hemoglobin disorders, molecular methods and protocols

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.. The HbS solution is further puri-fied on a DEAE sephac

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 ship between protein structure and physiological function In particular, X-rayanalysis of hemoglobin (Hb) crystals has been pivotal in the formulation ofbasic theories concerning the behavior of allosteric proteins Methemoglobin(MetHb) from horse was the first three-dimensional (3D) structure of liganded

relation-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 groupper subunit John Kendrew (myoglobin) and Max Perutz (Hb) received theNobel 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 inthe 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-raystructures 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 Hbconformation conformed to the MWC tense (T) state The source of the tension

in the T state was attributed to crosslinking salt bridges and hydrogen bondsbetween the subunits The relaxed (R) state has only a few intersubunit hydro-gen bonds and salt bridges

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 hemoglobin (HbCOA), and Shaanan (10) published the structure of human

carbonmonoxy-adult oxyhemoglobin (oxyHbA) Interestingly, the structure of oxyHbA wasdelayed 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 hasrevealed 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 betweenthat of R and R2 (unpublished results) The quaternary structural differencebetween T and R3 is as large as that of T and R2 However, R2 and R3 havevery different conformations The quaternary difference is determined bysuperimposing the α1β1 subunit interfaces and calculating the rotation anglebetween 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 phous Hb structure for further structural analysis The development of molecu-

isomor-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 grams existed Therefore, the atomic positions were refined visually against the

pro-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 structuraldifferences 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 tural changes and disease states, while Hb cocrystallized with small effectormolecules has advanced our understanding of the fundamental atomic-levelinteractions that regulate allosteric function of an important protein

struc-The general methodologies for isolating, purifying, crystallizing and crystalmounting for data collection follow The X-ray structure solution of Hb andvariants is routine and employs the techniques discussed above: isomorphous

Crystallization Conditions and Structural Properties of Selected Human Hbs

Name nary state form Crystallization condition Unit cell characteristicsa (Å) Reference DeoxyHbA T Normal 2.2–2.8 M NH4 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 Kphosph, pH 7.0 SG = P21212, AU = 1 tetramer RSR13-deoxy T Normal 2.5–2.9 M NH4 phosph/sulfate, pH 6.5 a = 63.2, b = 83.6, c = 53.9 Å, 1.85 27

DeoxyHbFc T Fetal 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 SG = P21, 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 = P41212, 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 = P41212, 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

0.12% BOG SG = P212121, 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 crystal (25).

d The quaternary structure of carbonmonoxy embryonic Gower II Hb lies between that of R and R2 states, though closer to the R2 state.

e Relaxed end-state structures (see text).

f The quaternary structures of Y and R2 state Hbs are similar.

Safo and Abraham

Table 2

Crystallization Conditions and Structural Properties of Selected Natural Mutant Human Hbs

Name nary state form Crystallization condition Unit cell characteristicsa (Å) Reference DeoxyHbA

Sickle cell T Glu6 βVal 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 Pro37 α-Glu- 2.2–2.8 M NH4 Phosph/sulfate pH 6.5 a = 63.2, b = 83.6, c = 53.8 Å, 1.7 30

Rothschild T Trp37 βArg 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 = P21212, AU = 1 tetramer Thionville T Val1 αGlu 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 a = 63.2, b = 83.6, c = 53.8 Å, 1.5 31

COYpsilanti Y Asp99 βTyr 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 = P3221, AU = 1 tetramer Cowtown R His146 βLeu 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 = P41212, AU = 1 dimer Knossosb T Ala27 βSer 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 SG = P21, AU = 1 tetramer 2.5 33

Grange- T Ala27 βVal 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 SG = P21, AU = 1 tetramer 2.5 33

Blancheb

Brocktonb T Ala138 βPro 2.2–2.8 M NH4 phosph/sulfate, pH 6.8 SG = P21, AU = 1 tetramer 3.0 34

Suresnesb T Arg141 αHis 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 SG = P21, AU = 1 tetramer 3.5 35

Kansas T Asn102 βThr 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 a = 63.4, b = 83.6, c = 53.9 Å, 3.4 36

β = 99.3 o , SG = P21, 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).

Table 3

Crystallization Conditions and Structural Properties of Selected Artificial Mutant Human Hbs

Name nary state form Crystallization condition Unit cell characteristicsa (Å) Reference

Y α42H T Tyr42 αHis 2.2–2.8 M NH4 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 Val96 αTrp 2.2–2.8 M NH4 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 Val96 αTrp 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 = P41212, AU = 1 dimer Deoxy-Hb β6W T Glu6 βTrp 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 = P212121, AU = 1 tetramer Deoxy-rHb1.1 T Asn108βLys 2.2–2.8 M NH4 phosph/sulfate, pH 6.5 a = 62.9, b = 82.0, c = 53.9 Å, 2.0 41

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 NH4 acetate, pH 5.0 SG = P212121, AU = 1 tetramer Deoxy- βV67T T Val67 βThr 2.2–2.8 M NH4 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-Arg141 αHbA 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 Kphosph, pH 7.0 SG = P21212, AU = 1 tetramer Bulltown T His146βGln 2.2–2.8 M NH4 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 Val1 βMet 2.2–2.8 M NH4 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

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 fusion (~500 mL) Sickle cell Hb (HbS) is purified from sickle cell blood, nor-mally obtained from homozygote sickle cell patients who receive blood-exchangetransfusions To avoid clotting, blood samples are normally stored with about1/10 vol of an anticoagulant agent, such as EDTA, heparin, or potassium citrate

trans-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 K2HPO4

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 deionizedwater 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

(NH4)2SO4, 1.5 vol of 2 M (NH4)2HPO4, and 0.5 vol of 2 M (NH4)H2PO4

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 FeSO4 (2 g) and Na citrate (1.5 g)

11 A few grains of Na dithionite

12 Test tube rack

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 flaskequipped 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

NaH2PO4 and 5 vol of 3.4 M K2HPO4 (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 alsoapplicable 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 lowmelting 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

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 ofglycerol

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 minorcomponents, such as fetal HbF (~1%) and hemoglobin HbA2 (2 to 3%), make

up the remainder The method described here for isolating HbA and HbS fromblood 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 Hband 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) fromthe centrifuge bottles and discard

4 Wash the RBCs three times with an excess volume of 0.9% NaCl, and then oncewith 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

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 ing cell stroma

remain-10 Pool the clear supernatant Hb solution into a chilled flask and discard the upy” pellet

“syr-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 HbA2 (lightband 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 forcrystal 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 HbA2 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

includes the high-salt crystallization of deoxyHbA and HbCO A and the salt crystallization of deoxyHbS The crystallization methods described are

low-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 impor-

tant precautions regarding setting up T- and R-state crystals, respectively.3.2.1 High-Salt Crystallization of T-State deoxyHbA

1 The materials in Subheading 2.2.1., with the exception of the stoppered glass jar

and the parafilm, are put in an antechamber of a glove box The vacutainer tubes

should be unstoppered, labeled as shown in Table 4, and arranged on a test tube

rack All containers, including those with solvents, should be left open

2 Alternately evacuate and fill the antechamber with nitrogen while stirring theHbA solution for 10–20 min to obtain completely deoxyHbA, water, and precipi-

tant solutions (see Note 10).

3 Purge the anaerobic chamber of the glove box with nitrogen to ensure a completeanaerobic condition

4 Transfer all materials from the antechamber to the anaerobic chamber

5 Add 25 mL of deionized water to the FeSO4 and Na citrate mixture and shake forabout 30 s

6 Allow the solution to settle and decant Use the supernatant (Fe citrate) solution

for all experiments (see Note 11).

7 Measure the volume of precipitant solution with a graduated cylinder, and add

water to restore to the original volume of 50 mL (3.6 M), if necessary.

8 Measure the volume of deoxyHbA solution with a graduated cylinder, and addwater to restore to the original volume of 12 mL (60 mg/mL), if necessary

9 Add a few grains of Na dithionite (or ~2 mM) to the deoxyHbA solution to reduce

any ferric heme that may be present

Table 4

High-Salt Crystallization of deoxyHbA

3.6 M NH4 phosph/ Deionized 0.5 M Fe 6g% deoxy- Final saltTube sulfate (mL) H2O (mL) citrate (mL) HbA (mL) conc (M)

10 Measure the precipitant solution and water and add to the vacutainer tubes as

indicated in Table 4.

