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Preference of oxygenation between α and β subunits of haemoglobin
J. Mol. Biol. (1983) I64 451-476
Preference of Oxygenation Between a and /I Subunits of Haemoglobin Results of Multidimensional
Spectroscopic
Observation
Department of Physics, Faculty of Science The University of Tokyo, Bukyo-ku, Tokyo 113, Japan (Received
8 October 1982)
The distribution of oxygen between the subunits of haemoglobin was studied spectrophotometrically. The difficulty in discriminating the spectral changes upon oxygen binding to the a or fl subunit can be surmounted by means of multidimensional spectroscopic observations and a correlation analysis of the data. M-type abnormal haemoglobins are used as a control against normal haemoglobin because only one type of its subunits can bind oxygen. A multidimensional spectroscopic measuring system, which has been developed in our laboratory, makes it possible to carry out simultaneous and continuous acquisition of a set of spectroscopic data at several wavelengths on one sample solution during the course of increasing or decreasing the partial pressure of oxygen. The data-storing function of a magnetic disk memory provides enough precision for a rigorous investigation of the correlation of oxygen equilibrium curves measured at several wavelengths. No chemical modification to enhance the spectral difference between subunits is necessary, In conclusion, by detecting slight differences between the oxygenation-sensitive bands of iz and /3 subunits. the fl subunits are found to have a higher affinity for oxygen than the u subunits.
1. Introduction Haemoglobin has been regarded as a typical model for allosteric proteins. Two a and two /3 subunits associate to construct an oligomeric structure cu2f12.The oxygen molecule plays the role of a homotropic allosteric effector by itself when it is bound to one of the haem groups, one of which is located in each subunit. It enhances the oxygen-binding affinity of neighbouring haems through haem-haem interaction,
and the well-known S-shaped oxygen equilibrium curve is produced. The work presented in this paper is a detailed study of the spectroscopic changes of haemoglobin during the course of oxygenation or deoxygenation in order to clarify which subunit, a.or /I, preferentially binds oxygen. The method used is highperformance multidimensional spectroscopy (Wada et al., 1980). It is specially t Author to whom all correspondence 0022~-2836/83/070451-26 $03.00/O
should be addressed. 151
0 1983 Academic Press Inc. (London) Ltd
‘H n.m.r.
Ahsorption of harm
M&hod
(‘o-Fe
hybrid
Proto-haem~m~so-harm hvbrid
02
0, “0
\‘B
(1’1) 7.4)
his-Tris
tlis-‘his
llis~‘l’rw
Phosphatr.
(i)H 7.3)
( IIH 7.3)
(pH 7.2)
( IJH i+b,
phosphate (pH T4)
Sodium his-Tris
i)
(pH 7
(pT) 6.x)
(pH
(pH S.&i + DPC 01’ THI’ f DPC: (pH 7%)
phosphatr 1)hosphatr
(~1) 6X) DPG OL’ IHP
his-Tris
Potassium Potassium
02 (‘2
5)
(pl)
his-Tris
phosphate phosphate+
(pH 6.5 5.9)
(pH i+k?] phosphate*
phosphate
(pH 7.0)
(‘(’
Potassium
Sodium
Phosphate
(pH 74)
phosphate
Sodium
7.0)
7.0) (pH 6-9)
(pH
(pH
Phosphate/borate his-Tris (pH 74)
Phosphate
Phosphatv
his-Tris (pH 7) Horatr (pH 9) Phosphate (pH 7) his-Tris+ DPO
(‘ondition
tetrnm,ers
0,
difference
in haemoglobin
S;odium Sodium
02
the first
0 2 binding
+leso)2/qproto)2 ~(proto)2~(meso)2
rnet~hybrid met-hybrid
(‘2
high~spin low-spin
(‘2 (‘2
02
rate
dissociatiotr
“2
hybrid
rate
associat,ion
affinity
“0
Ligand.
C’S met-hybrid
Mn-Fc
Modification
T.\KLE: 1
The oxygen af$nit?y of th,e + and /3 subunits
OXYGENATION
BETWEEN
Hb 1 ASD
p SUBUNITS
153
suited to our purpose since it permits us to distinguish the difference in the degree of oxygenation between unlike subunits by detecting small variations in spectroscopic properties (Philo et ul., 1981; Ueda et al., 1970) and comparing them with the aid of a digital data-storing system. A considerable number of reports have already been published on the preferential binding of oxygen to haemoglobin subunits, as summarized in Table 1. These previous studies can be broadly classified into two categories with respect, to the type of technique used, that is, optical spectroscopy of haemoglobins with a modified haem group such as cobalt-substituted haem, and nuclear magnetic or electron paramagnetic resonance studies on intact or spin-labelled haemoglobins. The optical measurements can be performed with a high signal-to-noise ratio, but it is so hard to differentiate the oxygenation of n and /3 that a modification of the haem group has been necessary to enhance the spectroscopic difference between them. The magnetic studies, on the other hand, can easily make this differentiation because of well-separated signals for IY and 8. while they have difficult’y in assigning the resonance peaks (Huang 8r Redfield, 1976). ln Table 1, it is interesting to find that the optical and magnetic methods have produced opposite results; namely, the optical method has found the /3 subunit to have the higher oxygen affinity, but it is the other way about in the magnetic studies. The modification of haem groups in the optical measurements might be a we have examined the detail of t’he source of this discrepancy. In this context. optical properties of haemoglobins without any modification of haem: t,he simultaneous and multidimensional data acquisition provides enough information for a precise analysis. The symbols
F,,,,(h. Y. AY),
O.ILi(A, Yi). so I) i(h. 1;). 0 I).,*&
Y 1,
o I).~(/\. I-, AY). AO.I).i(/\).
Ao.r) o(h).
used in this paper
are defined
below
fractional change of optical density of haemoglobin (N$~) at wavelength h and degree of oxygenation Y (defined in eqn (1) and expressed in eqn (12)). fractional saturation of haemoglobin with oxygen. fractional oxygen saturation of subunit i = x or 8. half of the difference between Y, and Y,. optical density of subunit, i in a haemoglobin tetramer at wavelength h and at Yi. optical density of an isolated i chain. change of optical density of subunit i caused by the subunit association. optical density of a haemoglobin tetramer. sum of 60 o.,(h. Y,) and 8o.~.&h. Ya). i.e. equation (8). oxy-minus-deoxy absorbance of the isolated i chain defined in equat’ion (5) oxy-minus-deoxy absorbance of the haemoglobin tetramer defined in equation (6). oxy-minus-deoxy difference of o.~)o(X. Y, AY) defined in equation (9).
454
.I. SASIJDA-KOUYAMA.
H. l’A(‘H1H~iX.i
AND
A. WAD;\
2. Principle of the Analysis (a) The fractional
change of optical
denaity
Hitherto the fractional oxygen saturation of haemoglobin has been expressed in terms of fractional change of optical density at the peaks of the oxy-minus-deoxy difference spectra in the visible region. such as 540. 560 or 576nm. In this procedure the equal affinity for oxygen of the different subunitas or the identity of their spectroscopic properties is implicitly assumed. The latter is contradictory to the spectroscopic evidence which shows a difference between t,he subunits. while the former has not been confirmed. In this section, therefore, we shall discuss first what conditions must be satisfied in order to express the fractional ox?;gen saturation of haemoglobin. Y; in terms of the fractional change of optical densit)?. F,, ,,(A, Yz, YD)* which is defined as:
when there exists a preferential oxygen binding to a or /3 subunits. Let us assume that the optical densities of isolated z and /3 subunits in haemoglobin t,etramers can be described by the sum of the opt’ical densities of isolated x and /I chains, and an additional term which describes the effect of the subunit association :
(h. Yj) = 0 l).i(~. Yj)+60 I).;(/\. Ii), () ‘) i mx2p2
i = ~ 01’ p.
(2)
where the degree of oxygenation of each subunit, is explicitly taken consideration. Hence the degree of oxygenation of haemoglobin molecules. related to Y, and YP as follows: I;+
Y/) = 2Y.
(3)
From equations (1) and (2). by assuming the characteristics of the subunits in a haemoglobin expressed as follows : F,,,j(A. Y,. YD) = C{[O.I)i(h.
additivity tetramer.
Yj)-O.l).i(h, 0)J + [60.1, &i, I’;) -60
do I) ,(A) = 0 I) ;(A, 1)-o ,#$)
= 0.1) &(A.
Here, an obvious relation between oxygenation of isolated chains : 0 I).i(h. Y;) -0
I),(/\, 0).
density
I).i(h, 0) = Llo.l).i(h) 1;.
are
i = i\ or j?.
1)-o.i~.,2~$.
the optical
of spectroscopic F,,,,(A. Y,. I’,{) is
I) i(‘\. O)~}/L10.1).,*p2(h)
where the quantities. do.~).,(h), do IQ(A) and Ao.~).,~,(h) amplitudes of the optical density change, that is:
d0.l)
into Y. is
maximum (5)
0). change
the
t(j) a.nd the degree ot
i = ‘I or /3.
(71
OSYGEKATIOK
is used. For simplification, as:
BETWEES
the quantities
o.D.&
Hb R AND
455
fi SCBUNITS
~.n.~(h. Y. AY) and Ao.~.,(h) are defined
Y, AY) = So.n,(h, Y,)+~o.D.&~, Y&,
Ao.~.~(h) = m.&
1)-on.&
(8)
0),
(9)
where AY = (Y, - Y,)/2. When Y = 1 or 0, the variable AY is equated to zero and omitted from the parenthesis in equation (9). Then equation (4) is written as: F,,,,(/\, Y. AY) = 2{[Ao.v.,(h)+Ao.~~.~(X)]Y+ [ -Ao.u.,(h)+Ao.r~,(h)]AY +o I).& I’, AY)-o LX& O)}/Ao D.,&),
(10)
where the variables Y and AY of F,,(X, Y,, Ya) are substituted equations (2), (5). (8) and (9), equation (6) is written as:
for Y, and Y,. From
Ao.~.,~,~(h) = S{Ao.n.,(h) + Ao.r).,(h) + Ao.I)&}.
(11)
Thus, a final equation for F,,,,(/\, Y, AY) is g’iven in terms of the spectroscopic properties of cxand /I subunits: F,,,,(L Y, AY) {do I).,(/\) + Ao.D.,(~)} Y + { -i Ao.D.,(/\) + Ao.~.~(h)}d Y + o.I).& Ao.r).,(h) + Ao.D.~(X)+ Ao.r).&)
Y. A Y) - O.D.&
0)
(121
When the effect of subunit association on absorbance spectra is small enough, the following relation can be adopted: o.D.& and in the following
Y. AY) < om,(h. Y,)+o II.,@. Y&,
(13)
discussion we assume: Ao.I).~(/\) 6 Ao.r).,(h) + Ao.I~.~(~).
