Journal of Clinical InvestigationVol. 43, No. 12, 1964

Oxygen Equilibria of Hemoglobin A2 and HemoglobinLepore *

GRACEG. EDDISON,t ROBIN W. BRIEHL,$ AND HELEN M. RANNEY(From the Departments of Medicine and Physiology of the Albert Einstein College of Medi-

cine and the Bronx Municipal Hospital Center, New York, N. Y.)

Human hemoglobin provides a model for studiesconcerned with the relationships of structure andbiologic function of proteins. Older evidence forconformational differences between oxygenatedand deoxygenated normal hemoglobin (1) hasrecently been confirmed and extended by X-raycrystallographic studies (2) and by comparison ofthe dissociation (3) and of the hybridization (4)of the oxygenated and deoxygenated pigments.

Normal and abnormal human hemoglobins havebeen utilized in the past by other workers for in-vestigation of relationships between structure andoxygen equilibria. Some of these observations,grouped on the basis of the structure of the he-moglobins studied, are briefly cited below.

1) Normal human, hemoglobins A (aA p2A),F (a,2A 'F), and A. (a2A 8A-) are tetramers con-taining apparently identical aA polypeptide chains,but differing in the non-alpha chains. Extensivestudies of the oxygen equilibria of hemolysates pre-pared from the erythrocytes of normal subjectsand of normal erythrocytes, containing almostentirely hemoglobin A, provide values with whichdata on other human hemoglobins may be com-pared. The increased oxygen affinity observedin the whole blood of newborn infants (5, 6) can-not be due solely to the presence of hemoglobin F,since solutions of hemoglobin F and of hemo-globin A exhibit the same oxygen dissociationcurves (7, 8). Furthermore, in the studies ofSchruefer, Heller. Battaglia, and Hellegers (8),

* Submitted for publication June 23, 1964; acceptedAugust 10, 1964.

Supported by grant G-6357 from the Life InsuranceMedical Research Fund, grant A-4502 from the Na-tional Institute of Arthritis and Metabolic Diseases, andgrant HE-07451 from the National Heart Institute.

t Recipient of fellowship (no. 5-F2-HE-18,557) fromthe National Heart Institute.

t Career scientist of the Health Research Council ofthe City of New York.

the oxygen equilibria of erythrocytes obtainedfrom an adult in whom hemoglobin F comprised69%o of the total pigment resembled the oxygenequilibria of normal adult blood rather than thatof cord blood. These workers (8) suggested thatdifferences between the fetal and adult red cellother than the type of hemoglobin must be con-cerned in the oxygenation function of whole bloodobtained from newborn infants.

Although hemolysates containing large pro-portions of hemoglobins F or A can be studied di-rectly, isolation procedures must be utilized toachieve appropriate concentrations of some of theminor components. For example, since hemo-globin A, (a2A SA2) accounts for only 5 or 6%oof the hemoglobin of subjects with thalassemiaminor, for functional studies at least partial iso-lation of hemoglobin A, is required. Rossi-Fanelli, Antonini, Benerecetti, and De Marco (9)isolated hemoglobins A and A2 electrophoreticallyand found no significant difference in the oxygendissociation curves. Meyering, Israels, Sebens,and Huisman (10) and Huisman, Dozy, Necht-man, and Thompson (11), however, employedcolumn chromatography for separations and foundthat hemoglobin A, exhibited increased oxygenaffinity as compared with hemoglobin A.