11 Measure 1- and 0.1-mL aliquots of deoxyHbA and Fe citrate, respectively, andadd to each vacutainer tube

12 Stopper each vacutainer tube, and tilt at least twice to mix the solution

13 Remove all the materials from the glove box and wrap parafilm around the per of each vacutainer tube

stop-14 Store the sealed vacutainer tubes in greased, stoppered glass jars filled withnitrogen Crystals normally appear within 3–10 d and vary in size from mi-croscopic to as large as 8 mm in any direction The crystals belong to spacegroup P21 with approximate unit cell constants of a = 63 Å, b = 83 Å, c = 53 Å,and β = 99°

3.2.2 High-Salt Crystallization of R-State HbCO A

1 Add a few grains of Na dithionite to 12 mL of HbA (40 mg/mL) in a bottomed flask (three to five times the size of the volume of the HbA solution)fitted with a stopcock adapter and connected to both a vacuum pump and a nitro-gen gas source with rubber tubing

round-2 Alternately evacuate and flush with nitrogen for about 10 min

3 Connect a CO source to a disposable pipet with rubber tubing (see Note 2).

4 Open the flask containing the deoxyHbA solution, and quickly bubble COthrough the solution to make the HbCO A derivative

5 Reconstitute the volume to 12 mL (40 mg/mL) with CO-purged deionized water

6 Bubble CO through the precipitant solution

7 Measure the precipitant solution and add to the vacutainer tubes as indicated in

Table 5.

8 Measure 1-mL aliquots of HbCO A and add to each vacutainer tube

Table 5

High-Salt Crystallization of HbCO Aa

3.4 M Na/K 4g% HbCO A Final saltTube phosph (mL) (mL) conc (M)

9 Add a drop or two of toluene to each vacutainer tube (see Note 12).

10 Slowly bubble CO through each vacutainer tube, stopper, and tilt at least twice tomix the solution

11 Seal the vacutainer tubes with rubber stoppers and store in greased, stopperedglass jars filled with nitrogen to minimize formation of MetHbA Crystals nor-mally appear within 3–10 d The crystals are octahedral and belong to space groupP41212, with approximate unit cell constants of a = 53 Å, b = 53 Å, and c = 193 Å.The method described to crystallize HbCO A is applicable to both oxyHbA and

MetHbA (see Note 13).

3.2.3 Low-Salt Crystallization of T-State deoxyHbS

1 Place all materials (except stoppered glass jar and parafilm) in the antechamber

of the glove box The vacutainers should be unstoppered and labeled as shown in

Table 6 All containers, including those of solvents, should be left opened (see

4 Add deionized water to restore the volume of the HbS to 1.2 mL, if necessary

5 Add a few grains of Na dithionite (or ~2 mM) to the HbS solution.

6 Add deionized water to restore the volume of the precipitant solution to 12 mL, ifnecessary

7 Measure the precipitant solution and deionized water and add to the vacutainer

tubes as shown in Table 6.

8 Measure 0.1 mL-aliquots of deoxyHbS and add to each vacutainer tube

9 Stopper each vacutainer tube and tilt at least twice to mix the solution

Table 6

Low-Salt Crystallization of deoxyHbS

50% PEG Deionize 0.2 M Citrate 12g% deoxyHbSTube 6000 (mL) water (mL) (mL) (mL)

10 Store the vacutainer tubes and contents as described in Subheading 3.2.1 (steps

13 and 14) Crystals grown by this method are twinned (23,24) and must be

sepa-rated before X-ray data can be obtained Final crystals have the symmetry of themonoclinic space group P21, with approximate cell constants of a = 53 Å, b = 184

Å, c = 63 Å, and β = 93° (see Note 15).

3.3 Crystal Preparation and Mounting

Hb crystals, like most other protein crystals, are fragile because of their highsolvent content and should be handled with care For room temperature datacollection, Hb crystals are mounted and sealed in a thin-walled glass capillaryabout twice the size of the crystal For cryogenic data collection, crystals aremounted in a thin fiber loop with a layer of suitable cryoprotectant aroundthe crystal

3.3.1 Room Temperature Data Collection

T-state crystals are prepared and mounted in the glove box, while R-statecrystals are mounted outside the glove box However, to minimize autoxida-tion, mount R-state oxyHbA crystals as described for T-state crystals

1 Select at least two 8-cm-long capillaries, and, using a soldering iron, melt a ring

of wax close to the middle of the capillary

2 Use a sharp tweezer to cut the bottom part of the capillary, just below the ring ofwax The top part of the capillary with the wide mouth is retained Seal the cut

bottom (with the ring of wax) with melted wax or epoxy (see Note 16).

3 Using a microscope, select a few good crystals by marking outside the vacutainertube where those crystals are

4 For R-state crystals, proceed to step 9.

5 For T-state crystals, place the materials in Subheading 2.3.1., in addition to the

prepared capillaries, in the antechamber of the glove box

6 Alternately evacuate and fill the antechamber with nitrogen for 5–10 min

7 Transfer all the materials to a nitrogen-purged anaerobic chamber (see Note 17).

8 With a blunt-end needle, introduce a small amount of mother liquor from thevacutainer tube into the upper third of the capillary (all the way to the top)

9 Using a disposable pipet with a rubber bulb, suck a suitable marked crystal uponto the solution in the capillary Allow the crystal to flow down to the air space

If the crystal is less dense than the mother liquor, invert the capillary to allow thecrystal to flow to the air space

10 Carefully push the crystal with a thin fiber or the blunt end of the needle into theair space

11 Remove the solution from the capillary with a syringe and needle

12 Carefully dry excess liquid from the crystal with a filter paper strip, a smaller cutcapillary, or even the tip of the blunt-end needle Leave a thin film of mother

liquor between the crystal and the capillary wall (see Note 18).

13 Reintroduce a small amount of mother liquor into the capillary, about 5 mm from

the crystal (~5-mm-long liquid) Do not fill all the way to the top (see Note 19).

14 Close the capillary with melted wax or epoxy

3.3.2 Cryogenic Temperature Data Collection

T-state crystals are prepared and mounted in the glove box, and R-state tals are mounted outside the glove box Slightly different procedures are used,

crys-so a protocol for each is given next

3.3.2.1 T-STATE DEOXYHBA CRYSTAL

1 Place the materials in Subheading 2.3.2.1 in the anaerobic glove box as already described in Subheading 3.3.1 (steps 5–7).

2 Submerge the cryovial in the Dewar liquid nitrogen using the cryovial tong

3 Prepare cryoprotectant solution by mixing 50 µL of mother liquor, 10–16 µL of

glycerol, and a few grains of Na dithionite (see Note 20).

4 Pick up a crystal with the disposable pipet, and place it into 5 µL of cryoprotectantsolution on a glass slide for about 30 s

5 Transfer the crystal to another 5 µL of cryoprotectant solution for another 30 s

6 Use a fiber loop to scoop the crystal

7 Plunge the loop containing the crystal and the drop of cryoprotectant directly into thecryovial which is submerged in the liquid nitrogen

8 Take the closed cryovial out of the glove box and mount the crystal on the

goni-ometer head in the cold nitrogen gas stream (see Note 21).

3.3.2.2 R-STATE COHB A CRYSTAL

1 Pick up a crystal with a disposable pipet, and introduce it into 5 µL of protectant solution on a glass slide for about 30 s

cryo-2 Transfer the crystal to another 5 µL of cryoprotectant solution for another 30 s

3 Scoop up the crystal, which has a protective cover of cryoprotectant liquid, with

a fiber loop

4 Place the fiber loop on the goniometer head in the cold nitrogen gas stream

4 Notes

1 For HbS purification, prepare an additional 3–5 L of buffer stock solution

con-taining 50 mM Tris buffer (pH 8.4) with EDTA The solution is made by mixing

50 vol of 0.1 M Trizma base and 17.2 vol of 0.1 N Trizma hydrochloride, and

adjusting the volume to 100 mL with deionized water containing 4 g of EDTA

2 CO should be handled with great care; it is extremely toxic All experimentsinvolving CO should be done in a fume hood in a well-ventilated room

3 Alternatively, a precipitant solution consisting of equal volumes of 3.4 M

NaH2PO4 and 3.4 M K2HPO4 (pH 6.7) may be used and 0.2 mL of distilled wateradded to each tube

4 EDTA helps prevent oxidation of ferrous heme to ferric heme by chelating anyheavy metals that may act as catalysts for the autoxidation process The final

concentration of the purified HbA will depend on the amount of buffer added tolyse the cell.