(14)
Incidentally, the relation, F,, ,,(A, Y, AY) = Y, which has been traditionally used, is a special case in equation (12) when A Y = 0 and the optical density change caused by the subunit association is proportional to Y: o.r).&
Y, AY)-o.I).&
0) = do.n.o(h)Y.
(b) F,, ,, (A, Y, AY) at several characteristic
(15)
wavelengths
In the previous section we correlated the fractional amount of spectroscopic change, F,, ,)(A, Y, A Y) with the degree of oxygenation of a haemoglobin tetramer (cx&), Y, and the difference between those of the ,!I and LXsubunits, AY. The structural change caused by the association is known to produce a slight change in the absorbance spectra of n and ,3subunits. The spectroscopic evidence reported by Ceda et al. (1970) is exhibited in Figure 1. It indicates that the oxy-minus-deoxy difference spectra of haemoglobin tetramers do not coincide with the mean of those of isolated (Yand /3 chains. 16
a)
In this section several characteristic wavelengths. written in a simple form by the intrinsic variables
at which ecluation ( 12) can INS E’ a.nd Al’. are discussed.
(1) hi (or Ai) : Thr wne~elength whew the spectrwr/ is insmsitiue i.e. o.D.&. Y, AY) = 0; (or hj where eyuat,ion (13) holds). At this wavelength,
equation
(12) can be reduced
I*‘,),)(hi* 1’. 0)‘)
= I’+
to xuhunit
nssocintiw~
t,o:
-&).I)
J/ii) + -~--do m&A,) d )d0 I) .(/\i)+dO I) a(h,)
(Itij
The wavelength at which the unbroken line (a2/12) is located at the mean position ot the dotted (a) and the dotted-broken (8) lines in Figure I sat’isfies this condition. Similarly. the associat,ion effect can be considered as negligible at the wavelength Ai, where the relationship of equation (14) holds. The condition is satisfied in a wavelength region at which an unbroken line is located in between t,he dotted and the dotted-broken lines. In this region, we have:
F,,,,(hj, Y,flY)
+ Y+
--do
I&(/q)
Lk.n.,(h;)
+A0
1)$(/i:) il l’
+flomp(A:)
cmQ(A;.
Y,
d Y)-0
+ A(,
I) .(h))+do
I) &.
O)-do -.--___ I) fi(/\;)
1) &I;), (17)
OXYGENATION
BETWEEN
Hh n, AND
157
fi RVBUNITS
The second term on the right side of equations (16) and (17) is the correction term for the difference in oxygen affinity between n and /3 subunits. The third term in equation (17) denotes the effect of the structural changes caused by the subunit association on the absorbanee spectra. (2) A,: ‘x-/3 equi-sensitive
wavelengths.
i.e. do.n.,(A,)
= Ao.I~.~(/\~); for
example
Ae = 540. 560 or 576 nm. At this wavelength: F,,,,(h,. From equation
Y. AY) =
2Ao.r~.,(h,)Y+o.r~.o(h,, Y, AY)-O.DQ(h,, 2AO.D.,(h,) +Ao.r).Q(h,)
0)
(18)
(15) we have:
F,,,,(/\,, Y.AY) = Y.
(19)
Thus, even if the structural change caused by the subunit association significantly affects the absorbance spectra, when on o(h,, Y, dY) changes linearly with the degree of oxygenation (Y), F,, ,,(A,, Y, AY) may be equated to Y. (3) Ass: fl-sensitive
wavelength,
At this wavelength.
equat,ion
F,, 1,(4,s 1’. AY) = Y+AY+
= Yp+
i.e. do.n,(hs,)
= 0.
(12) becomes:
~.~).~(h~,, Y, A Y)-on.&,.
0)-
Ao.l).p(has)+Ao
o.r@$),,
Y,AY)-o.I).Q(h,,, AO.I).&)
do.n.o(AsS)( Y+AY) l).(&)
O)-Oo.l).&ps)Y~
+Ao.r).Q(AsS)
of Therefore F,, ,) (A,,, Y. d Y) can be written in terms of the degree of oxygenation the #l subunit, Yfl = Y +A Y, and the correction term for t’he structural change caused by the association. (4) A,,: a-sensitive
wavelength.
In the same manner F,),,(h,,.
Y,AY)
i.e. Ao.I~.~(/\~~) = 0.
as equation = Y,+
(2(l), F,,,)(h,,, Y, AY) becomes:
O.I).Q(h,,, Y,AY)-o.l).a(A~s, AO.I).,(h,,) +A0
O)-Ao.l).Q(Aas)Y, I~.&,)
The equation contains the degree of oxygenation of subunit Y, = Y-A correction term for the structural change caused by the association. (c) The effect of subunits
association
(211 Y, and the
upon F,,,, (A. Y. AU)
At the upper side of Figure 2, enlarged and schematically drawn spectral lines 01 isolated Y and fi chains and the haemoglobin molecule (a2f12) in Figure 1 are shown. With regard to the effect of the structural change caused by the association on the absorbance spectrum, there can be considered to be three types of spectral tetramer ; these relationships between cx and p subunits in a haemoglobin
158
a ,no2pe
Pina2Pe \
:::t;-
i \ ! i \
FIG:. 2. Schrmatic illustrations of sevrral types of’ absorption tliflrrr~n~~~*hlw(,t ra ~.~llal,ged wound tlrc, lvavelengths of isohestic points of thr isolated z and b chains (0). Top: ohsrr,vcvl spwtra of’z,& (__ ). and isolated #I ( ) and j3 (~ - -) chains. Hottou: obser~~rtl sp.
relationships fall into two groups. The first is of the type shown in Figure d(a). which is sensitive to the quaternary structure formation. The second includes the types shown in Figure Z(b) and (c) where t)he spectrum is insensitive to t)he quaternary structure formation. In the latter case, according to the discussion in the [JIeViOUS section. the wavelength dependence of F,, ,) (h, Y. d Y) in the vicinity of 58.5 nm + hi is as in Figure 3. As the wavelength goes from X, to a region shorter than the isobestic point of ,I subunits, h,,, the contribution of the /3 subunit to the absorbance change increases
OXYGENATION
BETWEEB
Hb 7 ASl)
/3 SlTBITSlTS
15!)
kc:. 3. Enlarged difference spectra near .5%5 nm of typ b and type c of Fig. 1. The spectral wmtribution of the subunits. LIo.I).&I) and do I). and the effect of the subnnit associat~ion, do 11o(h). in equation (11). The length and the direction of arrows indicate t,he magnitude and the sign of do I) z. ocll~,andd~~n.o.(-)r,~z/~:(---)r:(---~-)~.
from an amount equal to that of a to an amount much larger than ~1. As the wavelength exceeds the isobestic point of/l subunits, A,,, to another A, in a longer wavelength region, the contribution of Y subunits to the absorbance change> decreases from a large amount to an amount equivalent to that of j3 values. In this context, the following experimental schemes were designed to examine the actual spectral types (see Materials and Methods, section (d) for details). First. the type of the difference spectra of the subunits in the tetrameric state in the vicinity of 585 nm, which corresponds to hi, is determined. To do this M-type abnormal haemoglobins, in which only one type of subunits can be oxygenated, are measured as a control. Secondly, when the results of the above experiments havcl shown that the spectral type is b or c, oxygen equilibrium experiments for F,, ,) (A. Y, A Y) of HbAt at A, and at a series of wavelengths in the vicinity of 585 nm \ = hf) are carried out. Thirdly. from the correlation diagrams of F,, ,,(A,,. Y, A Y) t!eTS%LsF ,,,, (A,, Y. AY) and F ,,,, (A,,, Y. A Y) z’ersus F,, ,,(A,, Y> d Y), information on the oxygen affinity difference between o( and /? subunits in the tetrameric state is obtained. ‘r Abbwviations
used: HbA. human adult haemoglobin:
c.d.. circular dichroism
460
.I. S~SI’I)A~liOU\iAblA.
H. TA(‘HlH.AS.1
3. Materials
.\SI)
.1 LVi\l),\
and Methods
(a) Xfflrritr/~~
Human adult, hiuvnoglobin was prepat’ed by thv method of Imai 8 Yonrt,ani (l!ViSo.h) with modifications. Ten ml of human blood were quashed several times with 0.9(:,, (wiv) sodium chloride. Packed red cells were then haemolysed by adding 1.5 ~(~1.distilled lvat,er and 0.5 vol. toluene (Drabkin. 1946). A clear haemoglo&n solution was obt,ained by centrifuging (1Ti.000 revs/min for 20 min) and freed from residual organic phosphates t,hrough a Sephadex 025 column (3.5 camx 60 cm) equilibra,ted with OG5 \q-Tris. HC’I. 0.1 st-KC’1 (pH 5.4) (Henesch et nl., 1968). Then the haemoglobin solution was ~ottc~cntratrd t,o about I mht-haem hy ultrafiltration through an Atnieon Y31 IO or ITIM10 mt~mbranc and dialysed against) thv buffer used for oxygenation clxperimrnts. On the day before the ~rtf~asttr.C~~~ertts. t,hc dialpet haemoglobin solution was adjustid to the desired concrntration. t,hr NAI)J’H-frrredoxitt reduction system was added. and the sol&m was incubated overnight (Hayashi rt (I/.. I!Ii:li. All the cornportents of the Inet,haemoglobin reducing system were purchased from Sigma (‘hemical C!o. (without, furt,her purification). The abovc~ operations wt’tv varried out at 4 C’ and all of thr measurements were tinished during the day following ittc~ubatiotr. ‘I%(. abnormal haetnoglobins, HbM Milwaukee and HbM Host,on wrrv gifts from I)r K. Sagat (Na.ra Medical l’nivrrsity) and I)r $1. Yamazaki (Kunra.ntc~t.o I’nivt:rsit.yl.
The apparatus used was developed t,o obtain sitttultattrously 3 spectroscaopic di~nc~trstotts. that is. absorbance, circular dichroism. fluorescence inttrnsity (not used in the present study) rt 01.. 1980). Thr partial oxygen pressure of the and Rayleigh scattered light intensity (Wada solution was mrasured con tinuouslp in t htx optical c*uvet)te by a ( ‘lark-type electrode made by Cnion Qiken (‘0. Ltd (Hirakata., of A,,,,wert’ estimat,ed as:
= / -0a48+0~016~
x J&A,,,,1
Therefore. in order to carry out a measurement of P;, 1jrecision. the following c:onditioti ttiust by satisfied :
and the wavelength
for measurements tnrtst 1~. IA- /!,,,,I> 12 n1n.
