Journal of Clinical InvestigationVol. 45, No. 6, 1966

Polycythemia Associated with a Hemoglobinopathy *

SAMUELCHARACHE,t DAVID J. WEATHERALL,ANDJOHNB. CLEGG(From the Departments of Medicine and Biophysics, Johns Hopkins University School of

Medicine and Johns Hopkins Hospital, Baltimore, Md.)

The efficiency of hemoglobin as an oxygen-carrying substance is dependent upon certain fea-tures of its molecular configuration. The func-tional integrity of the molecule depends not onlyon spatial relationships between the heme andglobin moieties, but on interrelations between thefour globin chains (1, 2). The functional sig-nificance of portions of the molecule can be in-vestigated by a study of hemoglobin with knownabnormalities of structure. Many structural ab-normalities have been reported, but functional ab-normalities are extremely rare.

Despite considerable differences in the aminoacid composition of the fi, 8, and y chains, the oxy-gen dissociation curves of purified solutions of he-moglobin A (a2Ih8), hemoglobin F (ay,), and he-moglobin A2 (a282) are quite similar (3-5). Incontrast, hemoglobins H (/4), Bart's (y,), and aA,in which interaction between a and , chains is im-possible, have very high oxygen affinities (1, 2,6). A Bohr effect is not present, and oxygendissociation curves are hyperbolic rather than sig-moid in shape, indicating that heme-heme interac-tions are absent.

Hemoglobin A treated with carboxypeptidase Aalso exhibits increased oxygen affinity and absenceof heme-heme interactions (7). Altered oxygenaffinity is thought to be a nonspecific result of in-ability of this modified hemoglobin to undergo aconformational change on deoxygenation.

Other abnormal hemoglobins with altered oxy-

* Submitted for publication December 3, 1965; ac-cepted February 8, 1966.

Presented in part at the Fifty-seventh Annual Meetingof the American Society for Clinical Investigation, May3, 1965, at Atlantic City, N. J.

This investigation was supported in part by U. S.Public Health Service graduate training grant 5T1AM-5260 and research grant HE-02799.

t Address requests for reprints to Dr. Samuel Char-ache, Johns Hopkins University School of Medicine,Dept. of Medicine, 601 N. Broadway, Baltimore, Md.21205.

gen affinity are characterized by substitutions of asingle amino acid. Oxygen affinity of the hemo-globins M appears to be reduced because of theirtendency to become oxidized to methemoglobin(8), and the amino acid substitutions responsiblefor these changes are thought to stabilize the ironof the heme moiety in the trivalent state (9).Hemoglobin Dpunjab has been reported to have in-creased oxygen affinity (10, 11), -and blood con-taining hemoglobin Zurich has increased oxygenaffinity (12). Hypotheses have not been ad-vanced to explain the mechanism of altered oxy-gen affinity, but alterations in both interchain re-lationships and electrostatic interactions are un-doubtedly involved.

In all of the hemoglobinopathies described, clini-cal consequences of altered oxygen affinity areeither overshadowed by other deleterious effectsof the disorder (hemoglobins H, Zurich, M, Bart's,and Lepore) or the magnitude of the alteration isso small that no significant clinical alteration isobserved (hemoglobin Dpunjab). The only hemo-globinopathy in which alteration of oxygen affinityhas been clinically significant is that described byReissmann, Ruth, and Nomura, in which a hemo-globin of low oxygen affinity produced impressivecyanosis (13). In the present investigation anew hemoglobin, hemoglobin Chesapeake, wasfound to be associated with significantly elevatedhematocrit levels in heterozygous carriers. Bloodoxygen affinity was increased, and increased oxy-gen affinity was shown to be a property of the iso-lated abnormal hemoglobin.

Methods

Hematologic and cardiorespiratory studies were per-formed by standard methods. Blood oxygen dissociationcurves were determined by the manometric method ofHellegers and his associates (14) .1 Details of theseanalyses will be published elsewhere (12). Electropho-

1 Blood oxygen dissociation curves were determined byDr. Andre Hellegers and his co-workers.

813

S. CHARACHE, D. J. WEATHERALL,AND J. B. CLEGG

resis, alkali denaturation, dissociation and recombinationexperiments, and incubation with oxidant dyes were per-

formed as previously described (15). Hemoglobin-glu-tathione complex was prepared for comparison of elec-trophoretic mobility by the method of Huisman andDozy (16).

