erlenmeyer or side-arm flasks in a walk-in refrigerator and chill to 4°C. 2. Centrifuge the RBCs at 600g for 20 min at 4°C. 3. Gently aspirate the supernatant solution (debris, plasma, and excess serum) from the centrifuge bottles and discard. 4. Wash the RBCs three times with an excess volume of 0.9% NaCl, and then once with 1.0% NaCl, each time centrifuging and discarding the supernatant solution. 5. Pool the RBCs into a chilled flask and lyse the cells by adding 1 to 2 vol of 50 mM Tris buffer, pH 8.6 (containing EDTA) (see Note 4). 6. Allow the mixture to stand on ice for 30 min with occasional gentle stirring. 7. Centrifuge the Hb solution at 10,000g for 2 h at 4°C. X-ray Crystallography of Hemoglobins 9 8. Pool the supernatant Hb solution, which is free of cell debris, into a chilled flask, and slowly add NaCl (40–60 mg/mL of Hb solution) while stirring the solution. 9. Centrifuge the Hb solution at 10,000g for 1 to 2 h at 4°C to remove any remain- ing cell stroma. 10. Pool the clear supernatant Hb solution into a chilled flask and discard the “syr- upy” pellet. 11. Dialyze the Hb solution against 50 mM Tris buffer, pH 8.6 (containing EDTA), at 4°C to remove NaCl or other low molecular weight impurities (see Note 5). 12. Further purify the dialyzed Hb by ion-exchange chromatography using DEAE sephacel to separate the HbA from other Hb components (see Note 6): a. Equilibrate the resin with 50 mM Tris buffer, pH 8.6. b. Run the Hb solution through the column with 50 mM Tris buffer, pH 8.6 (containing EDTA), to allow the various Hb bands to separate. HbA 2 (light band color) elutes first, followed by HbA (dark band color). The HbA frac- tions can be examined for purity by electrophoresis and only pure fractions (dark band) pooled together. 13. Concentrate the pooled fractions (40–100 mg/mL) with an Amicon stirred cell (Model 402) to a final HbA concentration of about 80–120 mg/mL (see Note 7). 14. Store the concentrated HbA, which is essentially the oxygenated form, at –80°C or freeze in liquid nitrogen. Hb stored at this temperature can remain suitable for crystal growth experiments for several years. 3.1.2. Purification of HbS HbS from homozygous sickle cell blood is isolated and dialyzed as described for HbA in Subheading 3.1.1. (steps 1–11). The HbS solution is further puri- fied on a DEAE sephacel ion-exchange column using a buffer gradient of 50 mM Tris buffer, pH 8.6 (containing EDTA), and 50 mM Tris buffer, pH 8.4 (con- taining EDTA) (see Note 1). 1. Elute first HbA 2 Tris buffer at pH 8.6, then HbS at pH 8.4. 2. Concentrate the pure HbS, identified by electrophoresis and store as indicated for HbA in Subheading 3.1.1. (steps 13 and 14). 3.2. Crystallization of Human Hb DeoxyHbA crystallizes from either high-salt or low-salt precipitants (7,21). The ligand-bound R-state Hbs, such as oxyHbA, HbCO A, and MetHbA; gen- erally crystallize under high-salt conditions (8–10,21), while the ligand-bound R2- or Y-state HbAs also crystallize mainly under low-salt conditions (11,13). The most common approach to crystallizing Hb is the Perutz’s (21) batch method. Alternatively, the vapor diffusion method of hanging or sitting drop (22) is used, especially when only a small amount of protein is available. Here, detailed crystallization is described for both T- and R-state human HbA and [...]... J., and Galactéros, F (1998) Determination of HbF levels: the routine methods Hemoglobin 22, 459–467 10 Huisman, T H J (1998) Separation of hemoglobins and hemoglobin chains by high performance liquid chromatography J Chromatogr 418, 277–304 11 Bisse, E and Wieland, H (1988) High-performance liquid chromatographic separation of human hemoglobins Simultaneous quantitation of fetal and glycated hemoglobins... 