2) Abnormal hemoglobins with single aminoacid substitutions. The functions of some of theabnormal hemoglobins that result from singleamino acid substitutions in one pair of polypep-tide chains have been studied. Among these arepl-chain variants, hemoglobin S (a2A 2 6 val), he-moglobin C (a2A /326lYs), hemoglobin DO, and ana-chain variant, hemoglobin I. Although de-creased oxygen affinity has been observed in wholeblood from patients with sickle cell anemia (12-15), data concerning the oxygen affinity of solu-tions of hemoglobins S are conflicting (16). Itseems probable, however, that large differences in

2323

G. G. EDDISON, R. W. BRIEHL, AND H. M. RANNEY

oxygen affinity do not exist for hemolysates ofhemoglobins A and S (8, 17, 18). No differencein the oxygen affinities of hemoglobins C and Ahas been observed in two studies (4, 18), but smallincreases in oxygen affinity for two hemoglobin Dvariants have been reported (18). HemoglobinI, an a-chain variant, had a normal oxygen

equilibrium (4). In the Mhemoglobins in whichoxidized heme iron is present in either the a or

,f polypeptide chains, there is obvious interferencewith the oxygenation function; Kiese, Kurz, andSchneider (19) have reported a case in which en-

zymatic reduction of hemoglobin M yielded a he-moglobin with a normal oxygen affinity. Reiss-mann, Ruth, and Nomura (20) described an un-

usual abnormal hemoglobin with a low oxygen

affinity; no structural studies of the pigment were

included in the report.3) Tetramers of one type of polypeptide chain.

Studies of functional properties of hemoglobinsthat are tetramers of one variety of polypeptidechain, i.e., H (ft4A) and Bart's (74F), have dis-closed striking abnormalities. Both hemoglobinH (21) and Bart's (22) exhibit extraordinarilyhigh oxygen affinities and absence of heme-hemeinteraction, and neither has a significant Bohreffect. It has therefore been suggested that in-teractions between a and chains are necessary

for these three aspects of hemoglobin function(21).

4) Hemoglobin Lepore. The structure of he-moglobin Lepore, as elucidated by Baglioni (23),represents a hitherto unrecognized class of he-moglobin abnormalities: normal a chains com-

bined with chains that are products of unequalcrossing over in the linked 8-,8 locus. Thus he-moglobin Lepore contains apparently normal aAchains and non-alpha chains, which include initialpeptides of 8 chains and the remaining peptidesfrom the ft-chain sequence. Hemoglobin Leporemay be designated a2A x2, where x representsthe chain resulting from the crossing over. Sincecrossing over may occur at different sites, dif-ferent examples of hemoglobin Lepore might ex-

hibit different lengths of 8 (or f) sequence. Huis-man, Still, and Nechtman noted an increased oxy-

gen affinity in an example of hemoglobin Lepore(18).

Thus, apart from certain special cases, e.g.,

the hemoglobin studied by Reissmann and co-

workers (20), hemoglobins that are tetramerscontaining two a chains linked to two or to twoy chains exhibit nearly the same oxygen affinityas does hemoglobin A. Data concerning the oxy-

gen affinity of hemoglobin A2 are conflicting (9-11).

The present studies were undertaken to re-

investigate the oxygen function of hemoglobin A2and to study the functional properties of anotherexample of hemoglobin Lepore.

TABLE I

Data from which the statistical analyses were calculated

Hemoglobin A Hemoglobin AWhole hemolysate concentration equiv- Isolated concentration equiv- Isolated

Normal whole containing hemo- alent to hemo- hemoglobin alent to hemo- hemoglobinhemolysate globin Lepore globin Lepore Lepore globin A2 A2

log Po0 pH log Pso pH log Pso pH log Ps0 pH log Ps0 pH log P50 pH

0.903 7.07 0.875 7.07 0.826 7.00 0.740 6.82 0.813 7.02 0.820 7.000.940 7.07 0.903 7.07 0.833 7.00 0.742 6.82 0.820 7.02 0.839 7.000.813 7.20 0.839 7.19 0.820 7.00 0.663 7.00 0.699 7.20 0.732 7.190.813 7.20 0.839 7.19 0.740 7.18 0.690 7.00 0.708 7.20 0.716 7.190.690 7.46 0.681 7.44 0.763 7.18 0.699 7.02 0.623 7.40 0.748 7.210.690 7.46 0.681 7.44 0.778 7.22 0.556 7.10 0.623 7.40 0.748 7.210.586 7.62 0.550 7.62 0.732 7.22 0.556 7.10 0.544 7.50 0.580 7.420.544 7.63 0.538 7.62 0.568 7.44 0.602 7.20 0.544 7.50 0.574 7.420.544 7.63 0.586 7.44 0.602 7.20 0.525 7.55

