Denatured hemoglobin in sickle erythrocytes.

T Asakura, … , M O Russell, E Schwartz

J Clin Invest. 1977;59(4):633-640. https://doi.org/10.1172/JCI108681.

To study the nature of numerous inclusion bodies seen in red cells from patients with sicklecell disease (Hb SS), we have prepared red cell ghosts free of oxyhemoglobin andanalyzed them by spectrophotometric and heme extraction methods. The absorptionspectrum in the visible region of the ghost suspensions was typical of hemichromes. Thespectrum was similar to that of denatured hemoglobin repared by treatment ofoxyhemoglobin S with mechanical shaking or heat. Similar treatment of cells containingonly normal hemoglobin (Hb AA) showed a very small amount of denatured hemoglobin,approximately one-fifth of the amount in Hb SS cells. The amount of denatured hemoglobindetermined after solution of membrane with 2.5% sodium dodecyl sulfate was 0.158+/-0.070% (1 SD) of the total cellular heme in Hb SS patients. In controls, the amount was0.030+/-0.016%. Persons with Hb AA and reticulocytosis did not have an elevated amountof membrane-associated heme. In patients with hereditary spherocytosis and autoimmunehemolytic anemia, denatured stromal hemoglobin was normal or slightly elevated beforeand after splenectomy. The increased amount of denatured hemoglobin in Hb SS red cellsmay be related to the instability of sickle oxyhemoglobin.

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Denatured Hemoglobin in Sickle Erythrocytes

TosmIo AsAKuRA, KAYOKOMINAKATA, KAZUHIKOADACHI, MARIE 0. RUSSELL,and ELIAS SCHWARTZFrom The Children's Hospital of Philadelphia and the Departments of Pediatrics and Biochemistry,University of Pennsylvania, Philadelphia, Pennsylvania 19104

A B S T RA C T To study the nature of numerous inclu-sion bodies seen in red cells from patients with sicklecell disease (Hb SS), we have prepared red cellghosts free of oxyhemoglobin and analyzed them byspectrophotometric and heme extraction methods.The absorption spectrum in the visible region of theghost suspensions was typical of hemichromes. Thespectrum was similar to that of denatured hemo-globin prepared by treatment of oxyhemoglobin Swith mechanical shaking or heat. Similar treatment ofcells containing only normal hemoglobin (Hb AA)showed a very small amount of denatured hemo-globin, approximately one-fifth of the amount in HbSS cells. The amount of denatured hemoglobin de-termined after solution of membrane with 2.5%sodium dodecyl sulfate was 0.158+0.070% (1 SD) ofthe total cellular heme in Hb SS patients. In con-trols, the amount was 0.030+0.016%. Persons withHb AA and reticulocytosis did not have an elevatedamount of membrane-associated heme. In patientswith hereditary spherocytosis and autoimmune hemo-lytic anemia, denatured stromal hemoglobin wasnormal or slightly elevated before and after splenec-tomy. The increased amount of denatured hemo-globin in Hb SS red cells may be related to theinstability of sickle oxyhemoglobin.

INTRODUCTION

In hemolytic anemia due to unstable hemoglobins,inclusions of denatured hemoglobin are commonlyfound in circulating red cells, particularly aftersplenectomy. The shortened red cell survival inthese disorders is due in part to increased rigidityof cells containing inclusions (1) and to membranedamage associated with intracellular precipitationof hemoglobin (2). Numerous small inclusions arealso commonly seen in red cells from patients withsickle cell disease under special conditions, either

Received for publication 19 April 1976 and in revisedform 14 December 1976.

by phase-contrast microscopy with high-intensityillumination or after acid elution of adult hemoglobinfrom cells (3). Sickle hemoglobin in the oxygenatedconfiguration is unstable under special conditions ofmechanical shaking or stirring, or with repeatedcycles of oxygenation and deoxygenation (4, 5).The present study deals with our attempts to deter-mine the nature and amount of denatured hemoglobinin red cell membrane fractions from patients withsickle cell disease and normal subjects after completeremoval of oxyhemoglobin.