OXYGENATION
BETWEEN
Hb z AXD
/3 SUBUNITS
46 I
(a) REPRODUCIBILITY
523 ms 0.0
1
:
50ms
I
; 233
: 9.5 s/l
TEST
T
125 ms/ 50samples * * I 246
-:
* 259
-:
? 272
1 -:
I
1: -: 285 nm
I
loop
233 nm * (b)
)
259nm
,
285nm -
,
285 nm’ 272 nm’ 259 nm ’ 2 0 11 6 .\
246 nm’ 233 nm’
FK:. 4. A test ofthe accuracy ofwavelength setting. (a) AI example of t.he time-course of a wavelength scanning programme. The absorption spectrum of R’-AMP in water,. (b) The fluctuation of observed optical densities measured at the points indicated by arrows in (a). The arwws at the right in this Figure denote the scanning direction to shorter (b) and longer (P ) wavelength. Each continuous line consists of Z(H) data points which are repeatedly measured accoxiing to the scanning programme indicated in (a) Since the observed optical density fluctuation at the slope of the spectral line is the same as those at its summit,
or through
the precision
of the wavelengt,h
setting
is estimated
to he 0.02 nm or better.
The half-intensity
bandwidth of the spectrometer in the present study was kept at 1.1 nm. which is the narrowest in the automatically slit-servo mode of our apparatus. (c) Experimuatal
Thr experiment,al
conditions are summarized
conditions
in Table 2.
(d) Exp:prrimrntal procedure X series of experiments to obtain a deoxygenation or an oxygenation curve whkh probed by mult,idimensional spectroscopic observahles consists of the following steps.
is
462
HbA
HbA+2
mwIH1’
Thr same as abovr
t At this wavelength $ At this wavelength
‘1‘1w Salllt’ it8 itho\ <’
WV also measured light-scattering intmsitiw we also measured rircular dichroistn
(i) I)eoxygenatkm (1 ) An oxy spectrum of’ a fully oxygenated sample solut,ion (I ml) is measured. (2) Gradual deoxygenation is started by flowing oxygerl-free nitrogen into an optival cuvette containing the solution. (3) A series of absorbance measurements at several wavelengths is c-arried out according to the computer commands which are programmed just prior to t.hc (Axperiment,. (4) The scanning over the wavelength axis is repeated during the
the air into
the cuvette.
a series of spertroscopic
measurements
of thr
OXYGENATION
BETWEEX
Hb x AND
p SI’BI’XITS
463
reoxygenation process is carried out in the same manner as the deoxygenation experiment. mentioned above. (2) When the sample is fully reoxygenated. a reoxy spectrum is measured. (3) By adding 2 mg Na&O,. another deoxy spectrum is measured. (4) The equivalences of the oxy and reoxy spectra (measured at (i)(l) and (ii)(2), and of the deoxy and Na&O,-deoxy spectra (at (i)(9) and (ii)(3)) are examined to determine whether any denaturation of auto-oxidation occurred during the course of the experiment. For HbA in phosphate buffer, only the deoxygenation experiment (series (i)) was carried out For M-type abnormal haemoglobins, the Na&O,-deoxy spectrum was not measured because the haem-iron of the abnormal subunits is also reduced by Xa,S20,.
4. Results (a) M-type
abnormal
haemoglobins
Figure 5 shows the correlations between the change of absorbance at 560 nm and those at other wavelengths designated in each diagram. The isobestic points are found to be 583.0 nm for HbM Milwaukee and 586.0 nm for HbM Boston. These two wavelengths are close to the isobestic points of the
isolated chains, 583 nm for 0:and 588 nm for /3. respectively shorter
and the other,
588 nm, is longer
than
the isobestic
(Fig. 1) ; one, 583 nm, is point
of haemoglobin
tetramers. Ai,, = 584.5 nm. The above evidence shows that the oxy-minus-deox) difference spectra of (Yand /3 subunits in the haemoglobin tetramer in the vicinity of HbM Milwaukee
583.5
..**
584
. .... . -
:* *. ..
. *
:*
..
FIG. 5. The results of HbM Milwaukee. Experimental conditions are shown in Table 2. (‘orrelation diagrams of the absorbance changes in the reoxygenation experiment. The ordinate is the fraction of absorbance change at the wavelength written in each diagram. and the abscissa is that at 560 nm (l--p equi-sensitive wavelength A,). Each point on this 2-dimensional surface indicates the absorbance at 2 wavelengths for a common oxygen pressure, that is common Y. The series of the points reveals a trace indicating a correlation of these 2 obsrrvables.
464
A. NASI’LIA-KOI’YAMA.
H. TA(‘HIB.L\NA
ANI)
;I. IVAI),\
585 nm are of type c, as defined in Figure 2. Therefore. with similar experiments on intact HbA the fractional oxygen saturation of z and /? subunits in haemoglobin tetramers can be obtained from the absorbance changes at the longer wavelength side of 584.5 nm, A,,. and at the shorter wavelength side of 584.5 nm. A,,. respectively. (i) Correlation
diayrnms
Figure 5 shows that the correlation diagrams. ‘!~,,Ao I) ix, ;,x,i PPI’SUSC?,,do I) ;I,i,l ti)r HbM Milwaukee, are nearly straight lines at 45” angles. (b) Human
adult haemoylobin
The time courses of oxygenation observed for HbA in bis-Tris buffer are shown in Figure 6. The evidence that the absorbance and scattering at 505 nm did not increase Because small confirms the absence of auto-oxidation and denaturation.
,....
. .__. :::::::7A::z:l::r;zL’f:::l .::::..::. .....:_::::;:::::“~::-:.:..::::::;::IE::;:: ..........._.........................,... .-““I ........................... .......~.....................................................~ . ...... ...................................................... .(” ..__ 586 ,.__. ....................................... ....i....................... ...... .._.................................. ................................................. .... ............................ 5 ($7 ................................... ........................ ,./ ......:... .: ........I...... ; +::.: lj 88 ................................ ......... ‘, .... . . 5 89 .........-...................... ..............I ........... scat ..................._............ ........................................,.. ........ ...............
584.5 585
I
Deaxygenotwn (h)
2
.. 0.5 log(p0,)
0.1 O.D. f 0.01 [6’]/degree 1000 count
i Reoxygenatlon
PIG:. 6. Kaw plots of thr time-cwrrsr of sptxt.ral and ox~gw ~~wssure ~~h;rt~gw of HbA in bw’l’r~~ buffer. Roth deoxygenation and reox~genation processes are shown. The ordinate scales are shown in the lower right bg an arrow which corresponds to 05 for logos). WI o I) for the absorbancr. 0~01[OIldegree for the ellipticity of circular dichroism (r.(k). and I(HHI photon counts/s for light scattering (Scat) intensity. The vertical bars attached to each raw I)lot, of absorbance indicate the position of half of the total change. An isobestir point is found at 5845 nm. Experimental conditions art’ shown in Table 2.
OXYGENATION
BETWEEN
Hb I AND
,¶ SI’BUSITY ,560
% A 0.D
/ ..: ...‘.
nm
I
,..’
,:’
+-----
,/’
..576
..’ . ..“‘.““”
;;,,,_...
......
.-588
__--
-__A-
r’
-1.
I;” ,f’ :’ ;: ..zi
:.
i’
,:-
._. _.___..
. ..
‘, 503
:: :I
.I ....____...--
i I ,._,,,_,
583-.
‘.....
..
:-588 ,:’
log POP
PIG:. 7. The normalized plots of spectroscopic oxygen equilibrium curves measured at sr\-wal wavelengths for HbA in potassium phosphate buffer. At the bottom, a comparison of the 2 curves which are probed through the 1 (588 nm) and fl (583 nm) subunits. Expwimental conditions arc shown in Table 2.
absorbance changes in the vicinity of the isobestic point (5845 nm) make the direct comparison of curves in raw data difficult. the data were expressed by means of normalized curves, i.e. o/oAo.~~.and O/oAc.d., versus partial oxygen pressure (i.e. the oxygen equilibrium curves). The results obtained with potassium phosphate buffer are shown in Figure 7. 1.n this Figure, vertical bars show the positions of PSO on the abscissa, which is the partial oxygen pressure at half of an absorbance change. Their numerical values are listed in Table 3. The oxygen equilibrium curves measured at 560 nm and 576 nm coincide, while those measured at 583 nm and 588 nm shift toward the lower and higher oxygen
pressures. respectively. As the shifts are very small. WV made a comparison I.,) superimposing them on thp common axis (the lower portion of Fig. 7). The oxygen equilibrium curves at 560 nm and 576 nm are located between those at, ,583 nm and 58X nm. The oxygen eyuilibrium curves of HbA in his-Tris buffer a,nd in his-Tris buffer with inositol hexaphosphate hare t’he following characteristics. (1) The oxygen equilibrium curves depend on the wavelength used: (2) t’hey shift toward higher oxygen affinity (lower oxygen pressure) in a wavelengtjh region shorter than 5845 nm. and toward lower oxygen affinity (higher oxygen pressure) in a longer
p50
mmHg
(al
-mmtig
(b)
585
590
FI(:, 8. T)ependrnw of the oxygen l~re~~ure at halfof’a slxx*ttwxol~ic~ measuring wavelengths. An BI’IWWon the ordinate directs the I’,, of (a) Intact haemoglobin in his-Tris buffer. (b) Intact haemoglobin in hexaphosphate (IHP). Experimental renditions are shown in Table
observable (fJ5,)) of H bA upon thv the absorbance change at 560 nn,. his-Tris buffer with added inosltol 2.
OXYGESATIOS
BETWEEN
Hb a ASD
,&?SITBUNITS
467
wavelength region ; (3) the shift becomes larger as the wavelength approaches the isobestic point at 584.5 nm. The above-mentioned characteristics are well demonstrated by plotting P 50 against wavelength (Fig. 8(a) and (b)). The value of P,, decreases gradually as the wavelength increases from 582 nm to 5845 nm. Beyond 584.5 nm it jumps up to a value higher than that at A,( = 560 nm) and then decreases again gradually to the P,, values at A,. (i) (‘orrelation
diagrams
Figure 9(a). (b), (c) and (d) shows the correlation of the fraction of absorbance changes at, A,, and at hsS(~&lo.n.(h,,) and %do.n(hPS)) to that at h,(%do.n (A,)). The fact tha,t the correlation diagram between h,s (Fig. 9(a)) shows a straight line with a 45” angle implies that equation (15) holds at h,s. On the other hand, the correlation diagrams, ~/o~o.~~.(583) vewus ~‘do.r).(h,) and %do.n (588) ?)erslls “/,don(h,), show convex and concave shapes, respectively (Fig. 9(a)). Two series of diagrams (Fig. 9(b) and (c)) measured in bis-Tris buffer and bisTris. inositol hexaphosphate have common characteristics. Namely (1) the correlation diagrams of the fraction of absorbance change at lower and higher wavelengths to that at A,( = 560 nm) deviate to opposite directions, that is. below 584.5 nm the curves deviate upward from the diagonal line and above 584.5 nrn
.’ . . : (.” ,/ ,/
583
I
588
583, 5 88 .