For studies of the oxygen affinity of purified hemo-globin Chesapeake, blood was obtained with heparin as

anticoagulant. Red cells were washed four times, hemo-lyzed by addition of 6 vol of 0.01 M phosphate buf-fer, pH 6.8, and centrifuged for 30 minutes in a refrig-erated centrifuge at 22,000 g. Carboxymethyl cellulose(CMC) chromatography was performed at 4° C on a 4-X 20-cm column by a modification of the technique ofHuisman and Meyering (17). A pH gradient from 6.8to 8.3 was established with 0.01 M phosphate buffersthat did not contain KCN. Flow rates were 3.5 to 4 mlper minute, and the chromatographic procedure was com-

pleted within 4 hours. Dialysis against 0.1 M phosphatebuffer was carried out overnight. Samples were dilutedwith 0.1 M phosphate, and oxygen affinity was measuredby the method of Allen, Guthe, and Wyman (18) withminor modifications of Ranney, Briehl, and Jacobs (2).The entire procedure was completed within 30 hours.Purity of samples used for these measurements was de-termined by electrophoresis.

Partial pressure of oxygen in millimeters mercury

(Po2) was plotted against proportion of oxyhemoglobin(y) according to the logarithmic form of Hill's equa-

tion (7, 19) :log [y/(l-y)] =n log Po2+k. Linearregression of y/(1 - y) on Po2 was calculated by themethod of least squares. The slope of the line obtained,n in the above equation, is a constant that reflects themagnitude of heme-heme interactions. Covariance analy-

ses of these data were perfornied by the method ofSnedecor (20).

Specimens of hemoglobin A and hemoglobin Chesa-peake prepared by the chromatographic procedure were

converted to their methemoglobin derivatives by over-

night dialysis against 0.067 M phosphate buffer, pH 6.5,or 0.067 M Tris buffer, pH 8.5, each containing 0.025 MK3Fe (CN) 6. Absorption spectra were measured witha Cary recording spectrophotometer. Heat precipitabilitywas determined by the method of Dacie and his associ-ates (21), using unaltered hemolysates.

For globin analysis, hemoglobin Chesapeake was

purified by starch block electrophoresis, and globin was

isolated with cold acetone that contained 2 ml of con-

centrated HCl per 100 ml. Alpha and j8 peptide chainswere separated on CMCby gradient elution chromatog-raphy in buffers containing urea and mercaptoethanol,and a chains were converted to aminoethyl derivatives(AE-a chains) with ethyleneimine. After desalting andtryptic hydrolysis, peptide mapping and amino acid analy-ses were carried out. Details of these procedures are

published elsewhere (22).

Results

Pro positus. An 81-year-old white man (F. M.)of German-Irish extraction sought medical advicebecause of mild angina pectoris. His past healthhad been good, except for an episode of melena atage 76 thought to be due to atrophic gastritis.At that time the hematocrit value had been 39%o,rising to 46% 1 month after discharge.

TABLE I

Hematologic data obtained from the family of the propositus, F. M.*

HbPedigree Ches Starch block electro-

no. Sex carrier Hb Hct RBC Retics MCV MCH MCHC phoresis

g/lOOml % /mm3 % A3 Aug % %A %Ches %A2II M + 19.9 58.0 6.35 91.0 31.5 34.0 63.2 34.9 2.012 F + 16.2 48.0 5.13 0.8 94.0 32.0 34.0Is F + 15.5 48.1 4.71 0.7 102.0 33.0 32.0 70.3 27.7 2.0I5 F + 18.0 53.5 5.54 0.9 97.0 33.0 34.0I7 F + 16.8 50.8 5.44 0.6 94.0 31.0 33.0Is0 F + 17.6 52.5 5.59 0.6 94.0 32.0 33.0114 F - 34.2

IIl M + 18.7 51.8 5.78 90.0 32.0 36.0 73.0 25.1 1.9113 M - 17.0 48.0 5.23 2.0 92.0 32.0 35.0IHI M - 15.3 43.3 4.70 1.3 92.0 32.6 35.0II8 F - 15.3 45.5 4.67 1.1 97.0 33.0 33.6IIi0 F - 44.7 1.0II12 M - 16.4 47.6 5.55 0.6 86.0 29.5 34.5I114 F - 15.7 47.0 5.02 0.2 94.0 31.5 33.5Ii16 F + 15.9 49.7 4.86 0.7 102.0 33.0 32.0 72.5 25.1 2.4I118 M + 17.2 52.1 5.41 0.4 96.0 32.0 33.0 70.0 28.0 2.0II20 F + 14.9 48.6 5.09 0.8 96.0 29.0 30.5 71.0 26.2 2.81126 F + 17.0 52.7 5.66 0.2 93.0 30.0 32.0 68.4 29.8 1.8