METHODSPatients. Patients with sickle cell disease ranged in age

from 3 to 19 yr old. Blood was drawn at the time of aroutine clinic visit and not during crisis or infection. Onlyone of these patients had a surgical splenectomy. The pa-tients with hereditary spherocytosis and autoimmunehemolytic anemia were children 4-6 yr in age. Thecontrols were mostly adult laboratory personnel, but alsoincluded several children with intact spleens.

Absorption spectra of red cell stromata prepared byvarious methods. Blood was drawn into heparin and redcells were washed three times with 0.85% saline solutionand were centrifuged. 0.2-ml aliquots of washed red cellswere hemolyzed with 40 ml of either (a) water, (b) 0.5mMEDTA, 5 mMpotassium phosphate buffer, pH 7.5,(c) 10 mMpotassium phosphate buffer, pH 7.5, or (d)20 mMpotassium phosphate buffer, pH 7.5, to determinethe best solution for removing native oxyhemoglobinfrom the stromal fraction. The hemolysate was centrifugedat 15,000 g for 20 min at 4°C. The stromata were washedfour more times with the same buffer used for initialhemolysis. The washed stromata were resuspended in 4ml buffer and the absorption spectra were recorded withan Aminco DW-2 UV-VIS spectrophotometer (AmericanInstrument Co., Inc., Silver Springs, Md.). The conversionof oxyhemoglobin to hemichrome during the washingprocedure was proven to be negligible by measuring thetotal oxyhemoglobin before and after the washing procedure.

Comparison of spectra of stromata after successivewashes. The purpose of this series of experimentswas to study the spectral changes of red cell ghosts asnative oxyhemoglobin was eluted with successive washes.1 ml of packed erythrocytes from normal controls or pa-tients with sickle cell disease was hemolyzed by adding19 ml of phosphate-EDTA buffer. After centrifugation at

TheJournal of Clinical Investigation Volume 59 April 1977-633-640 633

115

4 N'40U1 (Hemichrome)E

34150 7414E A0

w

wa obaie atrteteto3mlooxhogbiSwith1 u f4NNO ouin

zIfI~~~~~~I :~~~~537

ol

300 400 500 600 700WAVELENGTH(nm)

FIGURE 1 Absorption spectra of oxyhemoglobin and hemichrome. The hemichrome spectrumwas obtained after treatment of 3 ml of oxyhemoglobin S with 10 uld of 4 N NaOHsolution.

15,000 g for 20 min, 17 ml of the supernatant solution wascarefully removed (supematel). The absorption spectra ofthis supematant fraction were recorded between 250 and700 nm with a Perkin-Elmer-Coleman 124 double beamspectrophotometer (Perkin-Elmer Corp., Instrument Div.,Norwalk, Conn.) after appropriate dilution. The remain-ing 3 ml of hemoglobin and stroma mixture were gentlyhomogenized manually with a Teflon Potter-Elvehjemhomogenizer. A 0.1-ml aliquot of this mixture was resus-pended in 2.4 ml of the phosphate-EDTA buffer with aPotter-Elvehjem homogenizer. Care was taken not to producefoam, which might lead to artificial precipitation of hemo-globin. The absorption spectra of this stromal suspension(stroma,) were measured between 250 and 700 nm with anAminco DW-2 UV-VIS spectrophotometer. The rest(2.9 ml) of the original stromal mixture was diluted with 17.1ml of the phosphate-EDTA buffer and the procedure wasrepeated five more times to produce stromal fractions II-VI.

