FURTHER STUDIES ON HEMOGLOBIN- OXYGEN EQUILIBRIUM

The Japanese Journal of Physiology

14, pp.280-298, 1964

FURTHER STUDIES ON HEMOGLOBIN-

OXYGEN EQUILIBRIUM

Yasunori ENOKI AND Itiro TYUMA*

Second Department of Physiology, Nara Medical College,

Kashihara, Nara, Japan

In recent years, the correlation between the chemical structure and phy-

siological function of many functional proteins has been of more general and

wider interests (MoNoD et al. 1963). In this research field, hemoglobin may

be one of the best material in that 1) it can be easily prepared in large

quantity and in highly purified form and 2) the information on the chemical,i. e. primary to quarternary, structure has been increasingly accumulatingand 3) it bears an important physiological function in respiration.

The physiological function of hemoglobin has long been studied by virtue

of the oxygen equilibrium curve, of which the position (oxygen affinity and

Bohr effect) and the sigmoid character (heme-heme interaction) have attracted

the special attention of many physiologists, biochemists, and physi co-chemists.Previously, one of the present authors found that a considerable change

in both the shape and the position of the whole blood oxygen equilibrium

curve was induced in albino rats by exposing them to an acute and extremeoxygen lack (ENoKi, 1959a). The latter change could easily and largely be

explained in terms of metabolic acidosis, whereas the former one was argued

to be due to an alteration in the intracorpuscular electrolyte environment ofhemoglobin.

In this connection, the author intended to study the salt effects on hemo-

globin-oxygen equilibrium, especially on the heme-heme interaction (ENOKI,1959b), for no exhaustive and quantitative research had been made on this

problem since BARCROFT'S classical and incomplete works (BARCROFT andROBERTS, 1909). The results obtained clarified a quantitative relationship

between the magnitude of heme-heme interaction and the species and strengths

of various neutral salts and further suggested a possible important role of

charge distribution on the protein moiety in the heme-heme interaction.

Our later experiments, however, pointed out a possibility that the dialysis

of stock hemoglobin solution used in the above researches might be insufficient,

possibly making the above conclusion somewhat uncertain. The first aim of

Received for publication March 3, 1964.* 榎泰義, 中馬一郎

280

HEMOGLOBIN-OXYGEN EQUILIBRIUM 281

the present study, therefore, is to reinvestigate this problem. In addition,

further attempts are made to clarify the structural basis of hemoglobin-

oxygen equilibrium with special reference to the salt effects on the heme-heme

interaction.

MATERIALS AND METHODS

Preparation of hemoglobin solution. Ten ml. of fresh human blood was obtained by

venipuncture and poured into a glass centrifuge tube in which a sufficient amount of

potassium ammonium oxalate had been placed. After the separation of the plasma

by centrifugation, the red cells were washed 5 times with 0.9% saline. Finally the

packed erythrocytes were lysed with the equal volume of deionized water and the

hemolysate was left 2 hours at 2•Ž, then 0.5 volume of toluol was added and vigorously

shaken. The mixture was left further 5 hours in the cold and then the stroma and

toluol were spun off to discard. Next, the hemoglobin solution in Visking cellulose

casing (6.4 mm diameter) was exhaustively dialysed against frequent changes of

deionized water, the total amount being as much as 10 liters, under perpetual stirring

for 48 hours at 2•Ž. The material was then submitted to the high speed centrifuga-

tion (35,000 r. p. m., 30 minutes, 2•Ž) by a Spinco model L preparative ultracentrifuge

to secure the complete removal of residual stroma.

The final hemoglobin solution thus obtained was stored at 0 to 2•Ž and used for

the experiments within 5 days after the preparation.

Construction of oxygen equilibrium curve. Just before the use, the stock hemoglobinsolution was mixed with any concentrated salt solution and deionized water to attainany desired ionic strength and protein concentration. The mixture was then centri-fuged at 10,000 r. p.m. for 20 minutes and the clear supernatant was served for theexperiment. The final concentration of hemoglobin was usually 10% or so.

A spectrophotometric procedure (ENOKI, 1959b) was used for the construction of

hemoglobin-oxygen equilibrium curves. Oxy-hemoglobin solution was first deoxy-

genated in a cell-fused tonometer by repeating evacuations-nitrogen flushings and

temperature equilibrations (19. 5•Ž). After the complete deoxygenation, varied amount

of air was injected into the tonometer successively by a calibrated syringe furnished

with a finest hypodermic needle stuck into the syringe nozzle, and the tonometer

was rotated in a thermostatted water bath (19.5•Ž) to establish the temperature and

oxygen equilibration.

The oxygen pressure with which the hemoglobin solution is in equilibrium canbe calculated from the amount of air injected, room temperature, relative humidity,equilibrium temperature, barometric pressure, volume of gas phase in the tonometer,and a correction for the oxygen combined with hemoglobin.