IIIg F - 14.5 45.3 4.81 0.4 93.5 30.0 32.0TIIso M + 16.8 53.8 6.00 0.6 90.0 28.0 31.0 74.6 23.3 2.111131 F + 15.0III33 M + 17.4 54.2 5.58 0.7 92.0 31.0 32.0 69.7 27.7 2.6IIIii F + 15.9 48.8 5.20 0.8 94.0 30.6 32.0I1136 M - 14.5 43.6 4.88 0.4 89.0 30.0 33.5

113s7 M - 16.0 47.8 5.74 1.2 83.5 28.0 33.0IIIss M + 16.8 50.0 5.59 0.6 90.0 31.0 34.0 74.6 23.0 2.4

*Abbreviations: Hb Ches = hemoglobin Chesapeake; Hct = hematocrit; RBC = red blood cells; retics = reticulocytes; MCV= meancorpuscular volume; MCH= mean corpuscular hemoglobin; MCHC= mean corpuscular hemoglobin concentration.

814

POLYCYTHEMIAASSOCIATEDWITH A HEMOGLOBINOPATHY 815

A-N

(BART'S) I

A-HO2

A-CHES I

A-GSSG

AA

B...S-C

AGR STARCH GEL

HEMOGLOBINCHESAPEAKE

FIG. 1. HEMOGLOBINELECTROPHORESISON AGAR (pH 6.0) AND STARCH GEL (PH 8.6) (BROMOPHENOLBLUESTAIN). Hemoglobin Chesapeake is compared with hemoglobins N, Bart's (and F), Hopkins-2, synthetic he-moglobin-glutathione complex, S, and C.

1 2 34 56 7 8 9$10 12 13 14 15

1 2 3 4 5 6 7 8 9 l If 12 13 14 15 16 17 19 2021 22 23 24125 26 27 28 29

mEL r3)C MOI

* ABORTION

®3) DECEASED HEMOGLOBINCHESAPEAKE/ PROPOSITUS

FIG. 2. PEDIGREEOF THE PROPOSITUS, F. M. 0, female; I, male.

S. CHARACHE,D. J. WEATHERALL,AND J. B. CLEGG

X ray showed cardiomegaly of hypertensive con-figuration. Plasma erythropoietic activity wasless than 0.03 U per ml (23). An abnormal he-moglobin, designated hemoglobin Chesapeake, wasdetected by starch gel electrophoresis (Figure 1).

Treatment consisted of phlebotomies, to main-tain the patient's hematocrit value at less than50%o, and vasodilators. Angina disappeared, butrecurred 6 months later with its original severity.

Family study. Hemoglobin Chesapeake was de-tected in the blood of 16 of 26 members of F. M.'s

AFFECTED UNAFFECTED AFFECTED UNAFFECTEDMALES' MALES FEMALES FEMALES

FIG. 3. COMPARISONOF HEMATOCRIT VALUES IN AF-

FECTED AND UNAFFECTEDFAMILY MEMBERS. The verticalbars indicate mean + 2 SE.

On examination he appeared much younger thanhis stated age and was quite plethoric; no othersignificant physical abnormalities were present.

His hematocrit was 58%o, leukocyte count 5,100per mm3, and platelets 251,000 per mm3. Hemato-logic studies of members of his family are listed inTable I. No cardiopulmonary abnormalities suffi-cient to produce secondary polycythemia could bedetected: arterial oxygen tension was 72 mmHg(normal, 85 to 95 mmHg), and cardiac outputand oxygen consumption were normal. A chest

100

+ 0.5 -

oD -0.10

-0.2 -

-0.3 -

-0.4 -

-0.5 -

-0.6 -

/NI /N= 3

*- A-A pH 7.4

O-OA- Ches.pH 74

1.0 1.1 1.2 1,3 1.4 1.5 1.6 1.7- 1.8 1.9 2.0LOG p02

z 70-

: 60'

!as 50zU

C 40*w

30-

20

10

20 30pO2 mmHg

FIG. 4. OXYGENAFFINITY AT 38' CTHE PROPOSITUS (*) AND A NIECE (A).pared with data of Hellegers and Sclternal and fetal blood (24).