Heme concentrations in the supernatant solutions whichcontained mainly oxyhemoglobin were calculated by theuse of the millimolar extinction coefficient of mE577 = 15.0mM-1 cm-'. In stromal fractions which contained both theoxyhemoglobin and hemichrome forms, the total heme con-centrations were calculated from the optical density at theisosbestic points (the points of interaction) between thespectra of oxyhemoglobin and hemichrome (Fig. 1). Themillimolar coefficients used were mE525= 7.8 andmE588 = 4.83. The concentrations of heme were obtainedby averaging the calculations from the two points. Thismethod was used primarily to follow the elution of oxy-hemoglobin from suspensions of stromata, and to confirmthe presence of hemichrome and the absence of oxy-hemoglobin in the final stromal preparation. Although thevalues obtained by this method with individual samplesagreed within 10% experimental error on 10 duplicate ortriplicate experiments (Table I), the heme concentrationsdetermined by this method were always higher thanthose obtained by the extraction method described belowbecause of a slight elevation of the base line due to turbidity.The extraction method was therefore used for all finalquantitative determinations of heme content.

To determine if the hemichromes in the membranefraction were reversible, dithionite was added and car-bon monoxide was bubbled through the suspension. Thespectrum was unchanged after this treatment, indicating thatthe hemichromes were of the irreversible type. Thefractions of oxyhemoglobin in the mixture of oxyhemo-globin and hemichrome were calculated by the followingequations:

oxy-Hb() x, - 0.795 x 100,1.128where xl = E577/E525.The percentage of total proteins in the stromata was expressedas:

stromal protein (%) = stromanE280/total E280,

where n is the number of washings.

stroma,,E280 = E280 x 3 x x F,, and0.1

total E280 = (supernateIE280 x 17) + stromaIE280.

Corrections were made for the loss of 0.1-ml suspensionsout of 3-ml ghost suspensions used for spectrophotometeranalysis, i.e., F = (3.0/2.9)n-1, where F, is the correctionfactor for losses due to sampling for analysis.

The absence of oxyhemoglobin in the stromata was con-firmed by measuring the absorption spectra of the sus-pensions with an Aminco DW-2 UV-VIS spectrophotometer.Whenoxyhemoglobin remains in the stromata, the spectrumshows typical absorption peaks of oxyhemoglobin at 542and 577 nm.

Heme content of red cell stromata. The following ex-traction method was used for quantitative determination ofheme in the preparation of washed stromata. 0.5-1.0-ml ali-quots of washed erythrocytes were hemolyzed by mixingwith 40 ml of 5 mMof potassium phosphate buffer, pH7.5, containing 0.5 mMEDTA. After standing for 5 min,the hemolysate was centrifuged at 15,000 g for 20 min.

634 T. Asakura, K. Minakata, K. Adachi, M. Russell, and E. Schwartz

The stromata were washed two more times with the samebuffer. The pellets were washed once more by mixingfirst with 5 ml distilled water and then with 35 ml of thephosphate-EDTA buffer. The supernatant solutions werecombined. After confirmation of the absence of oxy-hemoglobin by analysis of spectra, the stromata weremixed with 2.3 ml of 2.5% sodium dodecyl sulfate (SDS)'solution, and left at room temperature overnight. Althoughfoaming due to SDS can be controlled by gentle handlingof the solution, the foaming can be effectively preventedby the addition of 1-2 ppm of SAG 10 (10% dimethyl-polysiloxane emulsion, Union Carbide Corp., New York).The heme content of the stromata was determined by theabsorbance at 404 nm of the resulting solution using 2.5%SDS as a blank. Millimolar extinction coefficients for de-natured hemoglobin were derived from oxyhemoglobinS precipitated by mechanical shaking (4, 5). The precipi-tate is known to have a hemichrome spectrum (5). Theprecipitate was gently dissolved in 2.5% SDS solution.The millimolar extinction coefficients of heme in 2.5%SDS are E404= 90.8, E600= 6.5 cm-', mM-1, and E418= 83.6and E,42= 11.5 cm-', mM-' in the presence of KCN. Theheme content in the supemate was measured as oxyhemo-globin.