Percentage oxygenation was computed from the optical density change upon the

oxygenation at one and fixed wave length, usually 760 mƒÊ, where the optical density

decreases as the oxygenation proceeds.

In a rough graphical estimation of the individual oxygen association constants,

K1 and r,4, special cares were taken for the determinations of oxygen equilibrium in

the lowest and highest regions of oxygen saturation.

Analysis of the oxygen equilibrium data. The data are analysed in terms of HILL'Sempirical equation (HILL, 1910),

282 Y. ENOKI AND I. TYUMA

(1)

where y and p denote the percentage oxygenation and the equilibrium oxygen pres-sure, respectively, and "n" and "K" are constants. The magnitude of heme-hemeinteraction is arbitrarily expressed by the value of "n" as obtained from the slopeof log {y/(100-y)} versus log p plot, for the above equation can be transformed to;

(2)

The oxygen affinity is represented by the constant K or more commonly and con-

veniently by, p50, the oxygen tension at which 50% oxygenation of hemoglobin present

occurs, for at P50 the equation (1) gives;

(3)

Measurement of ultraviolet absorption changes of hemoglobin upon the oxygenation. Thesame procedures as in the construction of oxygen equilibrium curve was used exceptthat the usual glass-cell fused tonometer was replaced by quartz-cell fused one inwhich the changes in the ultraviolet absorption spectrum could be determined inParallel with the trend of oxygenation.

After each experiment, hemoglobin sample used in oxygen equilibrium or ultra-violet absorption studies was taken out from the tonometer and the total concentra-tion was determined by a cyanmethemoglobin method. The samples were also checkedfor the absence of methemoglobin by MICHEL'S procedure (MICHEL et al., 1940) andof the denaturation by virtue of the decreased solubility in half-saturated ammoniumsulfate solution. The experiments were discarded in rare cases in which the presenceof oxidized or denatured hemoglobin was detected.

Alkali denaturation. Bovine hemoglobin, prepared from the fresh blood supplied by

a slaughterhouse, was chosen as the experimental sample for its high alkali resistance

to enable us to pursue the denaturation time course accurately for longer period.

Alkali denaturation kinetics of the deoxy-hemoglobin was determined in a Thunberg's

anaerobic tube fused with glass-cell. Four ml of oxyhemoglobin in 0.2 M sodium

chloride in the cell and 1 ml of 2.0 N sodium hydroxide in the side arm were com-

pletely deoxygenated by the repeated evacuations-nitrogen flushings and immersionin a water bath (38•Ž), and after cooling to room temperature they were mixed

thoroughly by inverting the anaerobic tube. Thereafter, the denaturation process

was followed by reading the absorbance change at 580 mƒÊ (Er) with appropriate time

intervals for 30 minutes. Next, the anaerobic tube was immersed in a water both

(38•Ž) for 20 minutes and after cooling to room temperature the absorbance reading

was made again at the same wave length (Ee). The control experiment was con-

ducted with the same procedure except that the sodium hydroxide in the side arm

was replaced by deionized water and the resultant reading at 580 mƒÊ was taken as

(Eb) after correcting for the hemoglobin concentration in the denaturation experi-ment. The hemoglobin concentrations were determined by the iron assays (THORP,1941). Thus, the percentage of still undenatured hemoglobin at a certain time is

given by;

(4)

Alkali denaturation behavior of oxygenated hemoglobin was determined as fol-

lows: Oxy-hemoglobin and sodium hydroxide or deionized water in the anaerobic


tube as referred to above was first deoxygenated and then re -oxygenated by air let -

ting in. Thereafter, the above denaturation or control procedure was adopted thoughth

e absorbance reading was made at 576 mƒÊ .

In these experiments , commercially available nitrogen gas was pre-purified bybubbling through a chromous chloride solution to serve for the deoxygenation ofoxy-hemoglobin (STONE and SKAVINSKI 1945) .

In all the above experiments , a Shimadzu photoelectric spectrophotometer typeQR-50 was used for spectrophotometric measurements .

RESULTS

Heme-heme interaction and various environmental salts . FIG. 1 shows theeffect of sodium chloride of various strengths on the oxygen equilibrium

curve. It is noticed that dialyzed hemoglobin exhibits a nearly hyperbolic

curve of the highest oxygen affinity and the increase of the salt strength

produces an increase in the sigmoid character and a decrease in the oxygenaffinity. These findings can be more clearly and quantitatively expressed inFIG. 2, in which the slope of each straight line drawn around the mid part

of each curve represents so-called HILL'S constant "n" , an empirical indexof the magnitude of heme-heme interaction .

The effect of various neutral salts on the constant "n" is summarizedin FIG. 3. One can obviously notice that hemoglobin shows little heme -hemeinteraction in salt-free medium as expressed by the "n" values of nearly

unity (1.0 to 1.2) and the increase in salt media . In the latter circumstance ,it is further noticed that the magnitude of the interaction is not only depen -dent on the ionic strength, but also on the nature of salt , especially thecharge of the constituent cation .