ADULT FIG. 5. OXYGENAFFINITY AT 380 C OF BLOODFROMTHE

PROPOSITUSAND A NIECE (0). Data are compared withdata from four controls (0) and plotted according toHill's equation, in which y = fraction of hemoglobinpresent as oxyhemoglobin and n = slope of the line. Ref-erence lines with slopes of 1 and 3 are shown in the up-

per left-hand corner of the Figure.

family (Figure 2). Inheritance followed an au-

tosomal dominant pattern. The hematocrit valuesof heterozygous carriers were significantly higherthan those of their normal siblings (p < 0.005 for

-.,-----..,.~ men, p < 0.001 for women) (Table I, Figure 3).40 50 Red cell morphology was normal in all family

members. Leukocytes and platelets appeared nor-OF BLOOD FROMData are com-

mal on blood smears. The arterial oxygen ten-

iruefer for ma- sion of one polycythemic woman (II18) was 99mmHg. No illness was associated with the pres-

60 -

58 -

V,w-J

r

0

wI

56 -

54 -

52 -

50 -

48 -

46 -

44 -

I I

816

0

0

I

POLYCYTHEMIAASSOCIATEDWITH A HEMOGLOBINOPATHY

ence of hemoglobin Chesapeake. Two female car-riers (II2,, III,,) had each had one spontaneousabortion. Each of these women subsequently hadnormal children, and one had had a child who diedwhile undergoing surgery for multiple congenitalcardiac defects.

Oxygen affinity of blood and purified hemo-globin. Blood samples from F. M. and from aniece (II,,) had oxygen affinities approximatelyas great as that of umbilical cord blood (Figure4). Data from these subjects and data from fournormal controls are plotted according to Hill'sequation in Figure 5. For hemoglobin Chesa-

100-

90

zw0

Ldat

z0

On

80

70-

60.

50

40.

30

20 -

10-

0 - Hb Chesapeake- HbA

I I,

0 1 2 3 4 5pO2 mm. Hg

6 7

FIG. 6. COMPARISONOF OXYGENAFFINITY AT 100 C OFHEMOGLOBINCHESAPEAKE(0) AND HEMOGLOBINA (e)ISOLATED FROMTHE SAME BLOOD SAMPLES. The "hemo-globin A" samples contained 30% hemoglobin Chesa-peake.

peake carriers, the slope (n) is 1.8; for normalblood n is 2.9. Covariance analysis indicated thatthe difference in slopes was significant at the 1%level.

The results of three experiments with purifiedhemoglobin at 10° C and pH 7.4 are plotted inFigure 6. Samples of hemoglobin Chesapeakeprepared by CMCchromatography contained lessthan 5% of hemoglobin A, whereas hemoglobin Asamples contained approximately 30% hemoglobinChesapeake. No methemoglobin absorption band

1 0~~~

o -I ~~~0-j-0.2]

*/ *-*4Hb.A pH7.4-0.4 0-0 Hb.Chesapeake0 ~~~~~~~~~~pH7.4

-0.6 0.

-1.2 -1.0 -0.8 -0.6 -0.4 -02 0 t0.2 tO.4 t.6LOG p02

FIG. 7. COMPARISONOF OXYGENAFFINITY AT 100 C OFHEMOGLOBINCHESAPEAKE(0) ANDHEMOGLOBINA (0),PLOTTED ACCORDINGTO HILL'S EQUATION. The "hemoglo-bin A" samples contained 30%o hemoglobin Chesapeake.

(between 610 and 650 mu) was detected in anysample after dialysis. The oxygen tension re-quired for half saturation of hemoglobin Chesa-peake (Pi = 0.3 mmHg) was higher than thatof hemoglobin A isolated from the same bloodsample (Pi = 1.2 mmHg). The data in Figure6 are plotted according to Hill's equation in Fig-ure 7. The slope of the regression line for hemo-globin Chesapeake is 1.1; that of hemoglobin Ais 2.0. Covariance analysis indicated that the dif-ference in slopes was significant at the 1 % level.

+1.0 -

+0.8 -

+0.6 -

+0.4 -

+0.2 -

LOG p 1/2 0 -

-0,2 -

-0.4 -

-0.6 -

-0.8 -

-1.0 -

6.4 6.8

*-* Hb.AO---o Hb. Chesopeake

7.2 7.6 8.0 8.4 8.8

pH

FIG. 8. VARIATION OF THE OXYGENPRESSUREAT 100 CREQUIRED FOR HALF SATURATION OF HEMOGLOBIN (P.)WITH PH. Data for hemoglobin Chesapeake (0) arecompared with data of Ranney and co-workers (2) forhemoglobin A (0).

817

S. CHARACHE,D. J. WEATHERALL,AND J. B. CLEGG

A.

* (±A). .. .:

.. .il . ... ..

*: ::S ::. ..: :: :.::...... ..::.. i.f..D.DE.:f- Xx '~ ~~~~~~~~.'i...........: t:. : .... ...... .