L/’
.i’ ..:... .:’ . . .:’.: (a)
FIG. 9(a).
.. ..-* .A
50;
a
I. ...’
i’
:. ..*
.. . .*
:
,:
507
.I .. :
1 !J 11 i.
590
I:
.:.
..
. ..
:
..
591
..*.
..,.
:
..
:.
:
.:-
.;
,
.. .
,:.
:
..
’
~qT--yiq ..
/ I
:. /
.*-
::
.:
507
....
.:.
:
:
I J
(b)
:I
/
589
. ..’ :. :’
:.
El .J
590
,/
i
:.
..
.*.
..
. .. ..
,:
. .. .. .
--
..9.’ .*’
.,:
:
: .:~
,i’
591
... .*
/
.. .
..: .:
..’ ..
:.
.:.’
.:.
/
: . .~
.:’
:. .*.
a Cc) F1c:. 9(b).
(e)
:
..
:.
OXYGEXATIOS 582
83
BETWEEN
Hb (t AND
. :*
584
’
tc3SUBITNITS
4ti9
’
L 586
t87
(d )
Fit:. 9. (‘orrelation diagrams between (@lo I) (A) or O/,dc.d.(h) (ordinate axis) and %Ao 11.(560) for HbA (abscissa axis). The number in each diagram denotes the measuring warelengt,h. (a) Obtained b) the deoxygenation experiment in potassium phosphate buffer. (b) Obtained by the deoxygenation experiment in bis-Tris buffer. (c) Obtained by the deoxygenation experiment in bis-Tris buffer with added inositol hexaphosphate (IHP). (d) Obtained by the reoxygenation experiment of the same sample as deoxpgenation in his-Tris buffer. Experimental conditions arr shown in Table 2.
downward. (2) The deviations from the diagonal line are larger as the wavelength approaches the isobestic point at 584.5 nm. (3) The correlation diagram between the circular dichroism, %dc.d.(560), and the absorbance, o/,Oo.n.(560), agrees with a diagonal line. The presence of inositol hexaphosphate enhances the above deviations. By comparing Figure 9(d) with Figure 9(a). the above findings are also found to be true in reoxygenation.
5. Discussion The present work again confirms that the oxygen affinity difference between unlike subunits is maintained even in the tetrameric state. In conclusion, the oxygen affinity of /3 subunits is found to be higher than that of (Ysubunits, and the effect of inositol hexaphosphate is to emphasize this tendency. The advantage of the present work is that intact (unmodified) haemoglobins could be used, so that we are freed from any undesirable and unpredictable effects which come from the chemical modification of the protein or the attachment of probes to it. The oxygenation and deoxygenation curves of high data-point density
470
A. NASUDA-KOCYAMA,
H. TA(‘HIKANA
?tNI)
A. W.&I):\
in several spectroscopic dimensions, at a variety of wavelengths, make it possible to provide a perspective view of molecular events produced by the oxygen binding. Slight differences between the spectroscopic properties of the n and the fi subunits can be detected by virtue of the functions of our apparatus. i.e. simultaneous data acquisition and data storing. Precise comparisons of the oxygen equilibrium curves and a detailed examination of their correlations are possible w&h t’his technique.
Previous spectroscopic studies with modilied haemoglobins have provided conclusions similar to those of the present study (Brunori et ~1.. 1970: (:ra~, k Gibson, 1971: Gibson, 1973; Imai et al., 1980: Maeda et a,Z.. 1972: Makino & Sugita. 1978; Nagai, 1977: Yamamoto Pr Yonetani. 1975). except, for the Mn-Fe tiybrid haemoglobin (Waterman &, Y onetani, 1970) and the a(Meso)2/?(Proto)2 hybrid haemoglobin (Yamamoto 8~ Yonetani. 1975). Imai et al. (1980) observed the oxygenation of k and fi subunits separately under, conditions similar to ours. They estimated the degrees of oxygenation of the subunits in an iron haemoglobin. YE and Y,, by referring to the microscopic oxygen equilibrium constants of Co-Fe hybrid haemoglobin. Using their numerical values, we calculated oxygen equilibrium curves and correlation diagrams of intact iron haemoglobin with 0.1 M-potassium phosphate buffer (pH7.4) at 15” as plotted in Figure 10. The results show curves reasonably similar to ours. A study on spin-labelled haemoglobin can provide detailed information about. the haem and residues which are functionally important (Asakura & Lau. 1978: Huestis & Raftery, 1972a,h.1973; Ogawa Kr MacConnell, 1967: Ogawa et nl.. 196s: Ogata 8: MacConnell. 1971,1972a,b). However. the effect of spin-labellinp
log PO2
FIG. IO. (a) Oxygen equilibrium CU~W reproduced from the data of Imai rt nl. (IWO). (~-. (..........) yei,: (-. -. -) y,. (b) (y? orrelation diagram of I’, and Ye to Y from the data of lmai (1980).
) 1’.
rt trl.
OXYGENATIOX
BETWEEN
Hb 1 AND
p SUBUNITS
471
modification on haemoglobin function is still not quite clear. It is necessary, therefore, to be assured how functionally equivalent the modified isolated chains are to the intact isolated chains. In the study on differently spin-labelled (SL) haemoglobins, CX(SL)~/~~and a’fl(SL)‘, the degrees of oxygenation obtained from the signal change of the probe, Y, and Y,, do not compensate for each other, so that their mean values are not equal to the fractional oxygen saturation of whole molecule, Y (Asakura & Lau, 1978). Nuclear magnetic resonance is a powerful method which can investigate several conformational aspects of haemoglobin simultaneously without modifying the haem groups. However, previous studies by nuclear magnetic resonance have not provided a unified conclusion (Huang & Redfield, 1976; Johnson & Ho, 1974; Lindstrom & Ho, 1972). The reasons for this lack of agreement are the difficulties in assigning the resonance peaks (Davis et aE.. 1971,1976; Huang & Redfield, 1976), and in estimations of the degree of oxygenation, which is obtained from a separate measurement of the optical absorbance change in the visible region. In the kinetic study, kinetic pathways from one state to another are not necessarily equal to the pathways of the fluctuation between the states in equilibrium. As a matter offact, the equilibrium constant evaluated by the rates of association and dissociation of oxygen is much different from that obtained in the oxygen equilibrium experiments (Gibson, 1973). Qualitatively, our result agrees well with that of Imai et al. (1980), whereas quantitatively the separation of our two experimental curves (Figs 7 and 9(a)) is narrower than theirs (Fig. 10). The values of PsO in the present study are a little higher than their values. These slight disagreements might be due to the higher temperatures in our experiments (Imai, 1979; Imai & Yonetani, 1975a,b). (b) Problems
in the present study on which further
discussion
is required
We must point out the problems that underlie the assumptions and the experimental conditions used in the present argument. The former concern the effect of the quaternary structural change and the quaternary structure formation. and the latter the effect of the heterotropic allosteric effector such as inositol hexaphosphate. The quaternary structure formation and the quaternary structural change are manifested indirectly by the spectral change o which reflects the association of subunits (Ueda et al., 1970; Philo et al., 1981) and the haem-haem interaction. However, the additivity equations, equations (2) and (4), take only the former factor into account. Therefore, if the haem-haem interaction exists, o.~.,(h, Y,) and So.n.,(X, Y,) should be affected by Y, and vice versa, so that the additivity no longer stands. An additional cross term of Y, and Ys should be involved in the equations in a strict sense. However, the fact that absorbance spectra retain their isobestic points throughout the oxygenation process (Enoki & Tyuma, 1964 ; and the present work) suggests that the major haemoglobin populations are the fully oxygenated and deoxygenated species, whereas partially oxygenated intermediates are minorities. Thus, the additivity assumed in equation (2) may be a reasonable assumption. D.Q
472
A SASL’DA-KOUYAMA.
H TA1’HIKASA
.1X1) .. . 1VAl1.4
As for the second problem, it has been pointed out that’ the allosteric effecters. such as organic phosphates and Cl -. influence the absorbance spect)ra of oxyhaemoglobin (Adams & Shuster, 1974: Imaizumi et n/.. 1978). According to these reports. the shape of the effector-induced difference spectra does not, depend upon the kind of effecters, while the magnitude of absorption decreases in the order of inositol hexaphosphate, 2,3-diphosphoglycerate and (‘I- (Imaizumi et al.. 1978). Unfortunately, one of the peaks of effector-induced difference spectra is close to t,hp isobestic point of oxy-minus-deoxp difference spectra. i.c>. 584.5 nm. However. in the following discussions, it will be shown t,hat t~his evidence does not affect our conclusion. In our experiments. 0.1 M-KU was always added. so that. if oxygenation produces the dissociation of Cl - which affects the optical absorption. the following result will be obtained. According t’o t,he report of Imaizumi et al. (1978). 560 nm and 585 nm are the wavelengths corresponding to a peak and a zero-point, in the difference spectrum, respectively. Therefore. a series of correlation diagrams between bhe absorbance changes at A,( = 560 nm) and those at, any wavelength in the ricinitjy of 585 nm should give similar profiles. However. correlation diagrams of the absorbance changes below and above 5X4.5 nm ex hihit. opposite features as shown in Figure 9(b) and (d). We therefore conclude tha.t thcs difference of o(- and &sensitivity exceeds the effect. of (‘1 - The binding sites for ( ‘1. are not considered to k~r in one particular type of subunits (Bonaventura et tsl.. 1976: Chiancone et al., 1974,1975,1976: Nigen & Manning. 1975: 0’J)onnell et (I/ 1979; Perutz et ~1.. 1980: Van Reek et al.. 1979). On the other hand, the effect of inositol hexaphosphate may not be ignored since it’ has a significant influence on oxygen binding affinity (Tynrna) et (II., 1973) and on the oxy spectra (Adams & Shuster. 1974: Imaizumi et crl.. 1978). The presence of 0.1 M-(“1 - reduces t,he effect of inositol hexaphosphat,e on t,he oxy spectrum. but it is still significant, (Imaizurni et 01.. 197X). To examine the effect of these ions. let us compare the correlation diagrams 01’ HbA with added 2 mr\l-inositol hexaphosphate and 0.1 LX-(“~-. As shown in Figure 9(c). there are large deviations from the diagonal line in bhe correlation diagrams between o;ldo.~).(p,) and ()&!o I) (A,) : especiall\- in t)he diagram between 584 nm and 560 nm. it, appears that p subunits are ’ fully oxygenated a,t half saturation of whole molecules. On t,he other hand. the deviations from the diagonal line in the correlation diagram between A,, and 560 nm (Fig. 9(c)) are smaller tha,rr those between A,, and 560 nm. These results may indicate the dissociation of inositol hexaphosphate bound to the p’P/32 Interface (&none & Perutz. 1971). In any case. the presence of inositol hexaphosphate emphasizes t.he difference in the binding affinity (Gray c(r Gibson, 1971 ). and this supports our oxygen interpretation.