The heme content in the stromata was expressed as afraction of total heme in erythrocytes: stromal heme (%)= (stromal heme)/(supematant heme and stromal heme)x 100.

RESULTS

Absorption spectra of red cell stromata. Theamount of hemoglobin remaining in red cell ghostsdepends on the concentration of the buffer with whichcells are treated (6, 7). Dodge et al. (7) reportedthat retention of hemoglobin is increased by hemolyz-ing the cells with phosphate buffer on concentrationsbelow 5 or above 20 mosmol. Our similar experi-ments with sickle erythrocytes confirm these re-sults. A considerable amount of hemoglobin was re-tained when the cells were hemolyzed with wateror with phosphate buffer at 10 mMor higher (Fig.2A). The maximum hemolysis was obtained with 5mMpotassium phosphate buffer containing 0.5 mMEDTA. The hemoglobin retained in ghosts after re-peated washings with water or with 20 mMphos-phate buffer was found to be mainly in its oxy-form as demonstrated by the prominent peaks at542 and 577 nm in the absorption spectra of theghost suspensions (Fig. 2A). In contrast, the redcell ghosts prepared with 5 mMphosphate buffercontaining 0.5 mMEDTA showed mainly the spec-trum of hemichrome. Hemichromes are derivativesof hemoglobin and other hemoproteins in which thesixth coordination position of heme-iron is boundto a nitrogen atom on the imidazole ring of histidinein position E7. Hemichromes are distinguishable bycharacteristic absorption spectra (8). Suspensions of

1Abbreviations used in this paper: ISC, irreversiblesickle cells; SDS, sodium dodecyl sulfate.

TABLE IReproducibility of Spectrophotometer Measurements

of Membrane-Associated Denatured Hemoglobin *

Differencebetween

Patient Patient high andnumber type Denatured Hb Mean low values

1 AA 0.10,0.10,0.11 0.10 0.012 AA 0.050, 0.053 0.058 0.0033 AA 0.077, 0.080 0.079 0.0034 AA 0.116, 0.116 0.116 05 SS 0.63,0.67,0.69 0.66 0.0066 SS 0.65, 0.69 0.67 0.047 SS 0.42, 0.39 0.41 0.038 SS 0.54, 0.59 0.57 0.059 Spherocytosis 0.031, 0.031 0.031 0

10 Koin 1.28,1.32 1.30 0.04

* The percentage of denatured hemoglobin was determinedfrom spectra of washed membranes as described in Methods.

precipitated hemoglobin prepared by mechanical shak-ing (4, 5) or by heating also show similar spectra(Fig. 2B).

There are distinct differences in the visual ap-pearance of red cell ghosts prepared by the differentmethods of lysis. The pellets prepared with waterexhibited a homogeneous pink color indicating theequal retention of oxyhemoglobin in each cell, whereasthe pellets prepared with 10 or 20 mMphosphatebuffer clearly showed two components: white ghostsand red erythrocytes. The pellets prepared by 5 mMphosphate containing 0.5 mM EDTA had twocomponents of dark brown and white, the ratio de-pending on the amount of denatured hemoglobinin the stromata.

Comparison of spectra of stromata from normaland sickle erythrocytes. Absorption spectra ofsuspensions of stromata from normal and sickleerythrocytes at each washing step are compared inFig. 3A and B. Initially the suspensions exhibit thespectrum of dissolved oxyhemoglobin. Upon repeatedwashings the fractions of hemichrome gradually in-crease as noted by the disappearance of the twosharp peaks of oxyhemoglobin at 577 and 542 nmand by the appearance of a hemichrome spectrumsimilar to that shown in Fig. 3A and B. The ratioof the absorbance at 280 nm to that at the Soretpeak (414 nm) also increases as soluble oxyhemo-globin is washed out. After washing three to fourtimes, these ratios become constant and the spec-trum is mainly that of hemichrome.