5 Strips of standard cellulose dialysis tubing that have been washed three or fourtimes and boiled for 10 min in deionized water are used for the dialysis This isdone to remove traces of impure compounds that may contaminate the HbA Thedialyzing buffer should be 50- to 200-fold of the HbA volume and should becontinuously stirred overnight If possible, the buffer should be changed every 2

to 3 h

6 Alternatively, HbA is dialyzed with 10 mM phosphate buffer, pH 7.0 The same

type of buffer is then used to purify the HbA, as described in the text, using G25Sephadex (fine) column

7 Alternatively, HbA is concentrated by ultrafiltration through an Mr 10,000pellicon cassette The concentration of HbA can be determined using the Perutz

(21) procedure The concentration is measured by taking 1 mL of HbA solution

and diluting it with 19 mL of deionized water and 80 mL of 0.07 M K2HPO4 Nadithionite powder (0.2 g) is then added to the solution to generate the fullyreduced deoxyhemoglobin derivative CO is then bubbled through the solution toproduce the COHb A derivative The extinction coefficient is measured at 540 nm,and the concentration of HbA is calculated by dividing the optical density by 8.03

8 All crystallization steps for deoxyHbA are performed under rigorous anaerobicconditions in a nitrogen atmosphere glove box It is critical that all crystallizationsolvents be purged of oxygen and stored under nitrogen These precautions arenecessary to prevent formation of oxyHbA or MetHbA

9 Human R-state COHb A , oxyHbA, and MetHbA crystallize isomorphously, andthe corresponding structures are very similar OxyHbA is very susceptible toautoxidation, which leads to formation of MetHbA during crystallization and data

collection To slow autoxidation, EDTA (1 mM) is added to the precipitating

agents to chelate traces of heavy metals that catalyze the autoxidation process.Autoxidation of oxyHbA proceeds very rapidly when deoxyHbA is present in thesolution; therefore, oxygen should be bubbled through the HbA solution to com-pletely oxygenate all the HbA In addition, crystallization should be performed at

a low temperature, preferably 4°C, to slow down autoxidation Even thoughHbCO A is fairly stable for a long period, the presence of oxygen leads to gradualoxidation of the ferrous heme Therefore, crystallization of HbCO A should beunder a CO atmosphere to avoid possible oxidation of the heme All solutions forHbCO A crystallization should be purged with CO before use

10 A simple glove bag or Plexiglas box with gloves can be substituted for a moreexpensive glove box If a glove bag or Plexiglas box is used, the HbA solutionhas to be deoxygenated outside the glove box The HbA solution is put in a round-bottomed flask (three to five times the size of the volume of the HbA solution)and then connected by rubber tubing to both a vacuum pump and a nitrogen gassource with a glass stopcock adapter The HbA is alternately evacuated andflushed with nitrogen for 30–60 min to obtain a deoxyHbA solution (For smallervolume, the deoxygenation time is decreased.) A larger flask prevents boiling

HbA solution from getting into the vacuum line during the evacuation cycle.Additionally, to avoid undue boiling and splashing of the HbA, the flask contain-ing the HbA solution may be cooled briefly in an ice bath before evacuation.Next, all materials are put into the glove bag or Plexiglas box With the exception

of the deoxyHbA solution, all other solution-containing flasks (precipitant, water,and buffer) should be left open Once all the materials are put in the glove bag orPlexiglas box, it is then purged continuously with nitrogen for at least 40 minbefore the flask containing the HbA solution is opened If the chamber is notairtight, it should be purged continuously with nitrogen during the crystallization

experiments (Subheading 3.2.1., steps 5–14).

11 Fe Citrate solution is prepared in situ from FeSO4 and Na citrate in the glove boxand used fresh because the compound is unstable and easily oxidizes to ferriccitrate Fe citrate is a mild reducing agent and helps prevent oxidation of the iron;

it also acts as an antimicrobial agent to prevent growth of bacteria and fungi

12 Toluene, like similar organic solvents, reduces the effective electrostatic ing between the macromolecules by decreasing the electrostatic properties of theprecipitating solutions This facilitates increased contact between the macromol-ecules and serves to induce crystallization The presence of toluene is also effec-tive in preventing microbial growth

shield-13 Recently, we have discovered two new crystal forms of HbCO A (R3 and RR2;

see Table 1) that grows under the same crystallization conditions One crystal

form is rectangular and needle-like and belongs to the space group P4122 Theother crystal form, which is also needle-like, belongs to the space group P212121

14 Alternatively, the HbA is deoxygenated outside the glove box as indicated above.For a small quantity of solution, the deoxygenation time is reduced accordingly

(see Note 10, and continue from Subheading 3.2.3., steps 4–10).

15 Crystals must be transferred to a stabilizing solution made of glutaraldehyde,which strengthens the crystals before cutting Glutaraldehyde stabilizes the crys-tals by crosslinking the subunits Soak the crystals for 1 d in a mixture of 35%

(v/v) PEG stock solution, 20% (v/v) 0.2 M citrate buffer (pH 5.6), 45% (v/v) of 2% Drabkin’s buffer, and 10 mM of Na dithionite The temperature of the solu-

tion is subsequently lowered to 3°C, and glutaraldehyde solution (50% [w/v]) isthen added The mixture is allowed to stand overnight at 3°C

16 Without the wax, the capillary may shatter when cut

17 If a glove bag or Plexiglas box is used, make sure that all necessary materials areput in the chamber and then purged continuously with nitrogen for at least 40 minbefore the vacutainer tube containing the crystals is opened If the chamber is notairtight, it should be purged continuously with nitrogen during the experiments

18 A large amount of mother liquor around the crystal may decrease the resolutionand increase mosaicity and background noise The crystal can also move freely

or slip While making sure that as much liquid as possible is removed, do notcompletely dry the crystal Excess drying will dehydrate the crystal, which mayresult in cracking, increased mosaicity, poor diffraction, disorder, and a largereduction in cell volume

19 Mother liquor in the capillary ensures that the crystal is kept in the saturated vapor

of the mother liquor during room temperature data collection to prevent drying

20 Paraffin oil (Hampton Research) can also be used as a cryoprotectant After ting the crystal in the paraffin oil, make sure that all excess mother liquor in theparaffin oil drop is removed by passing the crystal back and forth in the paraffinoil The drop should form a perfectly clear glass under the cold stream Whitepatches may lead to reduction in resolution and increase mosaicity

put-21 Simple freezing of the crystal will result in the formation of ice in the interior ofthe crystal and will render it useless The cryoprotectant forms a noncrystallineglass, which protects the crystal from freeze shock

References

1 Perutz, M F., Rossmann, M G., Cullis, A F., Muirhead, H., Will, G., and North,

A C T (1960) Structure of haemoglobin A three-dimensional Fourier synthesis

at 5.5Å resolution obtained by x-ray analysis Nature 185, 416–422.

2 Perutz, M F., Muirhead, H., Cox, J M., Goaman, L C., Mathews, F S.,McGandy, E L., and Webb, L E (1968) Three-dimensional Fourier synthesis of

horse oxyhaemoglobin at 2.8 Å resolution: (1) x-ray analysis Nature 219, 29–32.

3 Perutz, M F., Muirhead, H., Cox, J M., and Goaman, L C (1968) sional Fourier synthesis of horse oxyhaemoglobin at 2.8 Å resolution: the atomic

Three-dimen-model Nature 219, 131–139.

4 Ladner, R C., Heidner, E J., and Perutz, M F (1977) The structure of horse

methaemoglobin at 2.0 Å resolution J Mol Biol 114, 385–414.

5 Bolton, W and Perutz, M F (1970) The three dimensional Fourier synthesis of

horse deoxyhaemoglobin at 2.8 Å resolution Nature 228, 551, 552.

6 Monod, J., Wyman J., and Changeux J.-P (1965) On the nature of allosteric

tran-sitions: a plausible model J Mol Biol 12, 88–118.

7 Muirhead, H and Greer, J (1970) Three-dimensional Fourier synthesis of human

deoxyhaemoglobin at 3.5 Angstrom units Nature 228, 516–519.

8 Baldwin, J and Chothia, C (1979) Haemoglobin: the structural changes related

to ligand binding and its allosteric mechanism J Mol Biol 129, 175–220.

9 Baldwin, J (1980) The structure of human carbonmonoxy haemoglobin at 2.7 Å

resolution J Mol Biol 136, 103–128.

10 Shaanan, B (1993) Structure of oxyhaemoglobin at 2.1 Å resolution J Mol Biol.

171, 31–59.

11 Silva, M M., Rogers, P H., and Arnone, A (1992) A third quaternary structure of

human Hb at 1.7 Å resolution J Biol Chem 267, 17248–17256.

12 Smith, F R., Lattman, E E., and Carter, C W Jr (1991) The mutation β99

Asp-Tyr stabilizes a new composite quaternary state of human Hb Proteins 10, 81–91.

13 Smith, F R and Simmons, K C (1994) Cyanomet human Hb crystallized under

physiological condition exhibits the Y quaternary structure Proteins 18, 295–300.