::~~~~~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~::..:.... .. .:.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..........

C~~~t :~PL N 'E.S W"'Il

FIG. 9. GLOBIN ELECTROPHORESISIN STARCH GEL CONTAINING 6 M UREA. Globinfrom purified hemoglobin Chesapeake is compared with globin from hemoglobins Aand S and semipurified hemoglobin Hopkins-2 (amido black stain).

Similar experiments were carried out at pH6.8 and 7.6 with samples of hemoglobin Chesa-peake. No methemoglobin absorption. band (be-tween 610 and 650 mju) was detected in these sam-ples after dialysis. The two regression lines hadslopes of 1.1 and 1.2. The oxygen tension re-quired for half saturation of hemoglobin is plottedagainst pH in Figure 8. Included are data ofRanney and her associates for hemoglobin A, ob-tained under the same conditions (2). A Bohr ef-fect of approximately normal magnitude waspresent.

Physicochemical characteristics of hemoglobinChesapeake. On starch gel at pH 8.6, hemoglo-bin Chesapeake migrated more rapidly than eitherhemoglobin A or the synthetic hemoglobin-glu-tathione complex. Its mobility was approximatelythe same as that of hemoglobins Hopkins-2 or J.and less than that of hemoglobins Bart's, N, or I,suggesting that it had one less positive chargethan hemoglobin A at pH 8.6. HemoglobinChesapeake did not separate from hemoglobin Aduring citrate agar electrochromatography at pH6.0. It was not alkali resistant, it was not pre-

818

POLYCYTHEMIAASSOCIATEDWITH A HEMOGLOBINOPATHY

cipitated by incubation at 500 C for 2 hours, andno inclusion bodies formed in red cells after incu-bation with brilliant cresyl blue or new methyleneblue.

Dissociation and recombination with hemo-globin C, and electrophoresis of globin (Figure9), indicated that altered electrophoretic mobilitywas due to an abnormality of the a chain. Peptidemapping of tryptic digests of the AE-a chain ofhemoglobin Chesapeake showed that peptidesaAT10 and aAT11 were missing and that a newpeptide was present. The arginine stain revealedonly two (aAT4 and aAT14) of the three majorarginine positive peptides normally found on AE-achain peptide maps. These observations suggestedthat the C-terminal arginine in peptide aAT10 hadbeen replaced by a neutral amino acid, that thebond to peptide aAT11 was no longer susceptibleto trypsin, and that the new peptide was a conju-gate, aChesapeakeT 10-11.

Analysis of the new peptide (Table II) con-firmed this conclusion. With two exceptions, theamino acids found in the new peptide were thoseexpected from a combination of peptides aAT10and aAT11 (25). No arginine residues were de-tected, and the mole ratio of leucine was 2 instead

A-A

A-CHES

A-A

A-HO2

2 ~ ~~y 1 11

TABLE II

Mole ratios of amino acids in "new" peptide of hemoglobinChesapeake, compared with data for hemoglobin A (25)

aATIO+ afCmpmkaAT10 aATll aAT11 TIO-11

Lysine 1 1 1.2 (1)HistidineArginine 1 1Asparagine 2 2 2.0 (2)ThreonineSerineGlutamineProline 1 1 1.0 (1)GlycineAlanineCysteineValine 2 2 1.9 (2)MethionineIsoleucineLeucine 1 1 2.0 (2)TyrosinePhenylalanine 1 1 0.9 (1)Tryptophan*

* By staining reaction on paper.

of the anticipated 1. Substitution of leucine forthe only arginine in peptide aAT10 (position 92 ofthe a chain) satisfactorily accounts for the chargedifferences seen on starch gel electrophoresis ofhemoglobin Chesapeake. Full details of theseanalyses will be published elsewhere (26).

TRIS -C ITRATE-BORATEBENZIDINE STAIN

FIG. 10. STARCHGEL ELECTROPHORESISAT PH 8.6. The gel was overloaded and stained with benzidine in order todemonstrate variants of hemoglobin A2.

819

S. CHARACHE,D. J. WEATHERALL,AND J. B. CLEGG

>- .400 -

Inz

u .3000

-J

u .200

.100

MethemoglobinpH 6.5

500 600 700myU

Uf)zLLC)

-I

CL0T

.400-

.300 -

.200-

.100 -

MethemoglobinpH 8.9

500 600 700mjj

FIG. 11. COMPARISONOF VISIBLE ABSORPTION SPECTRAOF HEMOGLOBINCHESAPEAKEAND HEMOGLOBINA AS ACIDAND ALKALINE METHEMOGLOBINDERIVATIVES. The upperline represents hemoglobin Chesapeake.