The heme content, optical density at 280 nm, andfraction of hemichrome comparing total hemoglobinin stromal-stromav, are illustrated in Fig. 4. The

Denatured Hemoglobin in Sickle Erythrocytes 635

0.:

E

0cL0

0

'I

z

0

0

0)

4t

700

WAVELENGTH(nm)

412

Hb S Precipitates

( Suspension )

500 600

WAVELENGTH(nm)

FIGURE 2 (A) Absorption spectra of stromal suspensions prepared by various methods. Notethe oxyhemoglobin peaks at 542 and 577 nm in suspensions I, III, and IV. The spectrumof suspension II is mainly that of hemichrome. (B) Absorption spectrum of suspensionsof hemoglobin S precipitate. A precipitate of oxyhemoglobin S by mechanical shakingwas resuspended in 0.1 M potassium phosphate buffer, pH 7.5. The spectrum of the suspen-

sions was measured with an Aminco split-beam spectrophotometer. The spectrum is char-acteristic of hemichrome (8).

data shown are from one example of 10 experimentsin which the full calculations were done for atleast three spectral measurements of stromata (sam-ples after washes, e.g., I, III, VI) in experiments inwhich A and S cells were compared on the same

day. In these studies, the mean A28dtotal A280 (%)

value for Hb AA cells in washes IV-VI was 1.7+0.2 (1 SD) (12 determinations), and the value forHb SS cells was 3.4+0.55 (24 determinations).

Comparison by a paired value t test gives a signifi-cant difference (P < 0.001), as does a comparison ofthe means by the t test (P < 0.001). In four sets ofthese studies done on the same blood sample,the mean value of A28o/total A280 for stromata IV-VIwas 3.4+0.32. The relationship of A280/total A280to hemichrome is shown in Fig. 4, with values cal-culated from spectra (white bars in lower part offigure). As the red color disappears from the stromata

FIGURE 3 Absorption spectra of stromal suspensions and supematant solutions at variouswashing steps. (A and B) Stromal suspension of Hb A and Hb S. (C) Supematant washingsolutions of Fig. 3A. The washing procedures are described in the text. Note that only ofoxyhemoglobin is washed out.

636 T. Asakura, K. Minakata, K. Adachi, M. Russell, and E. Schwartz

400 500X (nm)

700637

4

300 600

100

I 500

I

0

0

0

0c

ao

II III IV V VISTROMA

FIGURE 4 Optical properties and hemichrome content ofsuspensions of stromata at various washing steps. Calcula-tions were made as shown in the text. The top panel, thepercentage of hemichrome in total hemoglobin asso-

ciated with stromata is shown for a patient with sicklecell disease (S) and a normal control (A). Note that the rela-tive amount of hemichrome increases as oxyhemoglobin iswashed out of stromata.

The bottom panel, the percentage of proteins in thestromata (A280 stroma) compared with the total protein inred cells (A280 Total), is shown for successive washingsteps. The height of each column represents the percentage.The columns with a cross-hatched base show resultsfrom normal cells (A), whereas those with a solid base are

from an experiment with cells from a patient with sicklecell disease (S). The clear bars were the expected E280due to hemoglobin calculated on the basis of heme ab-sorption at the Soret peak and represents the fraction ofprotein associated with heme (oxyhemoglobin and hemi-chrome). The cross-hatched or solid bars are the sum ofmembrane protein and hemefree globin.

by repeated washings, the fraction of hemichromegradually increases. This is clearly due to the washingout of oxyhemoglobin as seen in the successivespectra of the supernatant solutions (Fig. 3C). Al-though it is not known if any soluble stromal pro-

tein is washed out during washing, there is no de-tectable loss of denatured hemoglobin in thesupernate. After five to six washings, the stromatashowed the dark brown color of hemichromes.