All these results confirm our previous observations on the salt effects

FIG. 1. Oxygen equilibrium curves of human adult hemoglobin.

in NaCl solution of various ionic strengths . Percentage oxyhemo-

globin (y) is plotted against oxygen tension in mmHg (p) . Un-

buffered hemoglobin; 1.5•~10-3M, temperature; 19.5•Ž. Ionic

strength of the environmental salt is increased from left to right.


FIG 2. Oxygen equilibrium curves of human adult hemoglobin

in NaCl solution of various ionic strengths.

log y/ 100 - y

vs. log p plot from the FIG. 1.

FIG. 3. Dependence of the heme-heme

interaction upon the species and ionic strength

of the environmental salt. Unbuffered hemo-

globin; 1.5•~10-3M, temperature; 19.5•Ž.

Ordinate; "n" constant in HILL'S equation.

Abscissa; square root of ionic strength (ƒÊ).

○-○;NaCl,●-●;KCl,△-△;K2SO4,

□-□;MgCl2,■-■;CaCl2,▲-▲;MgSO4,

x-x; KH2PO4-K2HPO4 (pH 6.9)

(EN0KI, 1959b), although there are some quantitative differences which maybe attributed to the incomplete dialysis in the former experiments. In all

the above studies, though the buffer salts are not added, the influence of pH

alteration due to Haldane effect is ignored in the light of ALTSCHUL et al's

work which demonstrates it as trivial (ALTSCHUL et al. 1939). The circum-

stance is further validated by the present observations on the salt effects of


FIG. 4. Salt effect exhibited by

KH2PO4-K2HPO4 (pH 6.9) buffer upon the

human adult hemoglobin-oxygen equili-

brium. Hemoglobin; 1.5•~10-3M, tem-

perature; 19.5•Ž. •›-•›; in no salt,

•œ-•œ; in the phosphate buffer: from

left to right, ionic strength of 0.04, 0.067,

0.2, and 0.4.

phosphate buffer salt.

The results shown in FIG. 4 where the ionic strength of phosphate buffersalt (pH 6.9) is varied indicate the same salt effects as observed in the neutral

salts. The magnitude of the effect on the heme-heme interaction is almost

similar to that of the divalent cation salts (FIG. 3). In this figure, percentageof oxygenation (y) is plotted against logarithm of oxygen tension (log p) as

frequently done. In such a plot, alteration in the shape of the equilibrium

curves can be more clearly depicted than the usual y vs . p plot. The effectof phosphate and neutral salt is mutually additive .

Graphic estimation of the association constants , k1, and k4 of oxygen equilibrium.In all the studies above referred to , the salt effects on the heme-heme interac-tion are figured in terms of HILL's constant "n" which has been convention-ally used in this field of research . The constant, however, is of empiricaland arbitrary nature and for better understanding of this problem it is more

desirable to analyse the oxygen equilibrium data with any other parameter

of clearer physico-chemical meanings.

In this connection, SCATCHARD-EDSALL'S way of argumentation (EDSALL

and WYMAN, 1957) of "ligand binding by an equivalent set of groups withinteractions between them" appears to be somehow promising . As is wellknown, one molecule of hemoglobin bears four binding sites , namely fourhemes, for ligands such as oxygen , carbon monoxide etc.. Now, if hemoglobin,as it be assumed, binds the ligands , for example oxygen, in the four succes-sive steps, the successive association constants , K1, K2, K3, and K4, can bewritten as follows;

(5)

These constants are corrected for the statistical factor to give the intrinsic

microscopic association constants , K1, K2, K3, and K4;


(6)

Then, the average number of oxygen molecule bound per molecule hemoglobin,

can be described in the following way under the oxygen tension p which

is conventionally assumed to be identical with the activity of oxygen;

(7)

Here, we define a function, Q, by;

(8)

From equations (7) and (8), the relation (9) can be derived;

(9)

In the case of no interaction between the four binding sites where the sitesare all equivalent and independent, i. e. k1= k2= k3= k4, a plot of Q (or log Q)

against 5 will give a horizontal straight line. On the other hand, when the

sites interact each other in either positive (K1<K2<K3<K4) or negative (K1>K2

>K3>K4) manner, the plot will give a curve with either positive or negativeslope. As is easily understood from equation (9), the limiting value of Q as

p and hence 5 approaches zero is given by the relation,

(9')

On the other hand, as p becomes infinite and 5 approaches 4, i. e. 100% oxy-

genation, Q assumes the limiting form,

(9")

A set of carefully determined oxygen equilibrium data is depicted in log

Q versus 5 plot (FIG. 5). The upper two curves represent the results in -salt-

free medium and the lower two are those in sodium chloride (ƒÊ=1.00). In

each of these two pairs, the upper curve is the data at 13•Ž and the lower

is that at 23•Ž. The two different symbols, solid and open, constituting each

curve refer to the two experiments on the same sample under the same con-

dition but on different days. The reproducibility of the data is well satisfied.

