HEMOGLOBIN, ANHYDROHEMOGLOBIN,

AND OXYHEMOGLOBIN*
BY FELIX HAUROWITZ
(From the Department of Chemistry, Indiana University, Bloomington, Indiana)

(Received for publication, January 15, 1951)

When wet layers of reduced hemoglobin are exposed to low water vapor
pressures, the broad absorption band in the visible range of the spectrum
is replaced by two narrow bands of a typical hemochromogen spectrum
(l-3). If the water vapor pressure is raised again, the hemoglobin spec-
trum reappears. The absorption spectrum of oxyhemoglobin, in contrast

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to that of hemoglobin, does not undergo any change when the pressure is
lowered (2, 3). From these observations it was concluded that (a) hemo-
globin is an aquo compound, HbeH20, in which 1 molecule of water re-
places the oxygen molecule of oxyhemoglobin, Hb.02, that (b) hemoglobin
at low water vapor pressures loses the iron-bound water molecule and is
converted into anhydrohemogbbin, Hb, and that (c) the equilibrium be-
tween hemoglobin and oxyhemoglobin in an aqueous solution is repre-
sented by reaction (I)
(1) Hb.02 + Hz0 G= Hb-Hz0 + 0s
and not by the classical reaction (II).
(11) Hb-0 rtiHb+Oz
While the kinetic and thermodynamic constants of this reaction have been
determined by Hartridge and Roughton (4) and by Roughton et al. (5),
the purpose of the present investigation has been to examine the com-
bination of anhydrohemoglobin with oxygen and water, and to clarify
the r8le of the simplerreactions (II), Hb-O* --) Hb + 02, (III), Hb + Hz0
-+ Hb.H20, in the over-all reaction (I).

EXPERIMEN!I’AL

Spectrophotometry of Anhydrohemoglobed beef erythrocytes

were hemolysed by dialysis against water at 0”. The stroma was ad-
sorbed to alumina gel and removed by centrifugation. The supernatant
solution was concentrated in vacua at room temperature over KOH; its pH
was 7.1, its oxyhemoglobin content 28 per cent. The 8 mm. X 75 mm.
* This investigation was supported in part by a research grant from the National
Institutes of Health, United States Public Health Service. A preliminary report
was presented before the annual meeting of the American Society of Biological
Chemists at Atlantic City, April, 1950 (1).
443
444 HEMOQLOBIN

surface of a rectangular glass slide, G (Fig. l), was coated with 0.025 ml.
of the oxyhemoglobin solution and was placed into the horizontally fixed
tube T. The small tube C, containing dry calcium chloride and glass
wool, was then introduced into T. An adapter, A, was connected with
T by a rubber tube R. The tube T was evacuated by a Hyvac pump at-

-.-V

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-0

-i

-2

-3

-4
*s
-6car

FIG. 1. Tube for the measurement of absorption curves of dry layer8 of hemo-
globin derivatives.

tached at V. As soon as the bright red color of oxyhemoglobin was re-

placed by the purplish color of hemoglobin, the stop-cock S was closed.
In the evacuated tube, water slowly distilled at room temperature from
the hemoglobin layer, Hb, into C. After several minutes the purplish
color of the hemoglobin layer changed to an intense pink; simultaneously
the broad absorption band of hemoglobin was replaced by the two narrow
bands of anhydrohemoglobin. The evacuated tube was next detached
from the vacuum tubing at V and was placed vertically into the cell holder
of a Beckman spectrophotometer, so that the plane of G was perpendicular
 F. HAUROWITZ 445

to the direction of the light beam B. The absorption values found were
corrected by deducting those of T and G determined in a blank experi-
ment. After the spectrophotometric measurement, air was admitted by
opening S, A and C were removed, and the dry protein layer was dissolved
by filling T with 10 ml. of 0.05 per cent sodium carbonate solution. The
absorption spectrum of the oxyhemoglobin solution obtained was meas-
ured after removing G. The solution was then reduced by a trace of solid
sodium dithionite, Nan&Oa, and the absorption spectrum of the reduced
hemoglobin was measured.
The curves (Fig. 2) show that anhydrohemoglobin on addition of water
in the presence of air is converted into oxyhemoglobin and that hemo-

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E= 529
-0.6

FIG. 2. The solid line is the absorption curve of dry anhydrohemoglobin in vacua,
the broken line that of its aqueous solution containing oxyhemoglobin, the dotted
line that of the same solution after the addition of dithionite (hemoglobin spectrum).