A small amount of a hemoglobin with the elec-trophoretic mobility of hemoglobin S could be de-tected by starch gel electrophoresis with a Tris-citrate-borate buffer system, but not with othersystems (Figure 10). This fraction containedabout 1%o of the total hemoglobin present. Nostructural analyses of it were carried out.

Oxygenated solutions of hemoglobin Chesapeakehad normal visible and ultraviolet absorptionspectra, and deoxygenated solutions of hemoglo-bin Chesapeake had normal visible absorptionspectra. After conversion to acid or alkalinemethemoglobin, the optical density of solutions ofhemoglobin Chesapeake was somewhat greaterthan that of hemoglobin A between wavelengthsof 470 and 550 mju (Figure 11).

Discussion

Mild polycythemia in the propositus and inmembers of his family was associated with thepresence of an abnormal hemoglobin with in-

creased oxygen affinity. Blood containing 25 to35% of the abnormal hemoglobin had an oxygenaffinity approximately as great as that of umbilicalcord blood. Presumably, heterozygous carriershad an increased number of red cells because theirblood delivered less oxygen per red cell than didthe blood of their normal siblings. Increasedamounts of erythropoietin could not be demon-strated in the propositus's serum, a result similarto that found in polycythemic persons who havelived at high altitudes for long periods of time(23).

Carriers of hemoglobin Chesapeake were quitesimilar to some cases of "benign familial polycythe-mia" (27), particularly to individuals with mildlyelevated hematocrit values and without spleno-megaly. Hemoglobin, studied by paper electro-phoresis, was normal in the patients described byAuerback, Wolff, and Mettier (28). Blood fromtwo of the patients reported by Nadler and Cohn(29) has been restudied 2; both hemolysates werenormal after starch gel electrophoresis at pH 8.6.

The eight normal offspring of carrier femaleswere of particular interest. There was no sug-gestion that fetal oxygenation had been deficientduring the eight pregnancies in question, despiteabsence of the usual low oxygen affinity of ma-ternal blood relative to that of the fetus. Thepresence of high affinity for oxygen in the motherwas found to be compatible with the productionof term infants who developed normally, for therehad been only two spontaneous abortions among21 pregnancies. Similar observations have beenmade in another hemoglobinopathy: a female car-rier of hemoglobin Zurich was capable of bearingnormal offspring, although the affinity of her bloodfor oxygen was almost as great as that of carriersof hemoglobin Chesapeake (11). Adequacy offetal oxygenation is the result of many interrelatedfactors, including rates of uterine and umbilicalblood flow, size of placental capillary bed, and fe-tal blood volume. No such data are available inthis family.

The increased oxygen affinity of umbilical cordblood is not a property of the fetal hemoglobin itcontains (4). Heterozygous carriers of hemo-globin Chesapeake may have had an abnormal fe-tal hemoglobin while in utero, of composition

2 Through the courtesy of Dr. Nadler.

820

POLYCYTHEMIAASSOCIATEDWITH A HEMOGLOBINOPATHY

a2Chmy2. Unfortunately, we were unable to studyany blood samples that might have contained thishemoglobin. Such studies would be of consider-able interest, for the fetal analogue of hemoglobinChesapeake might have increased oxygen affinity,and the oxygen affinity of blood containing it mightbe very high.

The faint band of hemoglobin demonstrated onlyby discontinuous starch gel electrophoresis pre-sumably had the composition a2ChesS2. This "sec-ond A2 hemoglobin" contained about 17o of thetotal hemoglobin present. Carriers of hemoglobinChesapeake had relatively low proportions of he-moglobin A2 (a282) in their hemolysates (1.8 to2.6%o). Since approximately 30% of the majorhemoglobin component was hemoglobin Chesa-peake, one would expect about 17o of the secondhemoglobin A2, a prediction consistent with theobserved data.

Blood from heterozygous carriers, containingabout 30%7 hemoglobin Chesapeake, exhibited in-creased oxygen affinity and decreased heme-hemeinteraction (n = 1.8). Semipurified samples ofhemoglobin Chesapeake showed the same findings,as well as a Bohr effect of approximately normalmagnitude. Heme-heme interactions were di-minished (n = 1.1), but not abolished (n = 1.0).In studies' of hemoglobin Chesapeake, isolated bystarch block electrophoresis in barbital buffer, pH8.6, Nagel has obtained values for n of 1.3 on fourseparate measurements of oxygen equilibrium.He has also found lowered values for n in studiesof the whole hemolysate; preliminary measure-ments have indicated an n of about 1.8 (30).