14 Janin, J.,and Wodak, S J (1993) The quaternary structure of carbonmonoxy Hb

Ypsilanti Proteins 15, 1–4.

15 Rossmann, M G and Hodgkin, D C (1972) in The Molecular Replacement

Method (Rossmann, M G., ed.), Gordon & Breach, New York, pp 36–38.

16 Navaza J (1994) AMoRe: an automated package for molecular replacement Acta

Crystallogr D50, 157–163.

17 Luisi, B F and Nagai, K (1986) Crystallographic analysis of mutant human

haemoglobins made in Escherichia coli Nature 320, 555, 556.

18 Wireko, F C., Kellogg, G E., and Abraham, D J (1992) Allosteric modifiers ofhemoglobin 2 Crystallographic determined binding sites and hydrophobic bind-

ing/interaction analysis of novel hemoglobin oxygen effectors J Med Chem 34,

758–767

19 Brunger, A T., Adams, P D., Clore, G M., et al (1998) Crystallography & NMR

system: a new software suite for macromolecular structure determination Acta

Crystallogr D54, 905–921.

20 Murshudov, G., Vagin, A., and Dodson, E (1997) Application of maximum

like-lihood methods for macromolecular refinement Acta Crystallogr D53, 240–255.

21 Perutz, M F (1968) Preparation of haemoglobin crystals J Crystal Growth 2,

54–56

22 McPherson, A (1982) Preparation and Analysis of Protein Crystals (McPherson,

A., ed.), John Wiley & Sons, New York

23 Wishner, B C., Ward, K B., Lattman, E E., and Love, W E (1975) Crystal

structure of sickle-cell deoxyHb at 5 Å resolution J Mol Biol 98, 179–194.

24 Harrington, D J., Adachi, K., and Royer, W E Jr (1997) The high resolution

crystal structure of DeoxyHb S J Mol Biol 272, 398–407.

25 Fermi, G., Perutz, M F., Shaanan, B., and Fourme, R (1984) The crystal

struc-ture of human deoxyHb at 1.7 Å resolution J Mol Biol 175, 159–174.

26 Kavanaugh, J S., Rogers, P H., Case, D A., and Arnone, A (1992) tion X-ray study of deoxyhemoglobin Rothschild 37β Trp ∏ Arg: a mutation that

High-resolu-creates an intersubunit chloride-binding site Biochemistry 31, 4111–4121.

27 Safo, M K., Moure, C M., Burnett, J., Joshi, G S., and Abraham, D J (2001)High resolution crystal structure of deoxy T-state hemoglobin complexed with a

potent allosteric effector Protein Science 10, 951–957.

28 Frier, J A., and Perutz, M F (1977) Structure of human foetal deoxyhaemoglobin

J Mol Biol 112, 97–112.

29 Sutherland-Smith, A J., Baker, H M., Hofmann, O M., Brittain, T., and Baker,

E D (1998) Crystal structure of a human embryonic haemoglobin: the monoxy form of Gower II (α2ε2) haemoglobin at 2.9 Å resolution J Mol Biol.

carbon-280, 475–484.

30 Kavanaugh, J S., Moo-Penn, W F., and Arnone, A (1993) Accommodation ofinsertions in helices: the mutation in hemoglobin Catonsville (Pro 37α-Glu-Thr

38α) generates a 3(10) → α bulge Biochemistry 32, 2509–2513.

31 Vasseur, C., Blouquit, Y., Kister, J., Prome, D., Kavanaugh, J S., Rogers, P H.,Guillemin, C., Arnone, A., Galacterose, F., Poyart, C., Rosa, J., and Wajcman, H.(1992) Hemoglobin Thionville: An alpha-chain variant with a substitution of aglutamate for valine at NA-1 and having an acetylated methionine NH2 terminus

J Biol Chem 267, 12,682–12,691.

32 Derewenda, Z., Dodson, G., Emsley, P., Harris, D., Nagai, K., Perutz, M., andReynaud, J.-P (1990) Stereochemistry of carbon monoxide binding to normal

and Cowtown haemoglobins J Mol Biol 211, 515–519.

33 Huang, Y., Pagnier, J., Magne, P., Bakloute, F., Kister, J., Delaunay, J., Poyart,C., Fermi, G., and Perutz, M F (1990) Structure and function of hemoglobinvariants at an internal hydrophobic site: consequences of mutation at the beta 27

(B9) position Biochemistry 29, 7020–7023.

34 Moo-Penn, W F., Jue, D L., Johnson, M H., Olsen, K W., Shih, D., Jones, R T.,Lux, S E., Rodgers, P., and Arnone, A (1988) Hemoglobin Brockton [β138(H16)Ala → Pro]: an unstable variant near the C-terminus of the b-subunits with normal

oxygen-binding properties Biochemistry 27, 7614–7619.

35 Poyart, C., Bursaux, E., Arnone, A., Bonaventura, J., and Bonaventura, C (1980)Structural and functional studies of hemoglobin Suresnes (Arg 141α2 → His α2):

consequences of disrupting an oxygen-linked anion-binding site J Biol Chem.

255, 9465–9473.

36 Anderson, N L (1975) Structures of deoxy and carbonmonoxy haemoglobin

Kansas in the deoxy quaternary conformation J Mol Biol 94, 33–49.

37 Tame, J R H and Vallone, B (2000) The structures of deoxy human

haemoglo-bin and the mutant Hb Tyra42His at 120 K Acta Crystallogr D56, 805–811.

38 Puius, Y A., Zou, M., Ho, N T., Ho, C., and Almo, S C (1998) Novel mediated hydrogen bonds as the structural basis for the low oxygen affinity of theblood substitute candidate rHb(α96Val → Trp) Biochemistry 37, 9258–9265.

water-39 Harrington, D J., Adachi, K., and Royer, W E Jr (1997) Crystal structure ofdeoxy-human hemoglobin Gluβ6 → Trp Implication for the structure and forma-

tion 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 bio-physical characterization of the Valineβ67 (E11) → Threonine variant of hemoglo-

21

From: Methods in Molecular Medicine, vol 82: Hemoglobin Disorders: Molecular Methods and Protocols

Edited by: Ronald L Nagel © Humana Press Inc., Totowa, NJ

Analysis of Hemoglobins and Globin Chains

by High-Performance Liquid Chromatography

Henri Wajcman

1 Introduction

In recent years, high-performance liquid chromatography (HPLC) hasbecome a reference method for the study of hemoglobin (Hb) abnormalities.This technique is used in two distinct approaches The first is quantitativeanalysis of the various Hb fractions by ion-exchange HPLC, which is nowdone in routine hospital laboratories mostly by using fully automated systems.The second is reverse-phase (RP)-HPLC, which is of interest for more special-

ized studies (see Note 1).

2 Materials and Methods

2.1 Ion-Exchange HPLC Separation of Hbs

Cation-exchange HPLC is the method of choice to quantify normal and

abnormal Hb fractions (1–4) This is the method of reference for measuring

glycated Hb for monitoring diabetes mellitus It is also generally used for suring of the levels of HbA2, HbF, and several abnormal Hbs

mea-According to some researchers, this method could even replace

electro-phoretic techniques for primary screening of Hbs of clinical significance (3,5–7)

or, at least, should be an additional tool for the identification of Hb variants

(8) Automated apparatuses have been developed for large series measurement.

I describe the Bio-Rad Variant Hemoglobin Testing System (Bio-Rad, cules, CA), using the β Thalassemia Short program as an example of this type

Her-of equipment

2.1.1 Bio-Rad Variant Hb Testing System

The Bio-Rad apparatus is a fully automated HPLC system, using double length detection (415 and 690 nm) The β Thalassemia Short program is the mostwidely used system for HbA2 and HbF measurements, but other elution meth-ods, including specific columns, buffers, and software, are available from themanufacturer according to the test to perform This program has been designed

wave-to separate and determine, in 5 wave-to 6 min, area percentage for HbA2 and HbFand to provide qualitative determinations of a few abnormal Hbs Windows ofretention time have been established for presumptive identification of the mostcommonly occurring Hb variants The β Thalassemia Short program uses a 3.0 ×0.46 cm nonporous cation-exchange column that is eluted at 32 ± 1°C, with aflow rate of 2 mL/min, by a gradient of pH and an ionic strength made of twophosphate buffers provided by the manufacturer This material and procedurehave been used worldwide in many laboratories over the last several years Sincerecommendations for experimental procedure are fully detailed by the manufac-turer, I describe only a few additional notes of practical import

1 Blood is collected on adenine citrate dextrose (ACD)

2 Samples for analysis (about 0.2% Hb) are obtained by hemolysis of 20 µL ofblood in 1 mL of a buffer containing 5 g/L of potassium hydrogenophthalate, 0.5 g/L

of potassium cyanide, 2 mL of a 1% solution of saponine, and distilled water.This procedure for sample preparation, which is currently used for HPLC deter-mination of HbA1c, avoids some of the Hb components present in low amounts

(about 1%) eluted together with HbF in the HbF retention time window (8).