The absorption at 280 nm for sickle stromata ishigher than that of normal stromata, indicating an

increased protein content (Fig. 4). In stromav, ofnormal cells, about 10% of the absorption at 280nm is accounted for by denatured hemoglobin if

we assume that there is no heme-free globin. There-fore, about 90% of this absorption may be attributedto membrane protein. In stromav, of sickle cells,the percentage of denatured hemoglobin is at leastthree times the amount in normal cells. However,the increased stromal A280total A280 cannot be totallyaccounted for by the increased fraction of hemichrome.The remaining protein in sickle erythrocytes' stromatamust therefore be either heme-free globin or in-creased membrane protein.

Denatured hemoglobin content in normal and sicklecells. The amounts of denatured hemoglobin were

determined as total heme by dissolving the ghostsin 2.5% SDS solutions after removal of oxyhemo-globin. The results for red cells from patients withsickle cell disease, hereditary spherocytosis, auto-immune hemolytic anemia, and from controls are

shown in Fig. 5.In sickle erythrocytes, denatured hemoglobin was

0.158±0.070% (1 SD) of the total hemoglobin (range0.080-0.300). The highest values were in the twooldest patients (19 yr old) and in an 11-yr-old girlsplenectomized three yr before this study. A group

of children with hereditary spherocytosis and auto-immune hemolytic anemia was also studied to evalu-ate the effect of reticulocytosis or splenectomy on

0.300

z

co0

e! 0.200

I

0w

z

w 0.1000

FIGURE 5 Comparison of the amounts of membrane-associated denatured hemoglobin in various patients andnormal subjects. Sickle cell disease after splenectomy (-),spherocytosis before (0) and after (A) splenectomy, andautoimmune hemolytic anemia after splenectomy (A).

638 T. Asakura, K. Minakata, K. Adachi, M. Russell, and E. Schwartz

HEREDITARYSICKLE CELL SPHEROCYTOSIS

CONTROL ANDD I SEASE AUTOIMMUNE

HEMOLY. ANEMIA

0

0

000

0

o 00

0

0 0 0

I ~~~~~~00 0 a

0 0

0 00 A A &

g 0 0 A

00 0 00

8 _ _ _ _ _ _ _ _ _

denatured hemoglobin. Before splenectomy, thevalues were 0.030, 0.047, and 0.048, whereas in thesplenectomized group the mean was 0.063+0.018(range 0.041-0.090). The control group which in-cluded normal persons, several children with he-matologic disorders other than hemoglobinopathiesor hereditary spherocytosis, and one splenecto-mized child had a mean of 0.030+0.016 (range 0.010-0.055). To determine the amount of denatured hemo-globin in irreversible sickle cells (ISC), ISC wereprepared artificially by incubating sickle erythrocytesunder anaerobic conditions for 24 hr (9). Althoughmore than 70% cells were ISC, the increase in theamount of denatured hemoglobin in these cellswas negligible.

DISCUSSION

Although many previous investigations (6, 7, 10-13)have been concerned with the interaction of hemo-globin and the red cell membrane, little is knownabout the nature of hemoglobin in red cell ghosts.We have shown that there are at least two distincttypes of hemoglobin associated with red cell ghosts.One is hemichrome-type denatured hemoglobin andthe other is oxyhemoglobin. If the amount of hemo-globin in ghosts is determined as total heme, eitherby the pyridine hemochromogen method (7) or thecyanomethemoglobin method (1), the amount ofhemichrome-type denatured hemoglobin cannotbe distinguished from that of oxyhemoglobin remain-ing in the stromata. For this reason, the amount ofhemoglobin on ghosts measured by these methodsfluctuates with the variation in amount of oxyhemo-globin associated with the membranes due to differ-ences in methods of lysis and washing. The amountof hemichrome (denatured hemoglobin) should bemeasured after confirmation of the absence of oxy-hemoglobin by the direct measurement of suspensionsof ghosts with a split-beam spectrophotometer. Sincethe retention of oxyhemoglobin varies largely withwashing conditions, including ionic strength andpH, the binding of oxyhemoglobin to membranesappears to be nonspecific. On the other hand, the hemi-chrome-type denatured hemoglobin in red cellghosts remains bound to membranes with washing,and varies in amount in different disorders. Thespectrum of this hemoglobin is similar to that ofprecipitated hemoglobin formed by mechanical shak-ing or heat treatment (Fig. 2B), and represents atruly denatured, nonfunctional molecule. In theprocess of denaturation, hemoglobin forms twotypes of hemichromes, reversible and irreversible (8).The former can be converted to deoxyhemoglobinby reduction of hemichrome followed by anaerobicdialysis, whereas the latter cannot be renatured to