As evidently noticed, a strong positive interaction between the four hemes

is manifested in salt medium, whereas a much weaker positive interaction is

shown in salt-free medium. In the salt-free curves, however the substantial

nature cannot be understood, it is further noted a transient downward infle-

xion around 5=3, which contrasts with a steady and progressive course in

the salt medium. The first and fourth intrinsic association constants, Ki and

K4, are obtained from the extrapolated intercepts of the curves at the left

and right ordinates, respectively, and the values are summarized in TABLE 1,


FIG. 5. Scatchard plot for the oxygen equilibrium data of

human adult hemoglobin at two different temperatures and in

salt and no salt environment . See text for the explanation .

TABLE 1.

Graphically estimated intrinsic oxygen association constants, K1, and K4,

of human adult hemoglobin in relation to the usual oxygen equilibrium para-

meters, n and p50.

together with the usual oxygen equilibrium constants , n, and p50. The tableshows a parallel trend of "n" values and K 4/K1 ratios. The less (or more)the "n" value is, the less (or more) the ratio . In salt-free medium , K4, is only20

times as great as K1, whereas the ratio is 200 or so in the presence of 1 Msodium chloride.

Ultraviolet spectral changes on the oxygenation of hemoglobin . As regards theevolution of the characteristic oxygen equilibrium function of hemoglobin, ahypothesis has been advanced which lays a special emphasis on a conforma-


tional change of the protein moiety upon the oxygenation (WYMAN and

ALLEN, 1951, St. GEORGE and PAULING, 1951). The salt effects above stated

may be interpreted along this line and the measurement of changes in the

ultraviolet absorption upon the oxygenation may provide some experimental

criterion in this connection.

FIG. 6. Changes of ultraviolet absorption (E) of human adult

hemoglobin upon the oxygen or carbon monoxide coupling. Hemo-

globin; 2.6•~10-5M, medium; 0.1M phosphate buffer (pH 6.9), tem-

perature; 20.0•Ž. •œ-•œ; deoxyhemoglobin, •›-•›; oxyhemo-

globin, •¢-•¢; carboxyhemoglobin.

FIG. 6 represents the ultraviolet spectral changes on the attachment of

oxygen or carbon monoxide to hemoglobin in a phosphate buffer solution (0.1

M, pH 6.9). It is evident that the oxygenation and carbonmonoxygenation

are both accompanied with an enormous hyperchromicity in the medium

ultraviolet region (250 to 300 mƒÊ) and a concomitant slight shift of the absorp-

tion peak towards the shorter wave length. These changes are demonstrated

to be completely reversible and so it cannot be considered to originate from

an irreversible alteration, e. g. denaturation, of the protein.

Similar changes, at least qualitatively, can be observed in salt-free medium

as well as in the salt solution.

In order to clarify the nature of the above spectral changes, the difference

spectra are constructed between oxy- or carboxy- and deoxy-hemoglobin (FIG.

7). Below 300 mƒÊ, three difference peaks appear around 238, 275, and 290 mƒÊ

in oxy-versus deoxy-hemoglobin subtraction. The latter two are also observed

in carboxy- versus deoxy-hemoglobin difference spectrum.


FIG. 7. Ultraviolet difference spectra of human adult

oxy- or carboxy- vs. deoxy-hemoglobin . Hemoglobin;

2.6•~10-5M, medium; 0.1M phosphate buffer (pH 6.9), tem-

perature; 20.0•Ž. •œ-•œ; carboxy- vs. deoxy-hemoglobin,•›-•›; oxy- vs . deoxy-hemoglobin.

The interpretation of the above results is rather complicated since, accord-

ing to BEAVEN'S statement (BEAVEN et al . 1960), the heme moiety contributes

to the absorption spectrum of hemoglobin all over visible and ultraviolet

region and even at 290 mƒÊ its contribution is estimated as 66% of the total

absorbance. The positions of the difference peaks now observed, however,

coincide incidentally with those which have been usually assigned to aromatic

amino acid residues, phenylalanine , tyrosine, and tryptophan, in protein dif-

ference spectra. Thus, the above result may be interpreted as that the bind -

ing of ligands with the heme groups on hemoglobin molecule induces an

altered electronic state of the aromatic amino acid residue(s) in the protein

moiety in any way.

FIG. 8 and 9 depict a parallel trend of the heme-oxygen coupling and the

ultraviolet spectral changes of hemoglobin . FIG. 8 shows that the ultraviolet

absorption increases proportionately as the oxygenation does so. In FIG. 9,

it is shown that the percentage spectral changes at three different ultraviolet

wave lengths (264, 275 and 290 mƒÊ) are fairly comparable with the percentage

oxygenation computed from the absorption change at 370 mƒÊ where no con-

tribution of the globin part has been ascertained .