(c) Analysis generalized
of the wavelength,
concerted-transition
dependence
of correlatim
model and an assumption
diagrattrs
according
tv thr
about do u.,(h. Y. AS)
The generalized concerted-transition (GCT) model, which is an extended Monod. Wyman & Changeux (MWC’) model (Monod et al.. 1965). was proposed by Oga.ta &MacConnell (1971). They took the oxygen affinit,y difference between z and /3
OXYGENATION
BETWEEN
Hb a AND
/3 SUBUNITS
473
subunits into account on the assumption that the oxygen binding equilibrium constants of each subunit at the relaxed (R) state, KE and Ki, are equal to the experimentally measured affinities of isolated a and /3 chains. Their conclusion is that the affinity for oxygen of a subunits is higher than that of /I subunits for the tense (T) state, i.e. Klf > Kg. Let us try to interpret the wavelength dependence of correlation diagrams, F,,,,(A, Y.AY) versus F,,,,(h,, Y,AY), in the vicinity of hi,,( = 584.5 nm) for the condition that the absorbance change caused by the subunit association is proportional to Y, i.e. in accordance with equation (15). From equations (12) and (15). F,,,,(h. Y. AY) is written as: -AO.D.,(A) +Ao.D$(h) Ay Ao.Da(h)+Ao.I~.~(A)+Ao.D.Q(h) .
F,,,,(h, Y.AY) = Y+ The first derivative
(22)
of F,,,,(h, Y, AY) with respect to h is obtained as: AY + Ao.np(h) + Ao.D&}~
$&,,(A Y,AY)= {Am.,(h)
- [~Ao.D.&)
X
+ [~Ao.D.,(/\)
+
+
Ao.I,.~@)] & Ao.r).,(h)
do.~.~(A)]
$
+ [Ao.r).,(h) - Amp(h)] At A = ho,, by substituting
Ao.D.&~)
.
$ Ao.r,&)
(23)
= 0 into equation (23), we have:
Ao.Ix,@~,)
&Y ; F, 1)(A Y>A Yhs, =
1 {do D.p(ha,)+ Ao.I~.&,)}~ X
-
[~Ao.D.~(/\~,)
+do.~.~(h,J]
$
x Ao.r~,(h)),~~+ Ao.Ix,(~,,)
d - Ao.D.,(+d z Ao.~)&)l,~.
$Ao.D.,(X)I,.
I
> 0. where the relations, as illustrated -Ao.~.,&~)
(24’)
in Figure 1, among the absorbance changes: < Ao.~~.~(h,,) < 0 < Ao.~x~(h~J.
(25)
and their derivatives : & Ao.rxQ(h) < 0. & Ao.~.(h) + $ Ao.D.#)
i: $
Ao.r,.,(/\) < $ Ao.D.#)
< 0.
A/Js< h < Ls
(26)
474
il. ?;ASIlI)~-~C)I;Y~ill.~.
are used. Similarly.
-+,,(A;
Y.LlY)I,\
H. 7'.4('HIH.AS.4
ASI) .A \S'Ai,A
at h = A,, (00 I),~(,&) = 0) we have: 1
=-----
{do r).(h,,)+do.l).o(/\a,)j2 X
x
- & dO.l~.,(A)l,,~-tl;:Ali
do I).&,) i I) ,xlA,~+ 1;; A() I) Q(h)/,;\
I
I
+ IdO.l).,(h,,)-dO.I) (,(h,,)l (II, Do I) Q(h)j,,~ > 0. where the relations
among
(27) the absorbance
do.n.,(h,,)
changes:
< do I) Q(h,,) < 0,
(2X)
and their derivatives, i.e. equation (26): are used. From equations (24) and (27). the effect of the subunit association on the wavelength dependence of F,, ,) (A. Y. d Y) ‘I:ers?rs Y is found to be to produce a ~OIP convex curve below A,,,, and a less concave curve above A,,,,. respectivelg. than t,hose obtained when the effect of subunit association is neglected or happens to be zero. Since F,, ,, (A. Y. A Y) can be substituted by Y because of the perfect correlation at h,s, 560 nm and 576 nm, the above feature is also the case in the wavelengt,h dependence of the correlation diagrams. F,,,,(h. 1.. AY) WI’SUS F,,,,(h,. I’. A Y). A quantitative agreement between predicted values by the U(‘T model and values obtained by the experiments is examined as follows. First. do.u.o(h,, = 583) is assumed to be zero. because AC, 1).,(583) is nearly zero NS coinc-ides shown in Figure 1, and because the isobestic point of HbM Milwaukee with that of isolated Y chains: t,he latter fact, indicates t,he absence of an> contribution by n subunits to Ao.t).o(583). Therefore. F,,,,(583. I’. A Y) can br equated to YP. By comparing the correlation diagram of Y,, WTSIIS Y. which is obtained by the GCT model, with that of F,) ,)(5X3. I’. d Y) versus F,, ,)(560. Y. d k’ j. the ratio of the oxygen-binding equilibrium constants of n and /3 subunits in the ‘I’ state, Ki/Kz. can be estimated to he between 2.5 and 13 (the most probable value is 51). By using the most probable value. the ratio of ilo o o(588) and do.1).,(588) WII also be estimated to be l/2 from the deviation of ii’,, ,, (588, k’. A I’) from Y = 12 at P,*. Secondly, ilo.~)&) a.nd Ao.I).&) are obtained by assuming a linearity of t.hc absorbance change against the wavelength between 583 nm and 5X8 nm. Thirdly. do.~).o(A) is obtained by eyuation (11) by using do I) ,:,,(A) in Figure 6. These valur>s are summarized in the upper four rows of Table 4. By substituting the values of’ ~o.I).,(/\). Ao.I).~(/\) and o.IL~(X) into eyuation (22). t,he degrees of deviation of F ,,,, (A. Y. d Y) from Y = l/2 at P,, at various h values bet,ween 5X3 nm and 588 nm are estimated and summarized in the fifth row of Table 1. The degrees of the deviation of E’,, ,) (A. Y. A Y). which are estimated above. agree fairly well with those obtained by the experiments (c.ompare the lower two rows of Table 4). For other values of Ki/Kz. 2.5 and 13. the deviations of F,, l) (A. Y, A Y) are
OXY(:EX;ATION
RETWEES
Hb \ AXD
/l SUBTSITS
475
T..\HI,E 4 dependence of Ao.n .(A), Ao.II.~(/\), Ao.u.,(h), Ao.n.,Jh) F,,,, (A, Y, AY) from Y = l/Z at P,j,,
Wavelength
Wawlength
(nm)
5u3t
5x4
0f)00
- emu
5x4.5
5x5
and deviations
586
- 0~042 - 0~056 - 0084
,587
5uut
- 0.1 12
- @I10
:Lh I) I (0 I) ) :A(1 I)0
0.1 15
:do I) Q
0400
a() ‘) T2Q2 Ikiation of P,, ,) predicted (T/o)
0.1 15 651
12*3
-17+2
-7*1
-.5*1
-4*1
Drviation
6+1
1X&2
-lo+2
-7+1
-j*1
-4&l
of F,, ,, experiment
t The vnlurs arc obtained taxperimental values.
by setting
0~092 - 0004 wotio
the deviations
04~81
oa59
0946
- oG39
- 0.058
- oG67
owe
of F,,,,
of
0023
0900
- 047 1
- 0.070
- O-045 - 0~105 - 0.160
- 0210
to br equal to the corresponding
within the precision of the predicted values estimated for the most probable value, Ki/Kz = 51. Therefore, the conclusion of higher affinity of ,9 subunits than n subunits. which has been stated above, is true even when the effect of the quaternary structural formation of the tetrameric form is taken into account. We thank Dr T. Iizuka, Dr K. Imai, Dr K. Nagai, Dr H. Morimoto. Dr T. Shiga and Prof. M. Kotani for useful advice and discussions. We also thank Dr K. Nagai and Dr M. Yamazaki for their gift of M-type abnormal haemoglobins. REFERENCES Adams. 11. L. Xr Shuster, J. M. (1974). Riochem. Hiophys. Rrs. ~‘om,mun. 58. 525-531. Amone, A. & Perutz, M. F. (1974). lVuture (London), 249, 34-36. Asakura. T. & Lau, P. W. (1978). Proc. AVat. Acad. Sci.. I’.S.A. 75. 546%5465. Benesch, R., Benesch, R. E. 8r Yu, C”. I. (1968). Proc. Xut. Acccd. Sci., l:.S.A 59, 526-532 Bonaventura, J., Bonaventura. C., Sullovan, B.. Ferruzzi. G.. McC’urdg, Y. R.. Fox. ,J. & Moo-Penn. W. F. (1976). J. Biol. (‘hem. 251, 7563-7571. Brunori. AMM.,Antonini, G. 8 Wyman. J. (1970). J. Mol. Riol. 49, 461-471. (‘hiancone, E., Norne, J. E., Bonaventura, J., Bonaventura, (‘. Hr For&n. S. (1974). Riochim. Bioph.ys. ilcta, 336, 403-406. (‘hiancone, E.. Some, J. E., For&n. S., Bonaventura, .I.. Brunori, M.. Antonini. A. Ki Wyman. J. (1975). Eur. J. Biochem. 55, 385395. (‘hianconr. E.. Borne. J. E., For&n. S.. Mansouri. A. & Winterhalter. K. H. (1976). FF:BS Letters, 63. 309-312. Davis. D. G.. Lidstrom, J. R.. Baldrassare. J. ?J.. (‘hararhe. S.. Jones. R. T. & Ho, (‘. (1971). J. Mol. Hiol. 60. 101~111. l)ax-is. D. G.. Lidstrom, ,J. R., Baldrassare. J. ,J. Kr Winterhalter. K. H. (1976). l”fC:BA’ Letters, 63, 309-312. Drabkin, D. L. (1946). J. l?iol. (‘hem. 164, 703-723. Enoki. Jr. 8 Tyuma, I. (1964). Jap. J. Physiol. 14, 280-293. Gibson. Q. H. (1973). Proc. Xat. Acad. Sci.. 1T.S.A. 70, 1-4. Gray. R. D. XT Gibson. Q. H. (1971). J. Riol. Chem. 246, 5176-5178. Hayashi, A., Suzuki. T. & Shin, M. (1973). Riochim. Biophys. Acta. 207, 18-29. Huang, T. 8: Redfield, A. G. (1976). J. Biol. Chem. 251, 7114-7119. Huestis, W. H. 8r Raftery, M. A. (1972a). Biochem. Biophys. Res. Commun. 49, 1358-1365 Hwstis, VI;. H. Rr Raftery, M. A. (1972h). Biochemistry. 11, 1648-1654.