oxyhemoglobin. The hemichromes in the stromafraction in the present study were found to beirreversibly denatured.

Schneider et al. (3) observed Heinz bodies (intra-erythrocytic aggregations of denatured hemoglobin)in erythrocytes obtained from patients with sicklecell disease. They measured the hemoglobin con-tent of membranes after hemolyzing sickle cellswith water and found approximately 10% of thetotal hemoglobin in the ghost fraction in sickle eryth-rocytes in contrast to 2.6% in normal cells. Thepresent studies indicate that most of the remaininghemoglobin after lysis with water was in the oxy-form (Fig. 2A) and did not represent a constant orspecific form of membrane-associated hemoglobin.In the present study the amount of denatured hemo-globin in fresh erythrocytes from patients withsickle cell disease was only about 0.2% of the total,approximately five times higher than that in normalcells. It is more likely that the hemichrome-typedenatured hemoglobin discussed in this paper is thetype seen as red cell inclusions, because the spectrumof oxyhemoglobin changes to that of hemichrome witha variety of methods of precipitation, and also becausehemichromes were found in the form of Heinzbodies due to the presence of excess hemoglobinsubunits (14) or unstable hemoglobins (15). Althoughthe amount of denatured hemoglobin in normal andsickle erythrocytes with respect to the total hemo-globin in erythrocytes is small, the amount with re-spect to structural protein is large. Approximately5 x 105 hemoglobin molecules are denatured persingle sickle erythrocyte. The amount of denaturedhemoglobin in a sickle erythrocyte corresponds to itstotal structural protein. Since inclusion bodies causerigidity in erythrocytes, the spleen possibly detectsthis change and retains these cells.

Increased intracellular denaturation of hemo-globin in sickle cell disease does not seem to besolely due to reticulocytosis or the absence of splenicfunction. Patients with reticulocytosis in this studydid not have increased denatured hemoglobin, andthose who were splenectomized had only a slightor no increase above the control group (Fig. 5).The validity of the methods used in this study indetecting intracellular precipitation of oxidizedhemoglobin is indicated by 5-10-fold greater valuesthan normal found in red cells from patients withHb K6ln, Hb H disease, or severe glucose-6-phos-phate dehydrogenase deficiency.2

The mechanism of hemoglobin precipitation in-side erythrocytes containing Hb S is not known.It cannot be due to the gas-liquid surface denatura-

2Asakura, T., K. Adachi, and E. Schwartz. Unpublishedobservations.

Denatured Hemoglobin in Sickle Erythrocytes 639

tion which occurs during mechanical shaking. Re-peated passages of erythrocytes through very narrowcapillaries might cause sufficient agitation of oxy-hemoglobin to result in precipitation. Another possi-bility is the repetition of conformational changes be-tween oxy- and deoxyhemoglobin S during circulation.Experiments in our laboratory have shown that hemo-globin S denatures at a faster rate than does hemo-globin A during repeated changes of the gas phase fromair to nitrogen. The conformational changes be-tween the two states may provide sufficient molecularagitation to cause precipitation.