FIG. 8. The ultraviolet difference spectra (oxy vs. deoxy) in

varied degrees of oxygenation of human adult hemoglobin. Hemo-

globin; 3•~10-5M, medium; 0.1M phosphate buffer (pH 6.9), tem-

perature; 20.0•Ž. Percentage oxygenation; •œ-•œ: 16, • -• : 53,

•¢-•¢: 75, •›-•›: 100.

FIG. 9. Linear correlation between the

percentage oxygenation and percentage

ultraviolet spectral change of human adult

hemoglobin. Hemoglobin; 3•~10-5M, medi-

um; 0.1M phosphate buffer (pH 6.9), tem-

perature; 20.0•Ž. The % oxygenation is

computed from the absorption change at

370 mƒÊ.

Alkali denaturation behavior of oxy- and deoxy-hemoglobin. As is well known,alkali denaturation technique has been widely used to characterize humanadult and fetal hemoglobin. Although the exact nature of the reaction isnot yet fully understood, some structural difference in their protein part seemsto be responsible for their difference in the susceptibility to alkali. In thismeaning, the technique is adopted to detect any difference between bovine


FIG. 10. Marked difference in the alkali resistance

between bovine oxy- and deoxy-hemoglobin. Temperature;

21.0•‹C, final NaOH concentration; O. 57N. •œ-•œ; deoxy-

hemoglobin, •›-•› oxy-hemoglobin.

oxy- and deoxy-hemoglobin (FIG. 10). Semi-logarithmic plot of the denatura-

tion time course reveals a slight departure from the one component-first order

reaction, probably owing to heterogeneous character of the hemoglobin in

this species. Furthermore, it is clearly shown that the oxygenation induces

a considerably large alteration in the denaturation kinetics of the hemoglobin,

the half decay time being about 110 seconds in the deoxy- and 640 seconds

in the oxy-hemoglobin, respectively. The finding that oxy-hemoglobin bears

a higher resistance to alkali conforms to the result by HAUROWITZ, et al. at

2•‹C (HAUROWITZ et al. 1954).

Along the same line, a study is attempted to know whether or not the

oxygenation may modify the reactivity of hemoglobin towards p-chloromer-

curibenzoate. The coupling of them, as measured by absorbance change at

250mƒÊ, however, appears to be instantaneous in both oxy- and deoxy-hemo-

globin and no difference of their reactivity can be demonstrated.


DISCUSSION

The functional features of hemoglobin in its oxygen binding have been

best understood by constructing the oxygen equilibrium (dissociation) curve

and analysing it in terms of the three functional parameters heme-heme

interaction, BOHR effect, and oxygen affinity. The first of them, heme-heme

interaction, which is manifested in a sigmoid character of the oxygen equili-

brium curve, stimulated a great interest of many physiologists even in the

early stage of hemoglobin research. As early as 1909, BARCROFT and ROBERTS

showed that after three days' dialysis, the oxygen dissociation curve of

undialyzed dog hemoglobin lost its sigmoid character to become a nearly

rectangular hyperbolic curve with higher oxygen affinity and suggested that

the sigmoid character might be independent of inherent property of hemo-

globin molecule itself but be dependent on the electrolyte environment sur-

rounding the protein molecule (BARCROFT and ROBERTS, 1909). In this excel-

lent classical work, however, some ambiguities have been left in whether or

not the electrolyte environment is the only factor influencing on the equili-

brium characteristics. Any other corpuscular dialyzable consituent(s) may

participate in this phenomenon. A danger of denaturation or inactivation of

hemoglobin (met-hemoglobin formation), the latter of which was later shown

to make the curve hyperbolic (DARLING and ROUGHTON, 1942), is further

suspected in view of their rather drastic procedure, e. g. warming the protein

sample at 40•‹C for long period, in preparing the hemoglobin specimen.

These problems have long been left without any further elaborate investi-

gation except a few whose main concern was about the salt effect on the

oxygen affinity of hemoglobin (SIDWELL et al. 1938 TAKASHIMA, 1955) and

BARCROFT'S classical results have been commonly described in most of the

textbook of physiology as implying "salt effects " on the oxygen equilibrium

curve.

One of the present authors reported in the previous paper that by two

days' dialysis the oxygen equilibrium curve of concentrated (10%) human

hemoglobin was rendered hyperbolic without any sign of the denaturation or

met-hemoglobin formation and could be restored to normal sigmoid one by

adding any neutral salt, of which not only the strength but also the species

appeared to have a bearing with the magnitude of heme-heme interaction

evolved (ENOKI, 1959b). It was further recognized in that paper that the

oxygen affinity was much enhanced after the removal of salt and reduced by

the addition of salt. These results can be considered to clearly substantiate

the so-called "salt effects " on hemoglobin oxygen equilibrium and first esta-

blish the quantitative relationship between the magnitude of heme-heme

interaction and the strength and nature of several environmental neutral salts

(NaCl, KCI, MgCl2, CaC12, K2SO4, MgSO4). The related experiments with


almost similar results were presented a little later by ROSSI-FANELLI, and

collaborators (Rossi-FANELLI et al. 1961). One discrepancy, however, can be

pointed out in that their result shows the magnitude of heme-heme interac-

tion is only determined by the ionic strength but not by the species of environ-

mental salt.