37G
A. N.~Sl’i)A-liO~:~AMA.
H. ‘l’A(‘HIB.?iNA
ANl) :I. tVAlj.4
Huestis. W. H. K- Rafter?. 11. A. (1973). Biochrrnist~y. 12. 6:?31m %3S. Imai, K. (1979). J. Mol. Riol. 133, 233-247. Imai. K. H: Yonetani, T. (1975a). .I. Rio/. (‘hum. 250. 224i--2231. Imai. K. Kr Yonetani. T. (1975h). J. Hiol. Chem. 250. 7(339-7098. Imai, K.. Ikeda-Saito, hf., Tamamoto. H. K: Yonetani. T. (19X0). J. Mol. /
Preference of Oxygenation Between a and /I Subunits of Haemoglobin Results of Multidimensional
Spectroscopic
Observation
Department of Physics, Faculty of Science The University of Tokyo, Bukyo-ku, Tokyo 113, Japan (Received
8 October 1982)
The distribution of oxygen between the subunits of haemoglobin was studied spectrophotometrically. The difficulty in discriminating the spectral changes upon oxygen binding to the a or fl subunit can be surmounted by means of multidimensional spectroscopic observations and a correlation analysis of the data. M-type abnormal haemoglobins are used as a control against normal haemoglobin because only one type of its subunits can bind oxygen. A multidimensional spectroscopic measuring system, which has been developed in our laboratory, makes it possible to carry out simultaneous and continuous acquisition of a set of spectroscopic data at several wavelengths on one sample solution during the course of increasing or decreasing the partial pressure of oxygen. The data-storing function of a magnetic disk memory provides enough precision for a rigorous investigation of the correlation of oxygen equilibrium curves measured at several wavelengths. No chemical modification to enhance the spectral difference between subunits is necessary, In conclusion, by detecting slight differences between the oxygenation-sensitive bands of iz and /3 subunits. the fl subunits are found to have a higher affinity for oxygen than the u subunits.
1. Introduction Haemoglobin has been regarded as a typical model for allosteric proteins. Two a and two /3 subunits associate to construct an oligomeric structure cu2f12.The oxygen molecule plays the role of a homotropic allosteric effector by itself when it is bound to one of the haem groups, one of which is located in each subunit. It enhances the oxygen-binding affinity of neighbouring haems through haem-haem interaction,
and the well-known S-shaped oxygen equilibrium curve is produced. The work presented in this paper is a detailed study of the spectroscopic changes of haemoglobin during the course of oxygenation or deoxygenation in order to clarify which subunit, a.or /I, preferentially binds oxygen. The method used is highperformance multidimensional spectroscopy (Wada et al., 1980). It is specially t Author to whom all correspondence 0022~-2836/83/070451-26 $03.00/O
should be addressed. 151
0 1983 Academic Press Inc. (London) Ltd
‘H n.m.r.
Ahsorption of harm
M&hod
(‘o-Fe
hybrid
Proto-haem~m~so-harm hvbrid
02
0, “0
\‘B
(1’1) 7.4)
his-Tris
tlis-‘his
llis~‘l’rw
Phosphatr.
(i)H 7.3)
( IIH 7.3)
(pH 7.2)
( IJH i+b,
phosphate (pH T4)
Sodium his-Tris
i)
(pH 7
(pT) 6.x)
(pH
(pH S.&i + DPC 01’ THI’ f DPC: (pH 7%)
phosphatr 1)hosphatr
(~1) 6X) DPG OL’ IHP
his-Tris
Potassium Potassium
02 (‘2
5)
(pl)
his-Tris
phosphate phosphate+
(pH 6.5 5.9)
(pH i+k?] phosphate*
phosphate
(pH 7.0)
(‘(’
Potassium
Sodium
Phosphate
(pH 74)
phosphate
Sodium
7.0)
7.0) (pH 6-9)
(pH
(pH
Phosphate/borate his-Tris (pH 74)
Phosphate
Phosphatv
his-Tris (pH 7) Horatr (pH 9) Phosphate (pH 7) his-Tris+ DPO
(‘ondition
tetrnm,ers
0,
difference
in haemoglobin
S;odium Sodium
02
the first
0 2 binding
+leso)2/qproto)2 ~(proto)2~(meso)2
rnet~hybrid met-hybrid
(‘2
high~spin low-spin
(‘2 (‘2
02
rate
dissociatiotr
“2
hybrid
rate
associat,ion
affinity
“0
Ligand.
C’S met-hybrid
Mn-Fc
Modification
T.\KLE: 1
The oxygen af$nit?y of th,e + and /3 subunits
OXYGENATION
BETWEEN
Hb 1 ASD
p SUBUNITS
153
suited to our purpose since it permits us to distinguish the difference in the degree of oxygenation between unlike subunits by detecting small variations in spectroscopic properties (Philo et ul., 1981; Ueda et al., 1970) and comparing them with the aid of a digital data-storing system. A considerable number of reports have already been published on the preferential binding of oxygen to haemoglobin subunits, as summarized in Table 1. These previous studies can be broadly classified into two categories with respect, to the type of technique used, that is, optical spectroscopy of haemoglobins with a modified haem group such as cobalt-substituted haem, and nuclear magnetic or electron paramagnetic resonance studies on intact or spin-labelled haemoglobins. The optical measurements can be performed with a high signal-to-noise ratio, but it is so hard to differentiate the oxygenation of n and /3 that a modification of the haem group has been necessary to enhance the spectroscopic difference between them. The magnetic studies, on the other hand, can easily make this differentiation because of well-separated signals for IY and 8. while they have difficult’y in assigning the resonance peaks (Huang 8r Redfield, 1976). ln Table 1, it is interesting to find that the optical and magnetic methods have produced opposite results; namely, the optical method has found the /3 subunit to have the higher oxygen affinity, but it is the other way about in the magnetic studies. The modification of haem groups in the optical measurements might be a we have examined the detail of t’he source of this discrepancy. In this context. optical properties of haemoglobins without any modification of haem: t,he simultaneous and multidimensional data acquisition provides enough information for a precise analysis. The symbols
F,,,,(h. Y. AY),
O.ILi(A, Yi). so I) i(h. 1;). 0 I).,*&
Y 1,
o I).~(/\. I-, AY). AO.I).i(/\).
Ao.r) o(h).
used in this paper
are defined
below
fractional change of optical density of haemoglobin (N$~) at wavelength h and degree of oxygenation Y (defined in eqn (1) and expressed in eqn (12)). fractional saturation of haemoglobin with oxygen. fractional oxygen saturation of subunit i = x or 8. half of the difference between Y, and Y,. optical density of subunit, i in a haemoglobin tetramer at wavelength h and at Yi. optical density of an isolated i chain. change of optical density of subunit i caused by the subunit association. optical density of a haemoglobin tetramer. sum of 60 o.,(h. Y,) and 8o.~.&h. Ya). i.e. equation (8). oxy-minus-deoxy absorbance of the isolated i chain defined in equat’ion (5) oxy-minus-deoxy absorbance of the haemoglobin tetramer defined in equation (6). oxy-minus-deoxy difference of o.~)o(X. Y, AY) defined in equation (9).
454
.I. SASIJDA-KOUYAMA.
H. l’A(‘H1H~iX.i
AND
A. WAD;\
2. Principle of the Analysis (a) The fractional
change of optical
denaity
Hitherto the fractional oxygen saturation of haemoglobin has been expressed in terms of fractional change of optical density at the peaks of the oxy-minus-deoxy difference spectra in the visible region. such as 540. 560 or 576nm. In this procedure the equal affinity for oxygen of the different subunitas or the identity of their spectroscopic properties is implicitly assumed. The latter is contradictory to the spectroscopic evidence which shows a difference between t,he subunits. while the former has not been confirmed. In this section, therefore, we shall discuss first what conditions must be satisfied in order to express the fractional ox?;gen saturation of haemoglobin. Y; in terms of the fractional change of optical densit)?. F,, ,,(A, Yz, YD)* which is defined as:
when there exists a preferential oxygen binding to a or /3 subunits. Let us assume that the optical densities of isolated z and /3 subunits in haemoglobin t,etramers can be described by the sum of the opt’ical densities of isolated x and /I chains, and an additional term which describes the effect of the subunit association :
(h. Yj) = 0 l).i(~. Yj)+60 I).;(/\. Ii), () ‘) i mx2p2
i = ~ 01’ p.
(2)
where the degree of oxygenation of each subunit, is explicitly taken consideration. Hence the degree of oxygenation of haemoglobin molecules. related to Y, and YP as follows: I;+
Y/) = 2Y.
(3)
From equations (1) and (2). by assuming the characteristics of the subunits in a haemoglobin expressed as follows : F,,,j(A. Y,. YD) = C{[O.I)i(h.
additivity tetramer.
Yj)-O.l).i(h, 0)J + [60.1, &i, I’;) -60
do I) ,(A) = 0 I) ;(A, 1)-o ,#$)
= 0.1) &(A.
Here, an obvious relation between oxygenation of isolated chains : 0 I).i(h. Y;) -0
I),(/\, 0).
density
I).i(h, 0) = Llo.l).i(h) 1;.
are
i = i\ or j?.
1)-o.i~.,2~$.
the optical
of spectroscopic F,,,,(A. Y,. I’,{) is
I) i(‘\. O)~}/L10.1).,*p2(h)
where the quantities. do.~).,(h), do IQ(A) and Ao.~).,~,(h) amplitudes of the optical density change, that is:
d0.l)
into Y. is
maximum (5)
0). change
the
t(j) a.nd the degree ot
i = ‘I or /3.
(71
OSYGEKATIOK
is used. For simplification, as:
BETWEES
the quantities
o.D.&
Hb R AND
455
fi SCBUNITS
~.n.~(h. Y. AY) and Ao.~.,(h) are defined
Y, AY) = So.n,(h, Y,)+~o.D.&~, Y&,
Ao.~.~(h) = m.&
1)-on.&
(8)
0),
(9)
where AY = (Y, - Y,)/2. When Y = 1 or 0, the variable AY is equated to zero and omitted from the parenthesis in equation (9). Then equation (4) is written as: F,,,,(/\, Y. AY) = 2{[Ao.v.,(h)+Ao.~~.~(X)]Y+ [ -Ao.u.,(h)+Ao.r~,(h)]AY +o I).& I’, AY)-o LX& O)}/Ao D.,&),
(10)
where the variables Y and AY of F,,(X, Y,, Ya) are substituted equations (2), (5). (8) and (9), equation (6) is written as:
for Y, and Y,. From
Ao.~.,~,~(h) = S{Ao.n.,(h) + Ao.r).,(h) + Ao.I)&}.