The cause of most vaso-occlusive crises in sicklecell disease, and the differences in frequency ofcrises and severity of disease between patients, isnot clearly understood. Increased precipitation ofsickle hemoglobin inside the erythrocytes may bea contributing factor to the initiation of crises andchronic hemolysis. Intraerythrocytic precipitation ofhemoglobin would cause rigidity of red cells andwould contribute to stasis, sickling, and vaso-occlusion. The confirmation of this hypothesis re-quires extensive studies of many patients with sicklecell disease to determine the relationship -betweenthe amount of intraerythrocytic denatured hemo-globin and the clinical status of the patients.

REFERENCES

1. Jandle, J. H., L. K. Engle, and D. W. Allen. 1960.Oxidative hemolysis and precipitation of hemo-globin. I. Heinz body anemias as an acceleration of redcell aging.J. Clin. Invest. 39: 1818-1836.

2. Winterboum, C. C., and R. W. Carrell. 1974. Studies ofhemoglobin denaturation and Heinz body forma-tion in the unstable hemoglobins. J. Clin. Invest. 54:678-689.

3. Schneider, R. G., I. Takeda, L. P. Gustavson, and J. B.

Alperin. 1972. Intraerythrocytic precipitations of haemo-globins S and C. Nature (Lond.). 235: 88-90.

4. Asakura, T., P. L. Agarwal, D. A. Relman, J. A. McCray,B. Chance, E. Schwartz, S. Friedman, and B. Lubin.1973. Mechanical instability of the oxy-form of sicklehaemoglobin. Nature (Lond.). 244: 437-438.

5. Asakura, T., T. Ohnishi, S. Friedman, and E. Schwartz.1974. Abnormal precipitation of oxyhemoglobin S bymechanical shaking. Proc. Natl. Acad. Sci. U. S. A. 71:1594-1598.

6. Anderson, H. M., and J. C. Turner. 1960. Relation ofhemoglobin to the red cell membrane. J. Clin. Invest.39: 1-7.

7. Dodge, J. T., C. Mitchell, and D. L. Hanahan. 1963.The preparation and chemical characteristics of hemo-globin-free ghosts of human erythrocytes. Arch. Bio-chem. Biophys. 100: 119-130.

8. Rachmilewitz, E. A., J. Peisach, and W. E. Blumberg.1971. Studies on the stability of oxyhemoglobin A andits constituent chains and their derivatives. J. Biol.Chem. 246: 3356-3366.

9. Shu Chu Shen, E. M. Fleming, and W. B. Castle.1949. Studies on the destruction of red blood cells.V. Irreversibly sickled erythrocytes: their experimentalproduction in vitro. Blood. 4: 498-504.

10. Klipstein, F. A., and H. M. Ranney. 1960. Electro-phoretic compounds of the hemoglobin of red cellmembranes. J. Clin. Invest. 39: 1894-1899.

11. Winterboum, C. C., and R. W. Carrell. 1973. The attach-ment of Heinz bodies to the red cell membrane.Br. J. Haematol. 25: 585-592.

12. Fisher, S., R. L. Nagel, R. M. Bookchin, E. F. RothJr., and I. Tellez-Nagel. 1975. The binding of hemo-globin to membranes of normal and sickle erythrocytes.Biochim. Biophys. Acta. 375: 422-433.

13. Durocher, J. R., and M. E. Conrad. 1974. Erythrocytemembrane proteins in sickle cell anemia. Proc. Soc.Exp. Biol. Med. 146: 373-375.

14. Rachmilewitz, E. A., J. Peisach, T. B. Bradley, Jr.,and W. E, Blumberg. 1969. Role of haemichromes inthe formation of inclusion bodies in haemoglobin Hdisease. Nature (Lond.). 222: 248-250.

15. Rachmilewitz, E. A. 1974. Denaturation of the normaland abnormal hemoglobin molecule. Semin. Hematol.11: 441-462.

640 T. Asakura, K. Minakata, K. Adachi, M. Russell, and E. Schwartz