0.519 7.57 0.643 7.22 0.519 7.550.525 7.57 0.643 7.22

0.505 7.380.512 7.380.498 7.400.512 7.400.380 7.580.423 7.58

2324

OXYGENEQUILIBRIA OF HEMOGLOBINA2 AND HEMOGLOBINLEPORE

A. Methods

9%__ 1) Preparation of hemoglobin compoeic)tts. Blood

I o----Normal whole Hemolysate samples were collected in 3.2% sodium citrate from

901- -Whole Hemolysate containing Lepore 1) an 18-year-old male of Italian ancestry, heterozygous// for hemoglobin LeporeEinstein (24),' for isolation of he-

80 / moglobin Lepore (which comprised 12% of the totalA/ pigment), 2) a 58-year-old woman of Italian ancestry

70-with thalassemia minor, in whom hemoglobin A2 com-

70- /I prised 6% of the hemoglobin, for isolation of hemoglobin/1 A2, and 3) normal subjects, for preparation of control

60_ hemoglobin A samples. Hemolysates containing hemo-

/ l globin in concentrations of 10 g per 100 ml were pre-

50, pared by the method of Drabkin (25). Samples ofhemoglobin Lepore and of hemoglobin A2 were preparedfrom the appropriate hemolysate by electrophoresis in

40- / 0.05 M Veronal buffer on starch granules or on Pevikon/ / (26) by the method of Kunkel, Ceppellini, Milller-Eber-

30_ / / hard, and Wolf (27). Hemolysates from which con-30// trol hemoglobin A samples were to be prepared were di-

// luted to 1.2 g per 100 ml or to 0.6 g per 100 ml (for20- comparison with hemoglobin Lepore or A2, respectively)

/2' before electrophoretic separation. Electrophoretic sepa-10 -ail/,' ° rations of 15 hours duration at 40 C were carried out

II 8-"°on solutions of oxyhemoglobin and were completed in° I I I I less than 24 hours after the blood samples were taken.

.2 .4 .6 .8 1.0 1.2 14 At the conclusion of the electrophoretic separation,log P02 hemoglobin components were eluted with 0.1 M potas-

sium phosphate buffer, pH 6.5. Several different pH

1.o- values, ranging from pH 6.80 to 7.65, were achieved byI<> B dilution with, or in some cases dialysis against, the

0.9 _ ><= appropriate phosphate buffer. Solutions of hemoglobin*><^(2) A2, and of hemoglobin A that had been diluted to 0.6 g

Go8(2

per 100 ml before electrophoresis, were concentrated byultrafiltration (21) after elution. Values for pH were

0.7 - S 2)l2)obtained immediately before studies of oxygen equilibria.

0.7 Gu2) Characterization of isolated componcntts. For the50 >vS present studies, avoidance of methemoglobin formation

0.6- G O was considered more important than purity of the iso-I o----oNormal whole Hemolysate (2) lated components. Consequently no attempts were made

0.5- to improve the separations by prolonging the durationU*-*Whole Hemolysate containing Lepore of electrophoresis or by increasing the current. The

04_ average proportion of methemoglobin determined spec-trophotometrically on each separated component was

I I I 1 3.5%o, with a range of 0.5 to 6.0%. When the studies7.0 7.1 7.2 7.3 74 75 7.6 of oxygen equilibria were completed, the hemoglobin

pH solution was converted to carboxyhemoglobin and con-

A. Oxygen dissociation curve. No signifi- centrated by ultrafiltration, and a second electrophoreticifference in the oxygen affinities of normal whole 1 Preliminary studies of the structure of hemoglobinysate (line I) and whole hemolysate containing LeporeEinstein have shown no significant differences from

(line II) at pH 7.00, 200 C, was observed. B. the example of hemoglobin Lepore studied by Baglioniegression of log P5o on pH of normal whole he- (23).