Analysis of electrophoretic studies indicatedthat hemoglobin Chesapeake had one less positivecharge than hemoglobin A, at pH 8.6. The aminoacid substitution responsible for altered electro-phoretic mobility of hemoglobin Chesapeake wasreplacement of positively charged arginine by un-charged leucine in amino acid no. 92 of the a chain.This substitution corresponds to a one step muta-tion in the messenger ribonucleic acid codon forarginine, from cytosine-guanine- (adenine) to cy-tosine-uracil- (adenine), as outlined by Beale andLehmann (31). A substitution of this type mightproduce increased oxygen affinity either by directelectrostatic interactions or by altering interchainrelationships.

The effects of arginine and leucine would beexpected to differ from each other because of thedifference in their electric charges. The heme-linked histidine of the a chain is only five aminoacid residues away from the site of substitution(32), in such a position that an alteration ofcharge might affect the avidity of the heme ironatom for electrons donated by oxygen.

Charged amino acid side chains, like that of ar-ginine, tend to protrude outwards from the sur-face of the hemoglobin molecule in an aqueous en-vironment, whereas nonpolar groups, like that ofleucine, tend to be buried in the interior of themolecule (33). Amino acid no. 92 of the a chainis at the border between a and /8 chains (34), anarea in which the a and /3 chains slide past eachother during the spatial rearrangements of oxy-genation and deoxygenation. Altered structuralrelationships produced by substitution of a non-polar for a polar amino acid could mechanicallyimpede free equilibrium between the oxygenatedand deoxygenated forms of the molecule. Thishypothesis is favored by demonstration of im-paired heme-heme interactions, but data at presentare insufficient to strongly indicate either alterna-tive.

Summary

1. An abnormal hemoglobin, called Chesapeake,was associated with mild polycythemia in a Cau-casian family.

2. Blood from heterozygous carriers had an oxy-gen affinity as great as that of umbilical cord blood,and increased oxygen affinity was shown to be aproperty of purified hemoglobin Chesapeake.

3. Hemoglobin Chesapeake differed from hemo-globin A in substitution of leucine for arginine inthe ninety-second amino acid residue of the achain.

4. Hypotheses are presented relating the alteredfunction of hemoglobin Chesapeake to this dis-crete structural abnormality.

AcknowledgmentsWewish to express our thanks to Dr. Andre Hellegers

and his associates for performing the studies of oxygenaffinity of unaltered blood, to Dr. Alan Ross for assist-ance with biostatistical analyses, and to Dr. Helen Ran-ney for contributing both valuable advice and the useof her laboratory.

821

S. CHARACHE,D. J. WEATHERALL,AND J. B. CLEGG

References

1. Benesch, R. E., H. M. Ranney, R. Benesch, andG. M. Smith. The chemistry of the Bohr effect.II. Some properties of hemoglobin H. J. biol.Chem. 1961, 236, 2926.

2. Ranney, H. M., R. W. Briehl, and A. S. Jacobs.Oxygen equilibria of hemoglobin a' and of hemo-globin reconstituted from hemoglobin aA and H.J. biol. Chem. 1965, 240, 2442.

3. Allen, D. W., J. Wyman, Jr., and C. A. Smith. Theoxygen equilibrium of fetal and adult humanhemoglobin. J. biol. Chem. 1953, 203, 81.

4. Schruefer, J. J. P., C. J. Heller, F. C. Battaglia, andA. E. Hellegers. Independence of whole bloodand hemoglobin solution oxygen dissociationcurves from haemoglobin type. Nature (Lond.)1962, 196, 550.

5. Eddison, G. G., R. W. Briehl, and H. M. Ranney.Oxygen equilibria of hemoglobin A2 and hemo-globin Lepore. J. clin. Invest. 1964, 43, 2323.

6. Horton, B. F., R. B. Thompson, A. M. Dozy, C. M.Nechtman, E. Nichols, and T. H. J. Huisman.Inhomogeneity of hemoglobin. VI. The minorhemoglobin components of cord blood. Blood1962, 20, 302.

7. Antonini, E. Interrelationship between structure andfunction in hemoglobin and myoglobin. Physiol.Rev. 1965, 45, 123.

8. Kikuchi, G., N. Hayashi, and A. Tamura. Oxygenequilibrium of hemoglobin Mrwat.. Biochim. bio-phys. Acta (Amst.) 1964, 90, 199.