(11)
Thus, a final equation for F,,,,(/\, Y, AY) is g’iven in terms of the spectroscopic properties of cxand /I subunits: F,,,,(L Y, AY) {do I).,(/\) + Ao.D.,(~)} Y + { -i Ao.D.,(/\) + Ao.~.~(h)}d Y + o.I).& Ao.r).,(h) + Ao.D.~(X)+ Ao.r).&)
Y. A Y) - O.D.&
0)
(121
When the effect of subunit association on absorbance spectra is small enough, the following relation can be adopted: o.D.& and in the following
Y. AY) < om,(h. Y,)+o II.,@. Y&,
(13)
discussion we assume: Ao.I).~(/\) 6 Ao.r).,(h) + Ao.I~.~(~).
(14)
Incidentally, the relation, F,, ,,(A, Y, AY) = Y, which has been traditionally used, is a special case in equation (12) when A Y = 0 and the optical density change caused by the subunit association is proportional to Y: o.r).&
Y, AY)-o.I).&
0) = do.n.o(h)Y.
(b) F,, ,, (A, Y, AY) at several characteristic
(15)
wavelengths
In the previous section we correlated the fractional amount of spectroscopic change, F,, ,)(A, Y, A Y) with the degree of oxygenation of a haemoglobin tetramer (cx&), Y, and the difference between those of the ,!I and LXsubunits, AY. The structural change caused by the association is known to produce a slight change in the absorbance spectra of n and ,3subunits. The spectroscopic evidence reported by Ceda et al. (1970) is exhibited in Figure 1. It indicates that the oxy-minus-deoxy difference spectra of haemoglobin tetramers do not coincide with the mean of those of isolated (Yand /3 chains. 16
a)
In this section several characteristic wavelengths. written in a simple form by the intrinsic variables
at which ecluation ( 12) can INS E’ a.nd Al’. are discussed.
(1) hi (or Ai) : Thr wne~elength whew the spectrwr/ is insmsitiue i.e. o.D.&. Y, AY) = 0; (or hj where eyuat,ion (13) holds). At this wavelength,
equation
(12) can be reduced
I*‘,),)(hi* 1’. 0)‘)
= I’+
to xuhunit
nssocintiw~
t,o:
-&).I)
J/ii) + -~--do m&A,) d )d0 I) .(/\i)+dO I) a(h,)
(Itij
The wavelength at which the unbroken line (a2/12) is located at the mean position ot the dotted (a) and the dotted-broken (8) lines in Figure I sat’isfies this condition. Similarly. the associat,ion effect can be considered as negligible at the wavelength Ai, where the relationship of equation (14) holds. The condition is satisfied in a wavelength region at which an unbroken line is located in between t,he dotted and the dotted-broken lines. In this region, we have:
F,,,,(hj, Y,flY)
+ Y+
--do
I&(/q)
Lk.n.,(h;)
+A0
1)$(/i:) il l’
+flomp(A:)
cmQ(A;.
Y,
d Y)-0
+ A(,
I) .(h))+do
I) &.
O)-do -.--___ I) fi(/\;)
1) &I;), (17)
OXYGENATION
BETWEEN
Hh n, AND
157
fi RVBUNITS
The second term on the right side of equations (16) and (17) is the correction term for the difference in oxygen affinity between n and /3 subunits. The third term in equation (17) denotes the effect of the structural changes caused by the subunit association on the absorbanee spectra. (2) A,: ‘x-/3 equi-sensitive
wavelengths.
i.e. do.n.,(A,)
= Ao.I~.~(/\~); for
example
Ae = 540. 560 or 576 nm. At this wavelength: F,,,,(h,. From equation
Y. AY) =
2Ao.r~.,(h,)Y+o.r~.o(h,, Y, AY)-O.DQ(h,, 2AO.D.,(h,) +Ao.r).Q(h,)
0)
(18)
(15) we have:
F,,,,(/\,, Y.AY) = Y.
(19)
Thus, even if the structural change caused by the subunit association significantly affects the absorbance spectra, when on o(h,, Y, dY) changes linearly with the degree of oxygenation (Y), F,, ,,(A,, Y, AY) may be equated to Y. (3) Ass: fl-sensitive
wavelength,
At this wavelength.
equat,ion
F,, 1,(4,s 1’. AY) = Y+AY+
= Yp+
i.e. do.n,(hs,)
= 0.
(12) becomes:
~.~).~(h~,, Y, A Y)-on.&,.
0)-
Ao.l).p(has)+Ao
o.r@$),,
Y,AY)-o.I).Q(h,,, AO.I).&)
do.n.o(AsS)( Y+AY) l).(&)
O)-Oo.l).&ps)Y~
+Ao.r).Q(AsS)
of Therefore F,, ,) (A,,, Y. d Y) can be written in terms of the degree of oxygenation the #l subunit, Yfl = Y +A Y, and the correction term for t’he structural change caused by the association. (4) A,,: a-sensitive
wavelength.
In the same manner F,),,(h,,.
Y,AY)
i.e. Ao.I~.~(/\~~) = 0.
as equation = Y,+
(2(l), F,,,)(h,,, Y, AY) becomes:
O.I).Q(h,,, Y,AY)-o.l).a(A~s, AO.I).,(h,,) +A0
O)-Ao.l).Q(Aas)Y, I~.&,)
The equation contains the degree of oxygenation of subunit Y, = Y-A correction term for the structural change caused by the association. (c) The effect of subunits
association
(211 Y, and the
upon F,,,, (A. Y. AU)
At the upper side of Figure 2, enlarged and schematically drawn spectral lines 01 isolated Y and fi chains and the haemoglobin molecule (a2f12) in Figure 1 are shown. With regard to the effect of the structural change caused by the association on the absorbance spectrum, there can be considered to be three types of spectral tetramer ; these relationships between cx and p subunits in a haemoglobin
158
a ,no2pe
Pina2Pe \
:::t;-
i \ ! i \
FIG:. 2. Schrmatic illustrations of sevrral types of’ absorption tliflrrr~n~~~*hlw(,t ra ~.~llal,ged wound tlrc, lvavelengths of isohestic points of thr isolated z and b chains (0). Top: ohsrr,vcvl spwtra of’z,& (__ ). and isolated #I ( ) and j3 (~ - -) chains. Hottou: obser~~rtl sp
relationships fall into two groups. The first is of the type shown in Figure d(a). which is sensitive to the quaternary structure formation. The second includes the types shown in Figure Z(b) and (c) where t)he spectrum is insensitive to t)he quaternary structure formation. In the latter case, according to the discussion in the [JIeViOUS section. the wavelength dependence of F,, ,) (h, Y. d Y) in the vicinity of 58.5 nm + hi is as in Figure 3. As the wavelength goes from X, to a region shorter than the isobestic point of ,I subunits, h,,, the contribution of the /3 subunit to the absorbance change increases
OXYGENATION
BETWEEB
Hb 7 ASl)
/3 SlTBITSlTS
15!)
kc:. 3. Enlarged difference spectra near .5%5 nm of typ b and type c of Fig. 1. The spectral wmtribution of the subunits. LIo.I).&I) and do I). and the effect of the subnnit associat~ion, do 11o(h). in equation (11). The length and the direction of arrows indicate t,he magnitude and the sign of do I) z. ocll~,andd~~n.o.(-)r,~z/~:(---)r:(---~-)~.
from an amount equal to that of a to an amount much larger than ~1. As the wavelength exceeds the isobestic point of/l subunits, A,,, to another A, in a longer wavelength region, the contribution of Y subunits to the absorbance change> decreases from a large amount to an amount equivalent to that of j3 values. In this context, the following experimental schemes were designed to examine the actual spectral types (see Materials and Methods, section (d) for details). First. the type of the difference spectra of the subunits in the tetrameric state in the vicinity of 585 nm, which corresponds to hi, is determined. To do this M-type abnormal haemoglobins, in which only one type of subunits can be oxygenated, are measured as a control. Secondly, when the results of the above experiments havcl shown that the spectral type is b or c, oxygen equilibrium experiments for F,, ,) (A. Y, A Y) of HbAt at A, and at a series of wavelengths in the vicinity of 585 nm \ = hf) are carried out. Thirdly. from the correlation diagrams of F,, ,,(A,,. Y, A Y) t!eTS%LsF ,,,, (A,, Y. AY) and F ,,,, (A,,, Y. A Y) z’ersus F,, ,,(A,, Y> d Y), information on the oxygen affinity difference between o( and /? subunits in the tetrameric state is obtained. ‘r Abbwviations
used: HbA. human adult haemoglobin:
c.d.. circular dichroism
460
.I. S~SI’I)A~liOU\iAblA.
H. TA(‘HlH.AS.1
3. Materials
.\SI)
.1 LVi\l),\
and Methods
(a) Xfflrritr/~~
Human adult, hiuvnoglobin was prepat’ed by thv method of Imai 8 Yonrt,ani (l!ViSo.h) with modifications. Ten ml of human blood were quashed several times with 0.9(:,, (wiv) sodium chloride. Packed red cells were then haemolysed by adding 1.5 ~(~1.distilled lvat,er and 0.5 vol. toluene (Drabkin. 1946). A clear haemoglo&n solution was obt,ained by centrifuging (1Ti.000 revs/min for 20 min) and freed from residual organic phosphates t,hrough a Sephadex 025 column (3.5 camx 60 cm) equilibra,ted with OG5 \q-Tris. HC’I. 0.1 st-KC’1 (pH 5.4) (Henesch et nl., 1968). Then the haemoglobin solution was ~ottc~cntratrd t,o about I mht-haem hy ultrafiltration through an Atnieon Y31 IO or ITIM10 mt~mbranc and dialysed against) thv buffer used for oxygenation clxperimrnts. On the day before the ~rtf~asttr.C~~~ertts. t,hc dialpet haemoglobin solution was adjustid to the desired concrntration. t,hr NAI)J’H-frrredoxitt reduction system was added. and the sol&m was incubated overnight (Hayashi rt (I/.. I!Ii:li. All the cornportents of the Inet,haemoglobin reducing system were purchased from Sigma (‘hemical C!o. (without, furt,her purification). The abovc~ operations wt’tv varried out at 4 C’ and all of thr measurements were tinished during the day following ittc~ubatiotr. ‘I%(. abnormal haetnoglobins, HbM Milwaukee and HbM Host,on wrrv gifts from I)r K. Sagat (Na.ra Medical l’nivrrsity) and I)r $1. Yamazaki (Kunra.ntc~t.o I’nivt:rsit.yl.