molysates kline 1) and of whole hemolysates contain-ing Lepore hemoglobin (line II) is shown. No sig-nificant difference in oxygen affinities was observed.[The symbol (2) represents two identical determinations.The symbol 0 represents the coincident values fromone determination of the normal whole hemolysate andone determination of the whole hemolysate containing

Lepore hemoglobin.] The standard deviation from theregression for the unfractionated hemolysates was

0.0600, and for the hemolysates containing hemoglobinLepore it was 0.0424. The regression coefficients were

homogeneous (p > 0.5), and the regression lines did notdiffer significantly (0.3>p>0.1) from each other.

% 02

log P5

FIG.cant dhemolyLeporeThe r(

2325

100rI

G. G. EDDISON, R. W. BRIEHL, AND H. M. RANNEY

A.100o Hemoglobin A-concentrationz- equivalent to Hemoglobin Lepore A

9-0as Isolated Hemoglobin Lepore ,8

.

I I I I I.2 4 .6 X

log PO2

B.I ----Normal Whole HemolysateU- Whole Hemolysote containing Lepore

MO._--,HemogIobin A-concentrationequivalent to Hemoglobin Lepore

lre-- Isoloted Hemoglobin Lepore

(2)

I I I I *17.3 7.4 75 7.6

FIG. 2. A. Oxygen dissociation curve. A marked increase in the oxy-gen affinity of isolated hemoglobin Lepore (line IV) as compared to

hemoglobin A at a concentration equivalent to hemoglobin Lepore (lineIII) at pH 7.00, 200 C, was observed. B. The regression lines for isolated

hemoglobin Lepore (line IV) and for hemoglobin A at a concentration

equivalent to hemoglobin Lepore (line III) showed a marked increase in

oxygen affinity in isolated hemoglobin Lepore. Lines I, II, and III are ex-

tended to pH 6.8. The standard deviation of data of isolated hemoglobinA from the regression was 0.0775, and for hemoglobin Lepore it was

0.1539. The regression lines for isolated hemoglobins A and Lepore were

markedly different (0.0005 > p). Whether regression coefficients were ho-

2326

80 _

70k

601-

%02 50-

40[_

30k_

201-

1o0

1.0 t2 14

l.Ar

1.0-

Q9H

0.8kl0g P50 Q71-

0.6L

0.51-

0.4

6.8 6.9 7.0 7.1 7.2pH

--WI

OXYGENEQUILIBRIA OF HEMOGLOBINA2 AND HEMOGLOBINLEPORE

separation for quantification of components was carriedout. When hemoglobin Lepore was studied, the secondelectrophoretic separation was allowed to proceed for24 or more hours. It was found that the initial separa-tion of hemoglobin Lepore had yielded solutions con-taining an average of 70% (range, 62 to 81%o) Leporetogether with hemoglobin A.

Because large proportions of methemoglobin weresometimes encountered in solutions of hemoglobin A2prepared by electrophoresis on starch granules, Pevikonwas utilized as the supporting medium for the initialelectrophoretic separation of hemoglobin A2. The sepa-rations of hemoglobins A2 and A on Pevikon were ob-viously incomplete and 12 to 27% (mean, 24%o) con-tamination of A2 with A was found on the second(starch granule) electrophoretic separation after com-pletion of studies of oxygen function. Methemoglobinformation was, however, much less on Pevikon thanon at least some lots of starch. The data on isolatedhemoglobins Lepore and A2 in the present report werenot corrected for the observed contamination with he-moglobin A.