3 Twenty microliters of hemolysate is applied onto the column for analysis

Under these experimental conditions an excellent agreement is found betweenchromatographic measurement of HbF, down to 0.2%, and resistance to alkali

denaturation, up to 15% (9) Presumptive identification of the most commonly

occurring variants (Hb S, HbC, HbE, and HbD Punjab) is made using the tion time windows named S-Window, D-Window, A2-Window, and C-Window,which have been specified by the manufacturer Aged Hb specimens displaysome degraded products that are eluted in the P2 and P3 windows (e.g., glu-

reten-tathione-Hb) (Table 1).

Slight differences in the elution time of the various Hb components areobserved from column to column and from one reagent batch to another, whichshould be taken into account by a program supplied by the manufacturer Theelution time of an Hb component varies also slightly according to its concen-tration in the sample For a given column, a more accurate calibration than thatproposed by the manufacturer could be obtained using HbA2 as reference Theconcentration of this Hb, which varies between narrow limits, prevents signifi-cant modification of its elution time

Two methods are available for comparing data when the elution time ofHbA2 differs between two runs done with a different column or reagent batch.The first consists of slightly modifying the experimental procedure (tempera-ture or pH) to reproduce exactly the elution times of the previous runs Thesecond method consists of establishing a normalized retention scale taking asreferences two Hbs eluted within a linear part of the gradient.

The elution patterns of more than 100 variants have been published, but, in

my opinion, these data should be used as a confirmatory test for

characteriza-tion of a variant after a careful multiparameter electrophoretic study (8) rather

than as a primary identification method

2.1.2 Alternative Methods

When a dedicated machine is not available for Hb analysis, or when the matographic separation is done for “preparative” purposes, alternative techniqueshave to be used These procedures are suitable for conventional HPLC equip-ment Several anion-exchange and cation-exchange HPLC columns may be usedfor Hb separation; some are silica based and others are synthetic polymers These

chro-methods have been well standardized for several years (10,11).

PolyCat A (Poly LC, Columbia, MD) is one of the more popular phases for

Hb separations (6) It consists of 5-µm porous (100-nm) spherical particles of

silica coated with polyaspartic acid For analytical purposes, a 5.0 × 0.40 cmcolumn is used; elution is obtained at 25°C with a flow rate of 1 mL/min, bydeveloping in 20 min at pH 6.58 a linear gradient of ionic strength from 0.03 to

0.06 M NaCl in a 50 mM Bis-Tris, 5 mM KCN buffer The presence of KCN is

necessary to convert methemoglobin into cyanmethemoglobin, which displaysion-exchange chromatographic properties similar to those of oxyhemoglobin

(see Note 2).

Table 1

Analyte Identification Window a

Analyte name Retention time (min) Band (min) Window (min)

F 1.15 0.15 1.00 -1.30P2 1.45 0.15 1.30-1.60P3 1.75 0.15 1.50-1.90A0 2.60 0.40 2.20-3.30A2 3.83 0.15 3.68-3.98D-window 4.05 0.07 3.98-4.12S-window 4.27 0.15 4.12-4.42C-window 5.03 0.15 4.88-5.18

a Example provided by manufacturer.

2.2 HPLC Analysis of Globin Chains

2.2.1 Analysis of HbF Composition (see Note 3)

The solvent system, acetonitrile–trifluororoacetic acid (TFA), which is usedfor RP-HPLC, dissociates the Hb molecule into its subunits and removes theheme group This method is therefore used to analyze or separate the globinchains This kind of study may be useful in the investigation of many human

Hb disorders For instance, the determination of HbF composition (Gγ:Aγ ratio)

is of interest in several genetic and acquired disorders

A good separation is obtained between the Gγ and AγI, with most of the RPcolumns by using a very flat acetonitrile gradient By contrast, it is often muchmore difficult to separate Gγ from AγT, a frequent allele of AγI Among theprocedures that have been successfully proposed for this analysis, one of the

most popular is the RP-HPLC method described by Shelton et al (12) They

used a Vydac C4 column (The Separation Group, Hesperia, CA) eluted at aflow rate of 1 mL/min by developing in 1 h a linear gradient from 38 to 42%acetonitrile in 0.1% TFA with detection at 214 nm Under these conditions, thechains were eluted in the following order: β, α, AγT, Gγ, and AγI In recent years,

a modification introduced in the manufacturing process of this type of column

(13) made necessary the use the higher acetonitrile concentrations to elute the

γ-chains Unfortunately, it also resulted in the low resolution of AγT

2.2.1.1 RP PERFUSION CHROMATOGRAPHY

Perfusion chromatography involves a high-velocity flow of the mobile phase

through a porous chromatographic particle (14–16) The Poros R1® media(Applied Biosystems, Foster City, CA) used in this technique consists of10-µm-diameter particles These particles are made by interadhering under afractal geometry poly(styrene-divinylbenzene) leading to throughpores of6000- to 8000-Å-diameter microspheres with short, diffusive 500- to 1000-Å-diameter pores connected to them As a result, relatively low pressures areobtained under high flow rates The Poros R1® beads may be considered afimbriated stationary phase having retention properties somewhat similar to

those of a classic C4 support (15) The column (10 × 0.46 cm) is packed on aconventional HPLC machine at a flow rate of 8 mL/min using the Poros self-pack technology® according to the manufacturer’s protocol More than a thou-sand runs may be performed without alteration of the resolution

2.2.1.1.1 Sample Preparation

1 Samples containing about 0.1 mg of Hb/mL are obtained by lysis, in 1 mL water

(or 5 mM KCN), of 2–5 µL of washed red blood cells (RBCs)

2 Membranes are removed by centrifuging at 6000g for 10 min.

3 According to the HbF level, 20–100 µL of these hemolysates are applied onto thecolumn To avoid additional chromatographic peaks owing to glutathioneadducts, 10 µL of a 50 mM solution of dithiothreitol in water is added per 100 µL

of sample An in-line stainless steel filter (0.5-µm porosity) needs to be used toprotect the column

2.2.1.1.2 Equipment Any conventional HPLC machine can be used In the

method described here, the analyses were performed on a Shimadzu LC-6HPLC machine equipped with an SCL-6B system controller, an SIL-6Bautoinjector, and a C-R5A integrator (Shimadzu, Kyoto, Japan) A flow rate of3.0–4.5 mL/min was convenient for synchronization of injection, integration,and column equilibration

2.2.1.1.3 Experimental Procedure (see Note 4) Using a flow rate of 3 mL/min,

the various γ-chains are isolated by developing in 9 min a linear gradient from

37 to 42% acetonitrile in a 0.1% solution of TFA in water In practice, this isdone by using two solvents (A: 35% acetonitrile, 0.1% TFA in water; B: 50%acetonitrile, 0.1% TFA in water) and a linear gradient from 15 to 45% B.Before injection, the column is equilibrated by a 10 column volume wash withthe starting solvent, thus allowing completion of a cycle of analysis every 14 min.Elution is followed at 214 nm (wavelength at which double bonds absorb), andthe recorder is set to 0.08 AUFS Higher flow rates may be used, but the slope

of the gradient will need to be increased in proportion Keeping the same initialand final acetonitrile concentrations as above, elution is achieved in 6 min at aflow rate of 4.5 mL/min and in 4 min at a flow rate of 6.0 mL/min

2.2.2 RP-HPLC Analysis of Globin Chains (see Note 5)

Globin chain analysis is also important as an additional test that allows crimination between Hb variants for the identification of structural abnormali-

dis-ties Several RP-HPLC procedures have been proposed (10,14,17,18).