In the present re-investigation, special attentions are paid to secure the

complete removal of salts as far as possible by using Visking cellulose casing

in stead of cellophane bag in the previous study and stirring continuously

the external water with a magnetic stirrer, and so on. The results ob-

tained under these precautions, however, evidently confirm the previous

observations of the salt effects on the heme-heme interaction. Furthermore,

newly included data of phosphate buffer salt (FIGS. 4) allow us securely to

exclude a less probable possibility that the observed salt effects may be

originated from a salt-induced pH change of the isoionic protein solution.

In all the above experiments, the magnitude of heme-heme interaction.

has been evaluated by virtue of HILL'S constant "n" as is generally and con

ventionally used in this research field. The physico-chemical meaning of this

constant, however, has not yet been fully analysed and it is rather an arbi-

trary and empirical parameter. An attempt, therefore, has been made to

analyse the salt-induced evolution of heme-heme interaction in terms of the

successive oxygen association constants of individual hemes of homoglobin

molecule after SCATCHARD-EDSALL's way (EDSALL and WYMAN, 1958). The

argumentation along this line will enable us to understand the heme-heme

interaction on a sounder basis of clearer physico-chemical meaning.

As shown in FIG. 5 and TABLE 1, the association constant for the fourth .

and last step of oxygen binding by the hemes of hemoglobin molecule is

about 200 times as great as that for the first step in 1 M sodium chloride,

whereas this enhancement of the fourth association constant is only 20 fold

in salt free medium. It is further noticed that the mode of enhancement is

steady and progressive in the former case and somewhat unsteady as shown

by the downward inflection around ƒÒ=3 in the latter. The results in salt

medium conform to the more elaborate and accurate results by ROUGHTON

and his collaboraters (ROUGHTON, et al. 1955). The enhancement ratio, K4/K1

now determined in 1 M sodium chloride (TABLE 1), roughly agrees with that

obtained by them on adult sheep hemoglobin in 0.2 M borate (=283). These

results, although some reservations must be retained for the accuracy of the

association constants thus obtained (EDSALL and WYMAN, 1957), may imply

that in salt medium, the successive bindings of oxygen to the hemes of one

hemoglobin molecule enhances greatly, steadily, and progressively the affinity

of the unbound hemes for oxygen. Futhermore, the findings in salt-free

evironment reveal that the enhancement suffers some disturbance in this con-

dition.


In view of the current idea that the heme-heme interaction may originate

from a configurational change of the protein portion concomitant with the

oxygenation (WYMAN and ALLEN, 1951 ST. GEORGE and PAULING, 1951), the

above results can be interpreted as that the presence of salt appears to favor

such a conformation change as responsible for the evolution of heme-heme

interaction, possibly through the appropriate interaction with charged groupson the protein molecule.

Although a mass of evidence has been accumulated for the real existence

of such a conformation change (BENESCH and BENESCH, 1963), the exact natureof the structural alteration is not yet clearly known and in this respect our

previous experiment of the effect of strong urea on the hemoglobin oxygenequilibrium seems to be very implicative (ENOKI, 1959b). The result shows

that the magnitude of heme-heme interaction is only dependent on the strength

of any salt present, sodium chloride in that case, but independent of the

presence of 4M urea. On the contrary, ROSSI-FANELLI and colleagues inferthat strong urea induces a marked reduction of the heme-heme interactionand a remarkable increase of the oxygen affinity without any sign of altera-

tion of the visible spectrum and oxygen capacity though much more dilutehemoglobin is used in their experiments than in ours and also that these

changes are at least partially reversed to normal upon the removal of urea

by dialysis (ROSSI-FANELLI et al. 1959). As far as concerns with our results,

the sole effect of strong urea is to greatly enhance the oxygen affinity andin all cases where any reduction of the heme-heme interaction is observed,

the concomitant denaturation and oxidation of the protein is shown. These

changes of hemoglobin are particularly conspicuous in the presence of diva-lent cations such as Mg and Ca. Then, the implication of the facts maybe that the determinative role in the conformation change essential for the

heme-heme interaction be played by not hydrogen bonds but electrostatic

forces. Such a way of argument is compatible with the recently accumulat-

ing data which suggest that the heme-heme interaction may have an intimate

bearing with the interaction, probably electrostatic in nature, between the

four polypeptide subunits of the protein. A similar circumstance is also sub-

stantiated in PERLMAN'S studies on pepsin which conclude a relatively unim-

portant role of hydrogen bonds in maintaining the configuration of the pro-tein essential for its enzymic action (PERLMAN, 1961). At this instance, itmay be interesting to cite our recent result concerning the effect of some

organic and inorganic mercurials on the hybrids formation of heterologous

hemoglobins (ENOKI et al. unpublished). The result shows that the hybrids

are formed between adult human and canine hemoglobin in the presence of

more than five equivalents of mercuric chloride at neutral pH and sug-

gests a probable intimate bearing of the sulfhydryl groups, especially so-called unreactive ones, with the interaction between the subunit polypeptide


chains.