globin is formed on the further addition of dithionite. There is no evi-

dence for the presence of denatured hemoglobin (globin hemochromogen)
in the reduced solution. Rat and rabbit hemoglobin gave the same re-
sults as beef hemoglobin.
Spectrophotometry of DTY Oxyhemoglobin at Reduced Pressure--It was
necessary to dry oxyhemoglobin solutions very rapidly in order to prevent
extensive conversion into methemoglobin. The glass slide G (Fig. 1) was
coated with a layer of the concentrated oxyhemoglobin solution on its
lower surface and was placed horizontally over the rim of a Petri dish
containing phosphorus pentoxide. Drying was accelerated by placing the
dish in a vacuum desiccator and lowering the pressureto 80 to 120 mm.
of Hg. ’ Lower pressure was avoided because it led to deoxygenation.
The dried slide was rapidly transferred into the tube T (Fig. l), exposed
446 HEMOQLOBIN

to a high vacuum (p = 0.3 mm. of Hg), and the absorption spectrum

measured. The dry Hb.02 was then dissolved as described above, and a
second reading made.
542

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/--’ 600 550 500mu

FIQ. 3. The aolid lime is the absorption curve of dry oxyhemoglobin in V(ICUO,
the broken line that of its aqueous solution in the presence of air.

FIG. 4. Apparatus for the measurement of transition points in the hemoglobin-

anhydrohemoglobin system.

The curves (Fig. 3) show clearly that the absorption maxima of dry
Hb.02 are similar to those of the oxyhemoglobin solution and quite dif-
ferent from those of anhydrohemoglobin. The maximum at 626 w in-
dicates that a small amount of methemoglobm had been formed in spite
of the precautions observed.
 F. HAUROWITZ 447

Hemoglobin-Anhydrohemoglobin Equilibrium at Diferent Temperatures-

About 0.1 ml. of a 20 per cent oxyhemoglobin solution was placed in
the left part, Hb, of the double U-tube shown in Fig. 4. The right part,
W, contained 0.5 ml. of water. The apparatus was filled with nitrogen
gas through &, and the nitrogen was removed through Sz by evacuation
until the pressure dropped to 0.5 mm. of Hg. The filling with nitrogen
followed by evacuation was repeated ten times. After the last evacuation,
X1 and & were closed while S’s was connected to a McLeod manometer
gage. The left beaker, L, was next filled with water of a desired tempera-
ture, while the right beaker, R, was kept at a lower temperature, so that
water slowly distilled from Hb into W. As soon as the hemoglobin solu-

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Fro. 5. Transition points in the hemoglobin-snhydrohemoglobin system. The
water vapor pressure, p,, is measured in atmospheres. Different symbols are used
for different hemoglobin preparations.

tion became viscous, it was spread over the glass wall of the left U-tube
by gently tapping the tube. A light source was placed behind L and a
spectroscope in front of it. At a definite moment the two narrow bands
of anhydrohemoglobin became visible. This point was considered as the
transition point in the conversion of hemoglobin into anhydrohemoglobin.
The pressure at this point, pw, was measured by the McLeod gage. The
corresponding temperature, T (Fig. 5), was that of the water in L. By
raising or lowering the temperature of the water in R, the water vapor
pressure, pw, could be raised or lowered and the reaction Hb + Hz0 G
Hb.HzO repeatedly shifted to the left or right. Since the two narrow
absorption bands of anhydrohemoglobin are much more visible than the
broad absorption band of hemoglobin, the transition points recorded in
Fig. 5 were determined by measuring the reaction hemoglobin + anhydro-
hemoglobin. Examination of the reverse reaction gave similar, although
less precise, values.
448 HEMOGLOBIN

Hydration of Native and Denatured Oxyhemoglobin-l ml. of a 20 per

cent solution of oxyhemoglobin was placed into a flat weighing bottle and
dried over phosphorus pentoxide at -1 mm. of Hg pressure. The dry
preparation was weighed and placed into another desiccator containing
sulfuric acid; its temperature was kept at 24 f 1”. The water vapor
pressure, pw, in the desiccator was increased by adding water to the sul-
furic acid (6). After exposing the protein to constant water vapor pres-
sure for 24 hours, pw was determined by measuring the density of the
sulfuric acid; at the same time the water content of the protein was deter-

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+ RELATIVE HUMIDITY (7.J
FIG. 6. Water content of native (solid symbol) and heat-treated (open symbol)
oxyhemoglobin at 24” and different relative humidities. The water vapor pres-
sure was first, lowered (upper curve), then raised (lower curve).

mined gravimetrically. Since 24 hours are not sufficient for complete

equilibration, two different curves were obtained when pw was attained
by lowering or raising the pressure, as shown by the arrows in Fig. 6. The
water content of oxyhemoglobin which had been denatured by keeping
an aqueous solution at 95” for 2 minutes is also shown in Fig. 6. It is
remarkable that the extent of hydration is not changed noticeably by
denaturation.
DISCUSSION