3) Studies of oxygen equilibria. Studies of oxygenequilibria of the isolated components were completedwithin 30 hours after the blood samples were obtained.(Studies of whole hemolysates were completed within6 hours.) Determinations of oxygen equilibria werecarried out at 20° C on hemoglobin solutions of 0.1 g per100 ml (0.06 mMheme) concentration, by a modifica-tion of the method of Riggs (28) and of Allen, Guthe,and Wyman (29). The oxygen saturation was calcu-lated from measurements of optical density made in aBeckman DU spectrophotometer at 580 and 564 mu.Adequate deoxygenation of the hemoglobin solutionswas indicated by the following ratios for each sample:1) the ratio of the optical density of the deoxyhemo-globin at 564 my to the optical density at 580 myL was1.26 to 1.35 (mean = 1.31) and 2) the ratio of the op-tical density of the deoxyhemoglobin at 564 mA to the op-tical density of the oxyhemoglobin at 580 mtt was 0.79to 0.86 (mean = 0.82).

The range of value for n from Hill's equation (30),

Y 21VX1y P n1 - y Pso,

in which y represents the fraction of hemoglobin presentas oxyhemoglobin, P the oxygen tension, and P50 oxygentension necessary for 50%o saturation of the hemo-globin, was 2.3 to 3.1. Determinations that resulted invalues for n below 2.3 were discarded (see below).

4) Statistical analyses. The regression of log P5o onH was calculated from the data for each hemoglobin

studied by the method of least squares. To test thehomogeneity of the regression coefficients (regressioncoefficients are the slopes of the regression lines) andto compare regression lines of isolated components withhemoglobin A or with unfractionated hemolysates, ananalysis of covariance was made (31). If the regressioncoefficients of the oxygen equilibria of any two hemo-globins were homogeneous, the Bohr effects would be ofthe same order of magnitude. If the regression coeffi-cients were homogeneous, comparison of the levels of theregression lines would indicate alterations in oxygenaffinity. The results of the statistical analyses are sum-marized in the legends of Figures 1B, 2B, and 3B. (SeeTable I for the data.)

Results

1) Unfractionated hemolysates. Figures 1Aand 1B contain data on unfractionated hemolysatesprepared from erythrocytes containing normal he-moglobin (line I) and hemolysates containing12% hemoglobin Lepore (line II). In Figure1A, the difference between the oxygen dissociationcurves of these two hemolysates at pH 7.00 wasnot sufficient to be significant. In Figure 1B, therelationships between log P50 and different pHvalues were demonstrated. The regression lines(the slopes of which represent the Bohr effect) forlines I and II appeared about the same. The ab-sence of significant differences in oxygen func-tion between normal unfractionated hemolysatesand hemolysates containing 12%7 hemoglobinLepore was confirmed on statistical evaluation.

2) Isolated Lepore hemoglobin. In Figures2A and 2B, data on electrophoretically isolatedhemoglobin Lepore (line IV) were comparedwith normal hemoglobin that had been diluted toapproximately the concentration of hemoglobinLepore before electrophoresis (line III). Fig-ure 2A contains the oxygen dissociation curves ofthese two hemoglobins at pH 7.00; under thesecircumstances hemoglobin Lepore appeared tohave a greater oxygen affinity than did normalhemoglobin. From Figure 2B, it is clear thathemoglobin Lepore had a greater oxygen affinityat each of several pH values between 6.8 and 7.6.

mogeneous was unclear (0.10 > p > 0.05, p c 0.06), since tests for statisticalsignificance were at borderline values. The regression coefficients for iso-lated hemoglobin A and normal whole hemolysates were homogeneous(0.3 > p > 0.1), although the regression lines were different (0.0005 > p).The regression coefficients for isolated hemoglobin Lepore and whole he-molysates containing 12% hemoglobin Lepore were nonhomogeneous (0.01 >p > 0.005).