Although ultraviolet absorption spectra are widely taken as a criterion

of the structural changes of any protein , it has been rarely used in the struc-tural study of hemoglobin. The reason is that the interpretation of the results

may be rather complicated by a spectral contribution of the heme moiety

and the accurate extent of the contribution can hardly be estimated . Severalyears ago, some Japanese workers worked out extensive studies of the ultra-violet alterations which arose on the attachment of various ligand moleculesto the hemes in hemoglobin and concluded that the ligand binding including

oxygenation induced a conformational change of the protein molecule (NAKA-NISHI et al. 1955). Their conclusion as for the oxygen binding , however,bases on the fact that the reduction of oxy-hemoglobin with Rongalit accom -panies a considerable change in the ultraviolet absorption spectrum . In ourpresent absorption studies, the deoxygenation is carried out by a milder

physical procedure, i. e. repeated evacuations and nitrogen-flushings , ratherthan the use of the powerful reducing agent which may cause the ultraviolet

changes by itself. The ultraviolet changes are further examined by deriving

the difference spectrum which makes possible to detect a finest alteration inthe absorption. As is well known , the absorption spectra of proteins inmedium ultraviolet region are composed of the chromophoric contribution by

the constituent aromatic amino acid residues , namely phenylalanine, trypto-phan, and tyrosine. WETLAUFER and collaborators attempt a comparison ofthe difference spectra due to pH change in O-methyltyrosine and glycy1 -O-methyltyrosine and suggest that a relatively minor change in the configura -tion of protein may be sufficient to cause a larger alteration in the difference

spectra (WETLAUFER et al . 1958). As discussed recently by BENESCH andBENESCH, (BENESCH and BENESCH, 1963), there is little doubt that some enor-mous modification of the protein structure occurs on the oxygenation of hemo -globin. The ultraviolet changes above observed, therefore , can be interpretedas due to the alteration of the internal structure of the protein which induces

,in turn, an alteration of chromophoric properties of the constituent aromatic

amino acid residues. In this connection , however, further detailed investiga-tions are required to exclude the possible contribution by the heme which is

the primary site of change, i. e. oxygen binding , and the moiety responsiblefor the spectral alteration in visible region upon the oxygenation . Further-more, the linear correlation of the change in the ultraviolet absorption withthat in the oxygenation (FIG. 9) may deserve a further discussion , for thedielectric properties (TAKASHIMA and LUMRY, 1958) and the viscosity (MATsu-MIYA and LUMRY, 1961) of hemoglobin exhibit a characteristic non -linearalteration upon the oxygenation. The result does not agree also with our

own recent observation that the extent of the hybridization between adult

human and canine hemoglobin increases in a non-linear manner as their oxy -


genation proceeds (ENom et al. unpublished). In this case, the extent of the

hybridization may be considered to be a rather immediate criterion of the

strength of the interaction or linkage between the polypeptide subunits of

hemoglobin.

In their researches on denaturation of hemoglobins by alkali, HAUROWITZ

and his colleagues notice that deoxy-hemoglobins differ from oxy-hemoglobins

by the lower values for ƒ¢H=, and by more negative value for ƒ¢S= which are

calculated from the alkali denaturation kinetics and ascribe the difference to

configurational changes in the globin portion which accompany the oxygena-

tion of hemoglobin (HAuRowfrz et al. 1954). In their experiments with

deoxy-hemoglobin, however, the deoxygenation is made with sodium dithionite

which may be suspected of its denaturing action on the protein. Our present

results are evidently free from the suspicion, for the drastic chemical deoxy-

genation they used is replaced by milder physical one. The result (at 21•Ž)

shows that the oxygenation increases the resistance to alkali denaturation of

bovine hemoglobin (FIG. 10), which agrees with HAUROWITZ et al's result

with beef hemoglobin at 2•Ž but does not accord with their results at 28°C

where deoxy-hemoglobin shows higher resistance to alkali. The difference

between beef deoxy- and oxy-hemoglobin we observed at 21•Ž is fairly

reproducible and can also be verified with rabbit hemoglobin. Although

substantial nature of the alkali denaturation has been yet obscure in spite

of the extensive studies of previous investigaters, the above stated difference

between oxy- and deoxy-form may be reasonably accounted for as reflecting

a conformational difference between them.

SUMMARY

1. The dependence of the heme-heme interaction of human adult hemo-

globin on the species and strength of the evironmental salts was studied. In

a series of several neutral salt solution, the magnitude of the interaction was

dependent not only upon the ionic strength but also upon the species of the

environmental salt. In the latter of the two factors, the charge of the con-

stituent cation appears to have a primary importance. All these results

confirm our previous observations.