2.40 includes the high-salt crystallization of deoxyHbA and HbCO A and the lowsalt crystallization of deoxyHbS The crystallization methods described are modified batch methods by Perutz (21) and Wishner et al (23) and can also be applied to Hb mutants and Hb from other species See Notes 8 and 9 for important precautions regarding setting up T- and R-state crystals, respectively 3.2.1 High-Salt Crystallization... human hemoglobin chains J Chromatogr 321, 407–419 18 Wajcman, H., Riou, J., and Yapo, A P (2002) Globin Chains Analysis by RP-HPLC: recent developments Hemoglobin 26, 271–284 19 Clegg, J B., Naughton, M A., and Weatherall, D J (1966) Abnormal human hemoglobins: separation and characterization of the a and b chains by chromatography, and the detereminatioin of two new variants, Hb Chesapeake and Hb... chains by high pressure cation exchange chromatography Hemoglobin 9, 53–63 30 Wajcman Purification and Molecular Analysis of Hb by HPLC 31 3 Purification and Molecular Analysis of Hemoglobin by High-Performance Liquid Chromatography Belur N Manjula and Seetharama A Acharya 1 Introduction Hemoglobin (Hb) is a tetrameric protein (mol wt = 64,500) and is the major protein component of red blood cells... apparatuses have been developed for large series measurement I describe the Bio-Rad Variant Hemoglobin Testing System (Bio-Rad, Hercules, CA), using the β Thalassemia Short program as an example of this type of equipment From: Methods in Molecular Medicine, vol 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L Nagel © Humana Press Inc., Totowa, NJ 21 22 Wajcman 2.1.1 Bio-Rad... peptide mapping of globin chains is also carried out by RP-HPLC Preparative runs are generally carried out on an From: Methods in Molecular Medicine, vol 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L Nagel © Humana Press Inc., Totowa, NJ 31 32 Manjula and Acharya AKTA Protein Purification System (Amersham Pharmacia Biotech), which is also used for analytical runs Other... wavelength detection (415 and 690 nm) The β Thalassemia Short program is the most widely used system for HbA2 and HbF measurements, but other elution methods, including specific columns, buffers, and software, are available from the manufacturer according to the test to perform This program has been designed to separate and determine, in 5 to 6 min, area percentage for HbA2 and HbF and to provide qualitative... α-chains and two identical β-chains Besides HbA, human erythrocytes contain small amounts of other forms of Hb as fetal hemoglobin (HbF, α2γ2) and HbA2 (α2δ2), and products of posttranslational modifications as HbA1c HbS, the sickle cell Hb, is a genetic variant of HbA and is the most widely studied pathological form of Hb (1) Hb is a subject of active research not only for its molecular, genetic, and clinical... Headlee, M E., and Huisman, T H J (1984) Quantitation of hemoglobin components by high-performance cation-exchange liquid chromatography: its use in diagnosis and in the assessment of cellular distribution of hemoglobin variants Am J Hematol 17, 39–53 3 Rogers, B B., Wessels, R A., Ou, C N., and Buffone, G J (1985) High-performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias... structure and formation of the sickle cell fiber J Biol Chem 273, 32,690–32,696 40 Kroeger, K S and Kundrot, C E (1997) Structures of Hb-based blood substitute: Insights into the function of allosteric proteins Structure 5, 227–237 41 Pechik, I., Ji, C., Fidelis, K., Karavitis, M., Moult, J., Brinigar, W S., Fronticelli, C., and Gilliland, G L (1996) Crystallographic, molecular modeling, and biophysical . Press Hemoglobin Disorders Molecular Methods and Protocols Edited by Ronald L. Nagel, MD M E T H O D S I N M O L E C U L A R M E D I C I N E TM Hemoglobin Disorders Molecular Methods and Protocols Edited. by Ronald L. Nagel, MD X-ray Crystallography of Hemoglobins 1 1 1 From: Methods in Molecular Medicine, vol. 82: Hemoglobin Disorders: Molecular Methods and Protocols Edited by: Ronald L. Nagel © Humana. both T- and R-state human HbA and 10 Safo and Abraham includes the high-salt crystallization of deoxyHbA and HbCO A and the low- salt crystallization of deoxyHbS. The crystallization methods