2327

2328 G. G. EDDISON, R. W. BRIEHL, AND H. M. RANNEY

A,100-

mo--o Hemolbin A-concentrationequivalent to Hemoobin A2

90 -.* Isolated Hemoglobin A290-

80_

IS70 _

60

%°2 50 /

40 I

30 _

20-

10-

.2 .4 .6 .8 0 1.2 1.4log P02

1.0- I - -- Normal Whole HemolysateI . ._OHemoglobin A-concentration B

equivalent to Hemoglobin A20.9- MsN---*Isolated Hemoglobin A2

Q~~~rS ~~(2)'s0.7 s

log P50(2)0.6_

0.5_

0.4_

7.0 71 7.2 7.3 7.4 7.5 .7X6pH

FIG. 3. A. Oxygen dissociation curve. No significant differ-ence in the oxygen affinities of isolated hemoglobin A2 (line VI) andof hemoglobin A at a concentration equivalent to hemoglobin A2(line V) was observed. B. The regression of isolated hemoglobinA, (line VI) and of hemoglobin A at a concentration equivalent tohemoglobin As (line V) disclosed no significant differences in oxygenaffinities. The standard deviation for the regression of isolated he-moglobin A, was 0.0566, and for hemoglobin A at an equivalent con-

2329OXYGENEQUILIBRIA OF HEMOGLOBINA2 AND HEMOGLOBINLEPORE

The increased oxygen affinity of hemoglobin Le-pore was statistically highly significant. Figure2B also contains the plots for unfractionated he-molysates (lines I and II) from Figure lB. Thedifference between hemoglobin A isolated electro-phoretically in dilute solution (line III) andnormal unfractionated hemolysate (line I) is ofinterest: the increased oxygen affinity of the iso-lated normal hemoglobin as compared with wholehemolysate was statistically significant and pre-sumably resulted from alterations in the pro-tein occurring during the isolation procedure. InFigure 2B, the slopes of the regression lines ofisolated hemoglobin Lepore and of the isolatednormal hemolysates may be different, thus suig-gesting the possibility of an altered Bohr effectfor hemoglobin Lepore. This alteration, however,was of borderline statistical significance.

3) Isolated hemoglobin 2. Isolated hemo-globin A, (line VI) and normal hemoglobin di-luted to a concentration equivalent to that of thehemoglobin A, before electrophoresis (line V)are compared in Figures 3A and 3B. Figure 3Ashowed no difference in the oxygen dissociationcurves of these two hemoglobins at pH 7.00, andtheir regression lines demonstrated in Figure 3Bappeared the same. These conclusions were sup-ported by statistical analysis.

Discussion

In our studies, identical oxygen dissociationcurves of the minor component of normal hemo-globin, hemoglobin A2, and of the major compo-nent hemoglobin A were observed. Rossi-Fanelliand co-workers (9) obtained similar data for he-moglobins A, and A, whereas others (10, 11)have found hemoglobin A2 to have a higher oxy-gen affinity than hemoglobin A. No significantdifference in the Bohr effect of hemoglobin A2 ascompared to hemoglobin A was observed in theseor previous studies. The differing conclusions

concerning oxygen affinity of hemoglobin A2 mayhave resulted from differences in techniques ofisolating the components. For the present studiesand for those of Rossi-Fanelli and co-workers (9),zone electrophoresis was employed; our studiesof oxygen equilibrium were completed within 30hours after the blood was shed. Huisman and co-workers (10, 11), however, isolated hemoglobinA. by DEAE cellulose chromatography, and ap-proximately 72 hours elapsed before determina-tions of oxygen equilibria. In the present studies,a higher oxygen affinity of isolated hemoglobin Awas noted when hemoglobin A was diluted beforerather than after the electrophoretic separation.Therefore, control hemoglobin A was diluted tothe same concentration as hemoglobin A2 or he-moglobin Lepore before the electrophoretic sepa-rations.

The interaction constant, it, defined by Hill'sequation, is approximately 3 for normal humanhemoglobin. The oxygen dissociation curves ofhemoglobin A, and hemoglobin Lepore exhibitedno evidence of decreased heme-heme interactionwhen compared to similar data on isolated hemo-globin A. Values for n, however, were about 2.6for each of the three isolated components: A, A2,and Lepore. These changes in the interaction con-stant presumably resulted from changes in theprotein during the isolation procedure. Similarvalues for it were obtained by Rossi-Fanelli andco-workers (9). (Studies with a value for n of2.3 or lower probably represented excessive de-naturation and were discarded in the presentstudy.) The n values obtained by Huisman andco-workers (11) in studies of isolated hemoglobinA., ranged between 1.4 and 2.0.