Since anhydrohemoglobin is stable only in solid layers at low water

vapor pressure, its absorption spectrum cannot be examined in the dis-
solved state. The spectrophotometric analysis of layers of anhydrohemo-
globin indicates that the absorption spectrum (Fig. 2) is a typical
 F. HAUROWITZ 449

hemochromogen spectrum displaying a narrow intense absorption band

at 559 rnp and a less intense band at 530 mF.
The transition points of the system hemoglobin-anhydrohemoglobin de-
pend on the water vapor pressure, pw, and on the temperature as shown
by Fig. 5, where log pw is plotted against 1000/T. The total water con-
tent of oxyhemoglobin at t = 24” and different water vapor pressures is
shown in Fig. 6. The shape of the curves is the same as that of the curves
obtained when other proteins were investigated in the same manner by
Bull (6).
There is no doubt that most of the water of hydration is bound by the
protein moiety. Consequently, the water content of hemoglobin and

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anhydrohemoglobin cannot differ considerably from that of oxyhemo-
globin exposed to the same water vapor pressure. According to Fig. 5,
the conversion of hemoglobin into anhydrohemoglobin occurs when pw
is lowered to 0.016 atmosphere (log p w = - 1.8) at 24”. At the same
temperature and humidity, the water content of the protein is 10.8 f
0.2 per cent or 115 f 2 water molecules per subunit of the molecular
weight, 17,006 (Fig. 6).
While raising or lowering p w causes extensive changes in total hydration
(Fig. S), the spectral change occurs within a very narrow range of p,;
it involves certainly less than 4 to 5 water molecules and is reconcilable
with the assumption that each iron atom combines with a single water
molecule.
The manometric method used is not precise enough to decide whether
or not the hydration of 1 iron atom affects the hydration of the other 3
iron atoms of the same hemoglobin molecule. By neglecting this pos-
sible comphcation, and assuming that each iron atom combines with 1
molecule of water, the equilibrium between hemoglobin and anhydro-
hemoglobin is represented by Hb + Hz0 $ Hb.HsO. Accordingly, the
equilibrium constant is K = [Hb.Hz0]/[H20][Hb]. Since Hb and
Hb . Hz0 are present as solid phases, the constant depends only on p w
and is K = l/p,. The corresponding free energy change is AF = - RT
In ( l/pw) = RT In (p w) = 4.57 T log (p w). The AF values calculated
from Fig. 5 vary from -2.3 kilocalorie per mole at T = 301 “K., to -3.3
kilocalorie per mole at T = 282 “K. The slope of the straight line (Fig.
5) is equal to the heat of combination of the iron atom with a water mole-
cule; the value obtained is AH = - 15.4 kilocalorie per mole.
The thermodynamic constants for the dissociation of dry oxyhemo-
globin, Hb 9O2 + Hb + 02, cannot be determined experimentally because
no dissociation is observed, even at very low oxygen pressures (Fig. 3).
An approximate estimation of AH can be made by adding AH values for
450 HEMOGLOBIN

Reactions A to F, where g, 1, s, and aq stand for gaseous, liquid, solid, and

aqueous, respectively.
AE (kilocalories
per mole)

(A) Hb.O2(aq) + H20(1) = Hb.H,O(aq) + 02(aq) 9.4

(B) Hb.HzO(s) = Hb(s) + &O(g) 15.4
(Cl Hz0 (8) = H20(1) -10.7
(D) 02bd = 02(g) 3.8
W Hb.Oz(s) = Hb.Oz(aq)
(F) Hb.HzO(ap) = Hb.H20(s)

(‘3 Hb.O2(s) = Hb(s) + 02(g) 17.9

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The AH value in Reaction A is that found by Roughton et al. (5), that
for Reaction B is taken from Fig. 5, while those for Reactions C and D
are taken from the International Critical Tables. The heats of solution
of hemoglobin and oxyhemoglobin are unknown; however, they are most
likely of the samemagnitude, so that the values cancel each other and can
be omitted without affecting the final result. In view of these simplifying
assumptions, the summation of Reactions A to F cannot give more than
an approximate value for the heat of Reaction G. However, it is clear
that the dissociation of dry oxyhemoglobin (Reaction G) requires more
energy than the dissociation of oxyhemoglobin in the presence of water
(Reaction A).
The reaction Hb.Oz(aq) + HzO(Z) = Hb.HzO(aq) + O.(aq) leads
to the same end-products as the successivereactions Hb*Oz(aq) --) Hb(uq)
+ O&q>, and Hbh) + &WI --) Hb.H20(uq). The question arises,
therefore, whether the conversion of oxyhemoglobin into hemoglobin in
aqueous solution is a two-phase reaction involving the intermediary forma-
tion of anhydrohemoglobin.
The presence of intermediates in the oxygenation of hemoglobin and
in the reverse reaction is indicated by the fact that the heat of dissociation
of oxyhemoglobin (9.4 kilocalories) deviates widely from Ef - E,, where
El, the activation energy of the forward reaction, is 25 kilocalories, while
E,, the activation energy of the reverse reaction, is close to zero (4, 7).
Eley (7) has concluded from this discrepancy that oxygenation and de-
oxygenation must proceed through different activated complexes. While
it is difficult to formulate more than one activated intermediate for a
reaction of the type, Hb + 02 e Hb. 02, the formation of two or more
intermediates becomes obvious when hemoglobin is formulated as an aquo
compound, and when account is taken of the fact that oxyhemoglobin is a
diamagnetic complex, while hemoglobin is paramagnetic (8). With the
symbol Gb for globin, Py for a pyrrole nitrogen, a dash for an electron
pair in the diamagnetic complexes, and a colon for two of a larger number
 F. FIAUROWITZ 451