9. Gerald, P. S., and P. George. Second spectroscopi-cally abnormal methemoglobin associated withhereditary cyanosis. Science 1959, 129, 393.

10. Huisman, T. H. J., J. Still, and C. M. Nechtman.The oxygen equilibria of some "slow-moving" hu-man hemoglobin types. Biochim. biophys. Acta(Amst.) 1963, 74, 69.

11. Thompson, R. B., R. L. Warrington, and W. N. Bell.Physiologic differences in hemoglobin variants.Amer. J. Physiol. 1965, 208, 198.

12. Moore, W. M. O., F. C. Battaglia, and A. E. Hel-legers. Personal communication.

13. Reissmann, K. R., W. E. Ruth, and T. Nomura. Ahuman hemoglobin with lowered oxygen affinity andimpaired heme-heme interactions. J. clin. Invest.1961, 40, 1826.

14. Hellegers, A. E., G. Meschia, H. Prystowsky, A. S.Wolkoff, and D. H. Barron. A comparison ofthe oxygen dissociation curves of the bloods of ma-ternal and fetal goats at various pH's Quart J.exp. Physiol. 1959, 44, 215.

15. Weatherall, D. J., and S. H. Boyer IV. Evidence forthe genetic identity of alpha chain determinantsin hemoglobins A, A2 and F. Bull. Johns Hopk.Hosp. 1962, 110, 8.

16. Huisman, T. H. J., and A. M. Dozy. Studies on theheterogeneity of hemoglobin. V. Binding of hemo-

globin with oxidized glutathione. J. Lab. clin.Med. 1962, 60, 302.

17. Huisman, T. H. J., and C. A. Meyering. Studies onthe heterogeneity of hemoglobin I. The hetero-geneity of different human- hemoglobin types incarboxymethyl cellulose and in Amberlite IRC-50chromatography: qualitative aspects. Clin. chim.Acta 1960, 5, 103.

18. Allen, D. W., K. F. Guthe, and J. Wyman, Jr.Further studies on the oxygen equilibrium of he-moglobin. J. biol. Chem. 1950, 187, 393.

19. Hill, A. V. The possible effects of the aggregationof the molecules of hemoglobin on its dissociationcurves (abstract). J. Physiol. (Lond.) 1910, 40,iv.

20. Snedecor, G. W. Statistical Methods Applied toExperiments in Agriculture and Biology, 5th ed.Ames, Iowa State College Press, 1956.

21. Dacie, J. V., A. J. Grimes, A. Meisler, L. Steingold,E. H. Hemsted, G. H. Beaven, and J. C. White.Hereditary Heinz-body anaemia. A report ofstudies on five patients with mild anaemia. Brit.J. Haemat. 1964, 10, 388.

22. Clegg, J. B., M. A. Naughton, and D. J. Weatherall.An improved method for the characterization ofhuman haemoglobin mutants: identification ofa82295GLU haemoglobin NBaitimore'. Nature (Lond.)1965, 207, 945.

23. Waldman, T. A. Personal communication.24. Hellegers, A. E., and J. J. P. Schruefer. Nomo-

grams and empirical equations relating oxygentension, percentage saturation, and pH in ma-ternal and fetal blood. Amer. J. Obstet. Gynec.1961, 81, 377.

25. Konigsberg, W., and R. J. Hill. The structure ofhuman hemoglobin. V. The digestion of the a

chain of human hemoglobin with pepsin. J. biol.Chem. 1962, 237, 3157.

26. Clegg, J. B., and D. J. Weatherall. In preparation.27. Spodaro, A., and C. E. Forkner. Benign familial

polycythemia. Arch. intern. Med. 1933, 52, 593.28. Auerback, M. L., J. A. Wolff, and S. R. Mettier.

Benign familial polycythemia in childhood. Re-port of two cases. Pediatrics 1958, 21, 54.

29. Nadler, S. B., and I. Cohn. Familial polycythemia.Amer. J. med. Sci. 1939, 198, 41.

30. Nagel, R. Personal communication.31. Beale, D., and H. Lehmann. Abnormal haemoglobins

and the genetic code. Nature (Lond.) 1962, 207,259.

32. Perutz, M. F. Relation between structure and se-quence of hemoglobin. Nature (Lond.) 1962,194, 914.

33. Kendrew, J. C., H. C. Watson, B. E. Strandberg,R. E. Dickerson, D. C. Phillips, and V. C. Shore.The amino-acid sequence of sperm whale myo-globin. A partial determination by x-ray methods,and its correlation with chemical data. Nature(Lond.) 1961, 190, 666.

34. Perutz, M. F. Personal communication.

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