2. Similar salt effects were also verified in potassium phosphate buffer

(pH 6.9). The magnitude of the effect was roughly comparable to that of the

divalent cation neutral salts.

3. An attempt was made to graphically estimate the intrinsic constants

of the first asd fourth oxygen association (k1 and k4) by human adult hemo-

globin. The k4 value was 170 to 250 times as great as the Ki in sodium

chloride (ƒÊ=1.0), whereas the enhancement was only 14 to 25 fold in no salt


medium.

4. Oxygenation of hemoglobin was accompanied with a hyperchromicity

and a slight blue shift in the medium ultraviolet range. These changes were

completely reversible. The derived ultraviolet difference spectrum (oxy vs.

deoxy subtraction) showed three difference peaks around 238, 275 , and 290 mƒÊ.

A linear correlation was shown between the degree of oxygenation and the

magnitude of the ultraviolet spectral change.

5. Deoxygenation of bovine oxy-hemoglobin induced a remarkable reduction

in the alkali resistance.

6. Discussions were made of a probable correlation of the alterations in

the ultraviolet absorption and the alkali denaturation with a configurational

change of hemoglobin molecule upon the oxygenation.

This research has been made possible through the support and sponsorship of the U.S. Department of Army, through its Far East Research Office (Contract No . DA-92-557-FEC-36081) to which author's thanks are due.

REFERENCES

ALTSCHUL, A. M. AND HOGNESS, T. R. J. Biol. Chem. 129: 315, 1939. BARCROFT, J. AND ROBERTS, Ff. J. Physiol. 39: 143, 1909. BEAVEN, G. H., ELLIS, M. J. AND WHITE, J. C. Brit. J. Haematol. 6: 1, 1960. BENESCH, R. AND BENESCH, R. E. J. Mol. Biol. 6: 498, 1963. DARLING, R. C. AND ROUGHTON, F. J. W. Am. J. Physiol. 137: 56, 1942. EDSALL, J. T. AND WYMAN, J. Jr. Biophysical Chemistry Vol.1., Academic Press , New

York, p.626, 1958. ENOKI, Y. Nippon Seirigaku Zasshi 21: 1013, 1959a. ENOKI, Y. J. Nara Med. Assoc. 10: 345, 1959b. HAUROWITZ, F., HARDIN, R. L. AND DICKS, M. J. Phys. Chem. 58: 103, 1954. HILL, A. V. J. Physiol. 40: iv, 1910. MICHEL, H. O., HARRIS, J. S. AND DURHAM, N. C. J. Lab. & Clin. Med. 25: 445, 1940. MONOD, J., CHANGEUX, J. P. AND JACOB, F. J. Mol. Biol. 6: 306, 1963. MATSUMIYA, H. AND LUMRY, R. cited in Lumry, R. Seibutsu Butsuri 3: 1, 1961. NAKANISHI, S., NAKAJIMA, T., KAIINAGA, T. AND ARAYA, S. Bull. Tokyo Med. Dent.

Univ. 2: 123, 1955. PERLMAN, G. E. Biological structure and function, Goodwin, T. W. and Lindberg, O .

(ed.) , Academic Press, New York, p.59, 1961. ROSSI-FANELLI, A., ANTONINI, E. AND CAPUTO, A. J. Biol. Chem. 236: 397, 1961. ROSSI-FANELLI, A., ANTONINI, E. AND CAP UTO, A. Arch. Biochem. Biophys. 85: 540,

1959. ROUGHTON, F. J. W., OTIS, A. B. AND LYSTER, R. L. J. Proc. Roy. Soc. London. B144:

29, 1955. SIDWELL, A. E. Jr., MUNCH, R. H., BARRON, E. S. G. AND HOGNESS, T. R. J. Biol. Chem.

123: 335, 1938. St. GEORGE, R. C. C. AND PAULING, L. Science 114: 629, 1951. STONE, H. W. AND SKAVINSKI, E. H. Indust. Eng. Chem. (Anal . Ed.) 17: 495, 1945. TAKASHIMA, S. J. Am. Chem. Soc. 77: 6173, 1955.


TAKASHIMA, S. AND LUMRY' R. I. Am. Chem. Soc. 80: 4238, 1958.

THORP, R. H. Biochem. J. 35: 672, 1941.

WETLAUFER, D. B., EDSALL, J. T. AND HOLLINGSWORTH, B. R. J. Biol. Chem. 233: 1421,

1958.

WYMAN, J. Jr. AND ALLEN, D. W. J. Polymer Sci. 7: 499, 1951.

FURTHER STUDIES ON HEMOGLOBIN- OXYGEN EQUILIBRIUM

Documents

Transcript of FURTHER STUDIES ON HEMOGLOBIN- OXYGEN EQUILIBRIUM