On a conventional HPLC apparatus, a 20 × 0.46 cm column packed withLichrospher 100 RP8 (Merck, Darmstadt, Germany) is used Samples are pre-

pared as described in Subheading 2.2.1.1.1 Elution is obtained at 45°C with aflow rate of 0.7 mL/min using a 90-minute linear gradient of acetonitrile,

methanol, and NaCl made by a mixture of two solvents (18) Solvent A

con-tains acetonitrile, methanol, and 0.143 M NaCl, pH 2.7 (adjusted by a few drops of 1 N HCl), in the proportion of 24, 38, and 36 L/L, respectively Sol-

vent B is made from the same reagents but in the proportion of 55, 6, and 39 L/L,respectively The gradient starts with 10% B and ends with 70% B The design

of the gradient may be modified according to the machine, the geometry of thecolumn, and the separation to be achieved Elution can be followed at 214 or

280 nm Globin chains are eluted in the same order as on the Vydac C4 column

A kit for globin chain analysis with similar performance is also cially available from Bio-Rad (ref 270.0301).

commer-2.2.3 Scaled Up Methods for Chain Separation

For biosynthetic or structural studies, milligram amounts of globin chainsneed to be separated This can be achieved either by scaling up the RP-HPLCprocedure using semipreparative size columns or by cation-exchange -HPLCdone in the presence of dissociating concentrations of urea

2.2.3.1 SEMIPREPARATIVE SIZE RP-HPLC COLUMNS

2.2.3.1.1 Samples Globin solution rather than Hb solution is used Globin

is prepared from a 1% Hb solution obtained by hemolysing washed RBCs in

distilled water Stromas are removed by centrifuging at 6000g for 30 min, and

the globin is precipitated by the acid acetone method Usually, the sample ismade from 1 to 2 mg of globin dissolved in 250 µL of 0.1% TFA, whichrequires the use of a 500-µL injection loop

2.2.3.1.2 Chromotographic Procedure A 240 × 10 mm Vydac C4 column(ref 214TP510) is used Elution is obtained by a gradient of acetonitrile in0.1% TFA made by two solvents (solvent A contains 35% acetonitrile andsolvent B 45%) A typical elution program, using a flow rate of 1.2 mL/min,consists of a 10-min equilibration at 35% B, 70 min of a linear gradient from

35 to 55% B, 30 min of a linear gradient from 55 to 90% B, and 5 min of anisocratic step at 90% B for cleaning the column Elution of the column is fol-lowed at 280 nm with a full scale of 0.16 absorbance units (AU)

2.2.3.2 CATION-EXCHANGE HPLC IN PRESENCE OF 6 M UREA

USING A POLYCAT COLUMN

Procedures that are modified from the classic CM cellulose chromatography

described by Clegg et al (19) may be transposed to the HPLC technology (20).

The retention capacity of this type of column is higher than that of RP ports, allowing the handling of larger samples I describe here a method using

sup-a PolyCsup-at 300-Å, 10-µm particle column (150 × 4 mm)

2.2.3.2.1 Reagents and Buffers Two buffers are used Buffer A consists

of 6 M urea, 0.1 M sodium acetate, and 0.4% β-mercaptoethanol, with the

pH adjusted to 5.8 by acetic acid Buffer B consists of 6 M urea, 0.25 M sodium

acetate, and 0.35% β-mercaptoethanol, with the pH adjusted to 5.8 by aceticacid Both buffers need to be filtered through a membrane with 0.45-µmporosity before being used In addition, an in-line stainless steel filter (0.5-µmporosity) is needed to protect the column

2.2.3.2.2 Samples Up to 5–10 mg of globin, prepared by the acid acetone

method, is dissolved in 200–600 mL of buffer A

2.2.3.2.3 Chromatographic Procedure Elution is obtained by a gradient of

ionic strength developed with the two buffers A typical elution program, using

a flow rate of 1.0 mL/min, consists of a 10-min equilibration at 0% B, 5 min of

a linear gradient from 0 to 25% B, 50 min of a linear gradient from 25 to 100%

B, and 5 min of an isocratic step at 100% B for cleaning the column Elution ofthe column is followed at 280 nm with a full scale of 0.32 UA

3 Notes

1 Why should one method be preferred over another? The choice of a separationmethod between RP or ion-exchange chromatography depends on the purpose ofthe separation Ion-exchange is the only chromatographic method that allowspreparation of native Hb fractions The presence of cyanide ions in the buffers(or during sample preparation) will nevertheless hinder any further oxygen-binding study If the aim of the separation is to obtain Hbs suitable for functionalstudies, the technique will have to be modified accordingly by removing cyanidefrom all the steps It may be of interest in some cases to work with carbonmonoxy-hemoglobin, since Hb is very stable under this form and procedures are available

to return to the oxyform For several applications, salts in excess also need to beremoved RP separation methods always lead to denatured proteins that cannot

be used for functional studies Techniques involving an ionic strength gradientcan only be used for analytical purposes By contrast, using fully volatile buffers,such as the acetonitrile-TFA system, the isolated globin fractions can be vacuumdried and readily used for further structural studies such as mass spectrometrymeasurements

2 To isolate amounts of Hb in the milligram range, larger columns (15.0 × 0.46 cm)may be used According to the separation to be achieved, the dimensions of thecolumn, and the apparatus used, slightly different experimental conditions mayhave to be designed Elution is followed at 415 nm for analytical purposes or at

540 nm in preparative runs This buffer system is not suitable for ultraviolet (UV)detection The use of an in-line stainless steel filter (0.5-µm porosity) is recom-mended to increase the column life expectancy Reproducibility requires carefulpreparation of the buffers and temperature control Since in these chromato-graphic methods the elution is recorded at one of the wavelengths of absorption

of the heme, any factor modifying the absorption spectrum of the Hb moleculewill hinder accurate quantitative measurement For instance, unstable Hb vari-ants, which lose their heme groups or lead to hemichrome formation, will beunderestimated HbMs, which are hardly converted into cyanmethemoglobin,display a much higher extinction coefficient than oxyhemoglobin at 415 nm and

a lower one at 540 nm As a consequence, HbMs will be overestimated whenmeasured at the first wavelength, and underestimated at the second one A modi-fied experimental procedure allowing for a simultaneous measurement of HbF,glycated Hb, and several other Hb adducts has been proposed by using a combi-

nation of pH and ionic gradients (11).

3 In my laboratory, for routine determination of the γ-chain composition, wereplaced this procedure with an RP perfusion chromatography using a Poros R1®

column (Applied Biosystems) (14).

4 To obtain good reproducibility, we recommend using the same glassware forpreparing the solvents Solvents may be kept refrigerated at 4°C for a few days.Accurate balance of the TFA between both solvents is important to avoid baselinedrift Acetonitrile must be of HPLC grade with low UV absorbancy in the 210-nmregion With this Poros R1 column, the α-chain is eluted before the β-chain.Resolution may be improved by modifying the geometry of the column or thedesign of the gradient A 10 × 0.2 cm column may be used to improve separationbetween the various γ- or adult chains In this case, with a flow rate of 1 mL/min,after 5 min of equilibration at 5% B, the column is eluted using a 15-min lineargradient between 5 and 25% B of the described solvents This is followed by a 2-minisocratic elution at 25% B

5 Several columns may be used, but I have found that a method adapted from that

described in ref 17 leads to a good resolution Other columns or techniques may

nevertheless be more appropriate for some specific separations When graphic methods are used for globin chain quantification, it is important to con-sider the absorption coefficient of the various chains at the wavelength ofdetection In some cases, it may be identical, such as when comparing the variousγ-chains In other cases, the absorption may differ considerably; for example, at

chromato-280 nm, γ-chains, because of their 3 Trp residues, have a higher ε coefficient thanβ-chains (2Trp) and α-chains (1 Trp) Abnormal Hbs containing a number ofaromatic residues different from the normal may also display modified absorp-tion coefficient

tribution 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-performanceliquid chromatography in the diagnosis of hemoglobinopathies and thalassemias

Am J Clin Pathol 84, 671–674.

4 Samperi, P., Mancuso, G R., Dibenedetto, S P., Di Cataldo, A., Ragusa, R.,and Schiliro, G (1990) High performance liquid chromatography (HPLC): a

simple method to quantify HbC, O-Arab, Agenogi and F Clin Lab Haematol.

13, 169–175.

5 Shapira, E., Miller, V L., Miller, J B., and Qu, Y (1989) Sickle cell screening

using a rapid automated HPLC system Clin Chim Acta 182, 301–308.

6 Ou, C N and Rognerud, C L (1993) Rapid analysis of hemoglobin variants by

cation-exchange HPLC Clin Chem 39, 820–824.

7 Papadea, C and Cate, J C (1996) Identification and quantification of

hemoglo-bins A, F, S, and C by automated chromatography Clin Chem 42, 57–63.

8 Riou, J., Godart, C., Hurtrel, D., Mathis, M., Bimet, C., Bardakdjian-Michau, J.,Préhu, C., Wajcman, H., and Galactéros, F (1997) Evaluation of cation-exchangehigh-performance liquid-chromatography for presumptive identification of hemo-

globin variants J Clin Chem 43, 34–39.