of unpaired electrons in the paramagnetic complexes, oxyhemoglobin is

represented by (GbPyrFe-)02, hemoglobin by (GbPy,Fe:)HzO. The dis-
sociation of oxyhemoglobin and hemoglobin leads to the unstable inter-
mediates (GbPy4Fe-) and (GbPydFe:), respectively; other activated
complexes such as (GbPydFe-)HnO or (GbPydFe:)O* may be formed as
intermediates.
The hemochromogen-like absorption spectrum of anhydrohemoglobin
indicates that it is a diamagnetic complex like all hemochromogens (8),
and that all six coordinate positions are occupied by substituents. Since
no other substituents than polar groups of globin are available, anhydro-
hemoglobin is represented by (Gs) or (kbPyrFey), where the

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sixth valence of the iron atom is bound to the globin moiety of the same
or of an adjacent molecule respectively (3). The results of the thermo-
dynamic, kinetic, and structural analysis are shown by the accompanying
diagram.

\\ \?I
(GbPy,Fe-1 (Gal
Anhydrohemoglobin
1 AH = 16.4
kilocalories
t
= 66 lik-q? pGb’“ez) kilocalories
El AH = 9.4
kilocalories
(GbPy,Fe-JO2 1(
Oxyhemoglobin 1
The diagram illustrates (1) that different intermediates are formed in
oxygenation (c - d) and deoxygenation (a - b), (2) that deoxygenation
requires a high activation energy (I$) while oxygenation takes place with-
out noticeable activation, and (3) that dry oxyhemoglobin cannot dis-
sociate into oxygen and anhydrohemoglobin becausewater is indispensable
for reaction (b).

SUMMARY

1. The absorption spectrum of anhydrohemoglobin (Hb), obtained from

hemoglobin (Hb.H20) at low water vapor pressure, was measured. The
transition points of the system Hb e Hb.HzO were determined. The
thermodynamic constants for the combination of 1 molecule of water
with the iron of Hb are AF (at 20’7 = -2.6 kilocalorie per mole, AH =
- 15.4 kilocalorie per mole.
2. The hydration of the globin moiety at different water vapor pres-
sures was measured. At the transition point of the Hb + Hb.HzO sys-
t(em, globin contains 10.8 f 0.2 per cent water (t = 24”).
452 HEMOGLOBIN

3. Dry oxyhemoglobin does not give off oxygen at low pressures. The
deoxygenation of dry oxyhemoglobin is a highly endothermic reaction
(AH M 17.8 kilocalories per mole).
4. The structure of anhydrohemoglobin and its mode of formation from
hemoglobin are discussed.

BIBLIOGRAPHY

1. Haurowitz, F., Tune, S., and Cindi, R., Fe&ration PTOC., 9, 183 (1950).
2. van Zeynek, E., Nowiny Lekarske Poznan, 38, 10 (1926).
3. Hsurowitz, F., in Roughton, F. J. W., and Kendrew, J. C., Hemoglobin, London,
53 (1949).
4. Hartridge, H., and Roughton, F. J. W., Proc. Roy. Sot. London, Series A, 104,

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395 (1923); 107, 654 (1925).
5. Roughton, F. J. W., Adair, G. S., Barcroft, J., Goldschmidt, G., Herkel, W.,
Hill, R. M., Keys, A. B., and Ray, G. B., Biochem. J., !N, 2604 (1935); 30, 2117
(1936).
6. Bull, H. B., J. Am. Chem. Sot., 66, 1499 (1944).
7. Eley, D. D., TT. Faraday Sot., 39, 172 (1943).
8. Pauling, L., and Coryell, C. D., Proc. Nat. Acad. SC., 22, 159, 210 (1936).
 HEMOGLOBIN,
ANHYDROHEMOGLOBIN, AND
OXYHEMOGLOBIN
Felix Haurowitz
J. Biol. Chem. 1951, 193:443-452.

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