The increased oxygen affinity of isolated hemo-globin Lepore demonstrated in these studies re-sembled that observed by Huisman and co-work-ers (18). The expected and observed effect ofthis increase in oxygen affinity of hemoglobin Le-pore on the unfractionated hemolysate was so

centration it was 0.0360. The regression coefficients for these twohemoglobins were homogeneous (0.5 > p > 0.3), and the regressionlines were not significantly different (0.5 > p > 0.3). The regressioncoefficients for isolated hemoglobin A2 and unfractionated normalhemolysates were homogeneous (p > 0.5), although the regressionlines differed (0.0005 > p). The regression coefficients of hemoglo-bin A at a concentration equivalent of hemoglobin A2 and unfrac-tionated normal hemolysates were homogeneous (0.3 > p > 0.1);these regression lines were also different (0.0005 > p).

G. G. EDDISON, R. W. BRIEHL, AND H. M. RANNEY

slight as to be within experimental error. In ad-dition, the data were compatible with a small dif-ference in the magnitude of the Bohr effect ofisolated hemoglobin Lepore when compared withhemoglobin A at an equivalent concentration.Since denaturation in general increases the oxy-gen affinities of hemoglobins, it is possible thatthe increased affinity of hemoglobin Lepore is dueto an increased amount of denaturation. However,since the values for n were about the same forhemoglobin Lepore and hemoglobin A, thereis no evidence of any major difference in theamount of denaturation. Furthermore, the con-centrations of methemoglobin in the two hemoglo-bins were also approximately the same. SinceLepore amounts to only 12% of the total hemo-globin, comparison of oxygen equilibria of theisolated component with those of hemolysates orof erythrocytes containing hemoglobin Leporewould not provide crucial evidence concerningdenaturation.

Benesch, Ranney, Benesch, and Smith (21)pointed out that interactions between a and /3chains were apparently essential for three im-portant aspects of function of hemoglobin A: theBohr effect, the sigmoid shape of the oxygendissociation curve (n), and the oxygen affinity.From observations on dissociation and hybridiza-tion, it may be concluded that the interchain re-lationships between the a and -y chains of hemo-globin F (32, 33) and between the a and 8 chainsof hemoglobin A2 (34) are different from theinterchain relationships of hemoglobin A (a2Ap2A). These differences in interchain bonds donot, however, result in differences in the param-eters of oxygen function between hemoglobins F,A2, and A. In hemoglobin Lepore, two normala chains combined with two chains that have 8-likeN-terminal and /3-like C-terminal portions, an al-tered oxygen affinity was observed. The alteredoxygen affinity is apparently related to the pres-ence of the unusual non-alpha chains and suggeststhat the interchain interactions of hemoglobinLepore differ from those of either of the normalhemoglobins A2 or A.

Summary

The oxygen equilibria of hemoglobins A, Le-pore, and A2 isolated by zone electrophoresis

were studied. The oxygen equilibrium of hemo-globin A2 did not differ significantly from that ofhemoglobin A subjected to the same experimentalconditions. A significant increase in oxygen affin-ity was demonstrated in isolated hemoglobin Le-pore as compared with isolated hemoglobin A.The expected and observed effect of this in-creased affinity on unfractionated hemoglobinfrom a Lepore heterozygote was so slight as to bewithin experimental error. Isolated hemoglobinsA, A2, and Lepore appeared to have the sameheme-heme interaction and each exhibited a Bohreffect, although the Bohr effect of hemoglobinLepore might have differed from that of hemo-globin A. The increased oxygen affinity of hemo-globin Lepore may be an effect of altered inter-chain relationships resulting from the presence ofthe unusual non-alpha chains in the tetramer.

Acknowledgments

We thank Dr. Julien Hoffman for assistance withthe statistical analyses and gratefully acknowledge thetechnical assistance of Miss Eileen Berlad.

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2330

2331OXYGENEQUILIBRIA OF HEMOGLOBINA2 AND HEMOGLOBINLEPORE

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