9 Préhu,C., Ducrocq, R., Godart, C., Riou, J., and Galactéros, F (1998)

Determina-tion 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 ration of human hemoglobins Simultaneous quantitation of fetal and glycated

sepa-hemoglobins J Chromatogr 434, 95–110.

12 Shelton, J B., Shelton, J R., and Schroeder, W A (1984) High-performanceliquid-chromatographic separation of globin chains on a large-pore C4 column

J Liq Chromatogr 7, 1969–1977.

13 Vydac (1994–1995) HPLC columns and separation materials, Technical Bulletin

14 Wajcman, H., Ducrocq, R., Riou, J., Mathis, M., Godart, C., Préhu, C., andGalacteros, F (1996) Perfusion chromatography on reversed-phase column

allows fast analysis of human globin chains Anal Biochem 237, 80–87.

15 Afeyan, N B., Gordon, N F., Mazsaroff, I., Varady, L., Fulton, S P., Yang, Y.B., and Regnier, F E (1990) Flow-through particles for the high-performanceliquid chromatographic separation of biomolecules: perfusion chromatography

J Chromatogr 519, 1–29.

16 Afeyan, N B., Fulton, S P., and Regnier, F E (1991) Perfusion chromatography

material for proteins and peptides J Chromatogr 544, 267–279.

17 Leone, L., Monteleone, M., Gabutti, V., and Amione, C (1985) Reversed-phasehigh performance liquid chromatography of 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 humanhemoglobins: separation and characterization of the a and b chains by chromatog-raphy, and the detereminatioin of two new variants, Hb Chesapeake and Hb J

(Bangkok) J Mol Biol 19, 91–108.

20 Brennan, S O (1985) The separation of globin chains by high pressure cation

exchange chromatography Hemoglobin 9, 53–63.

31

From: Methods in Molecular Medicine, vol 82: Hemoglobin Disorders: Molecular Methods and Protocols

Edited by: Ronald L Nagel © Humana Press Inc., Totowa, NJ

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 majorprotein component of red blood cells (RBCs) In normal human erythrocytes,HbA composes about 90% of the total Hb It is made up of two identical α-chainsand two identical β-chains Besides HbA, human erythrocytes contain smallamounts 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, andclinical aspects, but also as a prototype of allosteric proteins Purification andcharacterization of Hbs has become easier and faster with the advent of high-pressure and high-performance instrumentation, high-sensitivity detectors, andthe availability of a wide variety of high-resolution column-packing materials.Methodological development using very small quantities of the protein is fea-sible, and the analytical methods are readily scalable Here, we describe threedifferent modes of high-performance liquid chromatography (HPLC) that areused in our laboratory for the purification and characterization of Hb, and modi-fied or mutant Hb

Hb is purified by ion-exchange chromatography (IE-HPLC), its size is lyzed by size-exclusion chromatography (SEC-HPLC) (under native and dis-sociating conditions), its globin chain separation is accomplished by reversephase HPLC (RP-HPLC), and tryptic peptide mapping of globin chains is alsocarried out by RP-HPLC Preparative runs are generally carried out on an

ana-AKTA Protein Purification System (Amersham Pharmacia Biotech), which isalso used for analytical runs Other instrumentation used for the analytical runsincludes a fast protein liquid chromatography (FPLC) system (AmershamPharmacia Biotech) for SEC and ion-exchange chromatography, and aShimadzu Liquid Chromatography System for RP-HPLC Examples of ion-exchange chromatographic purifications are given for analytical-scale runs(100 µg to 1 mg), small-scale preparative runs (up to 50 mg), and large-scalepreparative runs (up to 3 g) The analytical-scale runs are useful not only formethodological development, but also for characterization purposes and formonitoring the progress of a chemical modification reaction The SEC-HPLCruns are illustrated with analytical (1 mg) and semipreparative runs (100 mg).Examples of RP-HPLC are for analytical-scale runs (120 µg).

2 Materials

2.1 Purification of Human Hb by Ion-Exchange Chromatography (see Notes 1 and 2)

2.1.1 Anion-Exchange Chromatography

2.1.1.1 PURIFICATION OF HB ON DEAE-SEPHAROSE FAST FLOW: SMALL-SCALE

PURIFICATION (SEE NOTES 1 AND 2)

1 XK 16/10 chromatographic: column (Amersham Pharmacia Biotech)

2 DEAE-Sepharose Fast Flow anion exchanger: (Amersham Pharmacia Biotech)

3 Buffer A: 50 mM Tris-Ac, pH 8.5.

4 Buffer B: 50 mM Tris-Ac, pH 7.0.

5 Amersham Pharmacia Biotech AKTA Protein Purification System

2.1.1.2 PREPARATIVE-SCALE PURIFICATION OF HB ON Q-SEPHAROSE HIGH

PERFORMANCE CHROMATOGRAPHIC COLUMN

1 XK26/70 column (Amersham Pharmacia Biotech) (see Notes 1 and 2).

2 Q-Sepharose High Performance column-packing material (Amersham Pharmacia Biotech)

3 Buffer A: 50 mM Tris-Ac, pH 8.5.

4 Buffer B: 50 mM Tris-Ac, pH 7.0.

5 Amersham Pharmacia Biotech AKTA Protein Purification System

2.1.1.3 CATION-EXCHANGE CHROMATOGRAPHY: RECHROMATOGRAPHY

OF Q-SEPHAROSE HIGH PERFORMANCE PURIFIED HBA

ON CM-SEPHAROSE FAST FLOW

1 XK26/70 chromatographic column (Amersham Pharmacia Biotech)

2 CM-Sepharose Fast Flow cation exchanges (Amersham Pharmacia Biotech)

3 Buffer A: 10 mM potassium phosphate, pH 6.35, 1 mM EDTA.

4 Buffer B: 15 mM potassium phosphate, pH 8.5, 1 mM EDTA.

5 Amersham Pharmacia Biotech AKTA Protein Purification System

2.2 Ion-Exchange Chromatography as an Analytical Tool

2.2.1 Characterization of Recombinant Hb by Cation-ExchangeChromatography on a Mono S Column

1 Mono S HR5/5 column (1 mL) (Amersham Pharmacia Biotech) (see Notes 3

and 4).

2 Buffer A: 10 mM potassium phosphate, pH 6.5.

3 Buffer B: 15 mM potassium phosphate, pH 8.5.

4 Pharmacia FPLC protein purification system

5 Shimadzu UV-VIS detector at 540 nm

6 Shimadzu Chromatopac CR7A plus data processor

2.2.2 Monitoring Progress of a Chemical Modification Reaction

Analysis of Amidated HbS by Analytical Anion-Exchange

Chromatography on HiTrap Q Column

1 HiTrap Q, 1 mL column (Amersham Pharmacia Biotech) (see Note 3).

(pH 7.4), for evaluating the stabilization of the tetrameric structure of Hb

3 Pharmacia FPLC Protein Purification System

4 Detector: Shimadzu UV-VIS detector at 540 nm

5 Shimadzu CR7A plus data processor

2.3.2 Semipreparative SEC

1 Pharmacia XK26/70 column

2 Superose 12 prep-grade packing material (Pharmacia)

3 Buffer: PBS, pH 7.4

4 Pharmacia Biotech AKTA Protein Purification System

2.4 Globin Chain Analysis of Hb by RP-HPLC Analysis

1 Column: Vydac Protein C4 column (4.6 × 250 mm)

2 Solvent A: H2O, 0.1% trifluoroacetic acid (TFA)

3 Solvent B: acetonitrile, 0.1% TFA

4 Shimadzu Liquid Chromatography System consisting of two LC-6A pumps, anSPD-6A UV detector, an SCL-6B System Controller, and Class VP chromatog-raphy software

3 Methods

3.1 Purification of Human HbA by Ion-Exchange Chromatography

HbA is purified from erythrocytes obtained from adult human blood Theerythrocytes are gently washed with cold PBS, pH 7.4, and lysed with 4 vol-umes of water The lysate containing the Hb is separated from the cell debris

by centrifugation The lysate is dialyzed extensively against PBS, pH 7.4, tostrip the protein of 2,3-diphosphoglycerate

Because Hb can exist as an anion or a cation, depending on buffer tions, it can be purified by either anion- or cation-exchange chromatography,

condi-or a combination of the two Routinely, the erythrocyte lysate is first purified

on a Q-Sepharose High Performance column or on a DEAE-Sepharose FastFlow column followed by a second chromatography on a CM-Sepharose FastFlow column All purifications are carried out at 4°C

analytical-scale run of the same sample for which a preparative run is given in

Subheading 3.1.1.2 on a Q-Sepharose High Performance column (Fig 2).

Runs like this are useful for the evaluation of the run conditions prior to parative runs The total run time is 5 h 6 min, the total volume is ~612 mL, andthe gradient time/volume is 2 h/240 mL

pre-1 Pack the column (pre-1.6 cm × 6 cm, CV = 12 mL) according to the manufacturer’sdirections

2 Wash the column with 1 CV each of water, buffer A, and buffer B

3 Equilibrate the column with 10–25 CV of buffer A at a flow rate of 2 mL/min

4 Inject the sample and wash the column with 1 CV of buffer A to elute unboundprotein

5 Elute the bound protein with a linear gradient of 0–100% buffer B in 20 CV

6 Monitor the column effluent at 540, 600, and 630 nm (see Note 5).

7 Clean the column with 10 CV of buffer B

8 Reequilibrate with 20 CV of buffer A

3.1.1.2 PURIFICATION OF HB ON Q-SEPHAROSE HIGH PERFORMANCE:

PREPARATIVE RUN

A typical chromatographic profile of a human erythrocyte lysate (load: ~40 mL

containing ~3 g of Hb) is shown in Fig 2 The protein eluting at 1500 mL

(~65% buffer B) corresponds to HbA The fractions corresponding to this peak

are pooled, concentrated, and subjected to further purification by

cation-exchange chromatography on a CM-Sepharose Fast Flow column ing 3.1.2.) The following run takes about 25 to 26 h.

(Subhead-1 Pack the Q-Sepharose High Performance ion-exchange column at 4°C in aPharmacia XK26/70 column according to the manufacturer’s directions Typi-cally, a 2.6 × 58 cm column (~290-mL column volume) is used for the purifica-tion of 2.5–3 g of Hb

2 Wash the column first with 1 CV of water, followed by 1 to 2 CV each of 20%buffer B and 100% buffer B

3 Equilibrate the column with at least 10 CV of 20% buffer B, at a flow rate of1.5 mL/min

4 Dialyze the red cell lysate extensively against 20% buffer B, and filter through a0.2-µm filter

5 Load the lysate onto the column manually using line A18 of Pump A

6 Elute the protein with a linear gradient of decreasing pH consisting of 20–100%buffer B in 8 column volumes (2320 mL)

7 Monitor the column effluent simultaneously at three wavelengths; 540, 600, and

630 nm (see Note 5).

Fig 1 Small-scale anion-exchange chromatography of human red cell lysate on aDEAE-Sepharose Fast Flow column (1.6 × 6 cm) at 4°C Buffer A: 50 mM Tris-Ac,

pH 8.5; buffer B: 50 mM Tris-Ac, pH 7.0 The column was equilibrated with buffer A,

and a decreasing pH gradient of 0–100% buffer B over 20 CV was used for elution ofthe protein Protein load: 25 mg

3.1.2 Rechromatography of Q-Sepharose High Performance PurifiedHbA on a CM-Sepharose Fast Flow Column

A typical chromatographic profile is shown in Fig 3 The protein eluting at

1960 mL (~78% buffer B) corresponds to HbA Pool the HbA-containing tions, concentrate in an Amicon stirred cell to a concentration of 64–128 mg/mL,dialyze against the buffer of choice, and store either in liquid nitrogen or at –80°C

frac-1 Dialyze the HbA obtained from the Q-Sepharose High Performance column

(~1.3 g in 60 mL) against 10 mM potassium phosphate buffer; 1 mM EDTA,

pH 6.35 (see Note 6).

2 Load the dialyzed HbA on the CM-Sepharose Fast Flow column (2.6 cm × 59 cm),preequilibrated with the same buffer The large sample volume is not a consider-ation, since the protein binds to the column at the initial conditions Up to 3 g of Hbcan be purified on a 2.6 × 59 cm column

3 Elute the protein with a linear gradient of increasing pH, consisting of 0–100%buffer B over 8 column volumes (~2500 mL)

Fig 2 Preparative-scale anion-exchange chromatography of human red cell lysate

on Q-Sepharose High Performance column (2.6 × 58 cm) at 4°C Buffer A: 50 mM Tris-Ac, pH 8.5; buffer B: 50 mM Tris-Ac, pH 7.0 The column was equilibrated with

20% buffer B, and a decreasing pH gradient of 20–100% buffer B over 8 CV was usedfor elution of the protein Protein load: 3.0 g; fraction size: 20 mL

3.2 Ion-exchange Chromatography as an Analytical Tool

3.2.1 Characterization of HbA Expressed in Transgenic Swine byCation-Exchange Chromatography on a Mono S Column

The ion-exchange chromatographic procedures are also valuable as fast niques for the analysis of Hb variants and recombinant hemoglobins The elu-tion positions are dependent on the surface topology of the Hb The Mono Scolumn (Amersham Pharmacia Biotech) distinguishes between the correctly

tech-folded and mistech-folded forms of recombinant HbA (rHbA) (2–4) Adachi et al (2) have reported that the rHbA obtained from their yeast expression system

contains a misfolded form of HbA in addition to the correctly folded form Themisfolded and the correctly folded forms of rHbA exhibit distinct elution posi-

tions on a Mono S column Studies by Shen et al (3,4) have shown that rHbA

containing incorrectly inserted heme can be resolved from the species ing the correctly inserted heme on a Mono S column In our studies, the chro-matographic profile of the transgenic swine HbA on a Mono S column is

contain-identical to that of wild-type HbA (Fig 4), which, in conjunction with NMR

Fig 3 Repurification of HbA purified on Q-Sepharose High Performance column

(see Fig 2) by cation-exchange chromatography on a CM-Sepharose Fast Flow

col-umn (2.6 × 59 cm) at 4°C Buffer A: 10 mM potassium phosphate, 1 mM EDTA, pH 6.35; buffer B: 15 mM potassium phosphate, 1 mM EDTA, pH 8.5 The column was

equilibrated with buffer A and an increasing pH gradient of 0–100% buffer B over

8 CV was used for elution of the protein Protein load: ~1.3 g; fraction size: 12 mL Theeffluent was monitored at 540, 600, and 630 nm Elution profile for 540 nm is shown

and functional studies (5), has established the absence of misfolded forms in

this preparation

1 Equilibrate the Mono S column with 10 mM potassium phosphate, pH 6.5 (buffer A),

at a flow rate of 1 mL/min

2 Inject 1 mg of the HbA or TgHbA in 25 µL of buffer A

3 Wash the column with 2 CV of buffer A

4 Elute the protein with a linear increasing pH gradient consisting of 0–100% buffer

B over 45 CV

5 Monitor the column effluent at 540 nm

6 Regenerate the column in situ by washing first with ~5 CV of 100% buffer B

followed by reequilibration with 25 CV of buffer A (0% buffer B)

Fig 4 Comparison of elution profiles of wild-type HbA and HbA expressed intransgenic swine, on a Mono S HR5/5 column The flow rate was 1 mL/min Protein

load: 1 mg Buffer A: 10 mM potassium phosphate, pH 6.5; buffer B: 15 mM

potas-sium phosphate, pH 8.5 After injection of the protein, the column was washed with

2 CV of buffer A, and the bound protein was eluted with a linear gradient consisting of0–100% buffer B over 45 CV The effluent was monitored at 540 nm

3.2.2 Monitoring Progress of a Chemical Modificattion Reaction:Analysis of Amidated HbS by Analytical Anion-Exchange

Chromatography on HiTrap Q

3.2.2.1 PREPARATION OF AMIDATED HBS

HbS was amidated with ethanolamine, through a carbodimide and

sulfo-N-hydroxy-succinimide-mediated reaction, according to the previously described

procedures (6,7).

3.2.2.2 CHROMATOGRAPHY OF AMIDATED HBS ON HITRAP Q COLUMN

The chromatographic profile of an HbS preparation amidated with

ethanola-mine is illustrated in Fig 5 As can be seen, the amidated HbS can be separated

well from the unreacted HbS Thus, this profile illustrates the feasibility ofestablishing conditions for the separation of modified and unmodified HbSusing small amounts of the protein and within a short period of time Proteinloads as little as 100 µg are sufficient for such runs Thus, these columns arehighly useful for methodological development as well as for monitoring thetime course of a protein modification reaction

Fig 5 Chromatography of amidated HbS on a 1-mL HiTrap Q column at room

temperature Buffer A: 50 mM Tris-Ac, pH 8.5; buffer B: 50 mM Tris-Ac, pH 7.0 The

column was equilibrated with 10% buffer B The column was washed with 5 CV of10% buffer B after injection of the sample, and a decreasing pH gradient of 0–100%buffer B over 20 CV was used for elution of the protein Protein load: 1 mg Theeffluent was monitored at 540 nm