The apparatus used was developed t,o obtain sitttultattrously 3 spectroscaopic di~nc~trstotts. that is. absorbance, circular dichroism. fluorescence inttrnsity (not used in the present study) rt 01.. 1980). Thr partial oxygen pressure of the and Rayleigh scattered light intensity (Wada solution was mrasured con tinuouslp in t htx optical c*uvet)te by a ( ‘lark-type electrode made by Cnion Qiken (‘0. Ltd (Hirakata.
= / -0a48+0~016~
x J&A,,,,1
Therefore. in order to carry out a measurement of P;, 1jrecision. the following c:onditioti ttiust by satisfied :
and the wavelength
for measurements tnrtst 1~. IA- /!,,,,I> 12 n1n.
OXYGENATION
BETWEEN
Hb z AXD
/3 SUBUNITS
46 I
(a) REPRODUCIBILITY
523 ms 0.0
1
:
50ms
I
; 233
: 9.5 s/l
TEST
T
125 ms/ 50samples * * I 246
-:
* 259
-:
? 272
1 -:
I
1: -: 285 nm
I
loop
233 nm * (b)
)
259nm
,
285nm -
,
285 nm’ 272 nm’ 259 nm ’ 2 0 11 6 .\
246 nm’ 233 nm’
FK:. 4. A test ofthe accuracy ofwavelength setting. (a) AI example of t.he time-course of a wavelength scanning programme. The absorption spectrum of R’-AMP in water,. (b) The fluctuation of observed optical densities measured at the points indicated by arrows in (a). The arwws at the right in this Figure denote the scanning direction to shorter (b) and longer (P ) wavelength. Each continuous line consists of Z(H) data points which are repeatedly measured accoxiing to the scanning programme indicated in (a) Since the observed optical density fluctuation at the slope of the spectral line is the same as those at its summit,
or through
the precision
of the wavelengt,h
setting
is estimated
to he 0.02 nm or better.
The half-intensity
bandwidth of the spectrometer in the present study was kept at 1.1 nm. which is the narrowest in the automatically slit-servo mode of our apparatus. (c) Experimuatal
Thr experiment,al
conditions are summarized
conditions
in Table 2.
(d) Exp:prrimrntal procedure X series of experiments to obtain a deoxygenation or an oxygenation curve whkh probed by mult,idimensional spectroscopic observahles consists of the following steps.
is
462
HbA
HbA+2
mwIH1’
Thr same as abovr
t At this wavelength $ At this wavelength
‘1‘1w Salllt’ it8 itho\ <’
WV also measured light-scattering intmsitiw we also measured rircular dichroistn
(i) I)eoxygenatkm (1 ) An oxy spectrum of’ a fully oxygenated sample solut,ion (I ml) is measured. (2) Gradual deoxygenation is started by flowing oxygerl-free nitrogen into an optival cuvette containing the solution. (3) A series of absorbance measurements at several wavelengths is c-arried out according to the computer commands which are programmed just prior to t.hc (Axperiment,. (4) The scanning over the wavelength axis is repeated during the
the air into
the cuvette.
a series of spertroscopic
measurements
of thr
OXYGENATION
BETWEEX
Hb x AND
p SI’BI’XITS
463
reoxygenation process is carried out in the same manner as the deoxygenation experiment. mentioned above. (2) When the sample is fully reoxygenated. a reoxy spectrum is measured. (3) By adding 2 mg Na&O,. another deoxy spectrum is measured. (4) The equivalences of the oxy and reoxy spectra (measured at (i)(l) and (ii)(2), and of the deoxy and Na&O,-deoxy spectra (at (i)(9) and (ii)(3)) are examined to determine whether any denaturation of auto-oxidation occurred during the course of the experiment. For HbA in phosphate buffer, only the deoxygenation experiment (series (i)) was carried out For M-type abnormal haemoglobins, the Na&O,-deoxy spectrum was not measured because the haem-iron of the abnormal subunits is also reduced by Xa,S20,.
4. Results (a) M-type
abnormal
haemoglobins
Figure 5 shows the correlations between the change of absorbance at 560 nm and those at other wavelengths designated in each diagram. The isobestic points are found to be 583.0 nm for HbM Milwaukee and 586.0 nm for HbM Boston. These two wavelengths are close to the isobestic points of the
isolated chains, 583 nm for 0:and 588 nm for /3. respectively shorter
and the other,
588 nm, is longer
than
the isobestic
(Fig. 1) ; one, 583 nm, is point
of haemoglobin
tetramers. Ai,, = 584.5 nm. The above evidence shows that the oxy-minus-deox) difference spectra of (Yand /3 subunits in the haemoglobin tetramer in the vicinity of HbM Milwaukee
583.5
..**
584
. .... . -
:* *. ..
. *
:*
..
FIG. 5. The results of HbM Milwaukee. Experimental conditions are shown in Table 2. (‘orrelation diagrams of the absorbance changes in the reoxygenation experiment. The ordinate is the fraction of absorbance change at the wavelength written in each diagram. and the abscissa is that at 560 nm (l--p equi-sensitive wavelength A,). Each point on this 2-dimensional surface indicates the absorbance at 2 wavelengths for a common oxygen pressure, that is common Y. The series of the points reveals a trace indicating a correlation of these 2 obsrrvables.
464
A. NASI’LIA-KOI’YAMA.
H. TA(‘HIB.L\NA
ANI)
;I. IVAI),\
585 nm are of type c, as defined in Figure 2. Therefore. with similar experiments on intact HbA the fractional oxygen saturation of z and /? subunits in haemoglobin tetramers can be obtained from the absorbance changes at the longer wavelength side of 584.5 nm, A,,. and at the shorter wavelength side of 584.5 nm. A,,. respectively. (i) Correlation
diayrnms
Figure 5 shows that the correlation diagrams. ‘!~,,Ao I) ix, ;,x,i PPI’SUSC?,,do I) ;I,i,l ti)r HbM Milwaukee, are nearly straight lines at 45” angles. (b) Human
adult haemoylobin
The time courses of oxygenation observed for HbA in bis-Tris buffer are shown in Figure 6. The evidence that the absorbance and scattering at 505 nm did not increase Because small confirms the absence of auto-oxidation and denaturation.
,....
. .__. :::::::7A::z:l::r;zL’f:::l .::::..::. .....:_::::;:::::“~::-:.:..::::::;::IE::;:: ..........._.........................,... .-““I ........................... .......~.....................................................~ . ...... ...................................................... .(” ..__ 586 ,.__. ....................................... ....i....................... ...... .._.................................. ................................................. .... ............................ 5 ($7 ................................... ........................ ,./ ......:... .: ........I...... ; +::.: lj 88 ................................ ......... ‘, .... . . 5 89 .........-...................... ..............I ........... scat ..................._............ ........................................,.. ........ ...............
584.5 585
I
Deaxygenotwn (h)
2
.. 0.5 log(p0,)
0.1 O.D. f 0.01 [6’]/degree 1000 count
i Reoxygenatlon
PIG:. 6. Kaw plots of thr time-cwrrsr of sptxt.ral and ox~gw ~~wssure ~~h;rt~gw of HbA in bw’l’r~~ buffer. Roth deoxygenation and reox~genation processes are shown. The ordinate scales are shown in the lower right bg an arrow which corresponds to 05 for logos). WI o I) for the absorbancr. 0~01[OIldegree for the ellipticity of circular dichroism (r.(k). and I(HHI photon counts/s for light scattering (Scat) intensity. The vertical bars attached to each raw I)lot, of absorbance indicate the position of half of the total change. An isobestir point is found at 5845 nm. Experimental conditions art’ shown in Table 2.
OXYGENATION
BETWEEN
Hb I AND
,¶ SI’BUSITY ,560
% A 0.D
/ ..: ...‘.
nm
I
,..’
,:’
+-----
,/’
..576
..’ . ..“‘.““”
;;,,,_...
......
.-588
__--
-__A-
r’
-1.
I;” ,f’ :’ ;: ..zi
:.
i’
,:-
._. _.___..
. ..
‘, 503
:: :I
.I ....____...--
i I ,._,,,_,
583-.
‘.....
..
:-588 ,:’
log POP
PIG:. 7. The normalized plots of spectroscopic oxygen equilibrium curves measured at sr\-wal wavelengths for HbA in potassium phosphate buffer. At the bottom, a comparison of the 2 curves which are probed through the 1 (588 nm) and fl (583 nm) subunits. Expwimental conditions arc shown in Table 2.
absorbance changes in the vicinity of the isobestic point (5845 nm) make the direct comparison of curves in raw data difficult. the data were expressed by means of normalized curves, i.e. o/oAo.~~.and O/oAc.d., versus partial oxygen pressure (i.e. the oxygen equilibrium curves). The results obtained with potassium phosphate buffer are shown in Figure 7. 1.n this Figure, vertical bars show the positions of PSO on the abscissa, which is the partial oxygen pressure at half of an absorbance change. Their numerical values are listed in Table 3. The oxygen equilibrium curves measured at 560 nm and 576 nm coincide, while those measured at 583 nm and 588 nm shift toward the lower and higher oxygen
pressures. respectively. As the shifts are very small. WV made a comparison I.,) superimposing them on thp common axis (the lower portion of Fig. 7). The oxygen equilibrium curves at 560 nm and 576 nm are located between those at, ,583 nm and 58X nm. The oxygen eyuilibrium curves of HbA in his-Tris buffer a,nd in his-Tris buffer with inositol hexaphosphate hare t’he following characteristics. (1) The oxygen equilibrium curves depend on the wavelength used: (2) t’hey shift toward higher oxygen affinity (lower oxygen pressure) in a wavelengtjh region shorter than 5845 nm. and toward lower oxygen affinity (higher oxygen pressure) in a longer
p50
mmHg
(al
-mmtig
(b)
585
590
FI(:, 8. T)ependrnw of the oxygen l~re~~ure at halfof’a slxx*ttwxol~ic~ measuring wavelengths. An BI’IWWon the ordinate directs the I’,, of (a) Intact haemoglobin in his-Tris buffer. (b) Intact haemoglobin in hexaphosphate (IHP). Experimental renditions are shown in Table
observable (fJ5,)) of H bA upon thv the absorbance change at 560 nn,. his-Tris buffer with added inosltol 2.
OXYGESATIOS
BETWEEN
Hb a ASD
,&?SITBUNITS
467
wavelength region ; (3) the shift becomes larger as the wavelength approaches the isobestic point at 584.5 nm. The above-mentioned characteristics are well demonstrated by plotting P 50 against wavelength (Fig. 8(a) and (b)). The value of P,, decreases gradually as the wavelength increases from 582 nm to 5845 nm. Beyond 584.5 nm it jumps up to a value higher than that at A,( = 560 nm) and then decreases again gradually to the P,, values at A,. (i) (‘orrelation
diagrams
Figure 9(a). (b), (c) and (d) shows the correlation of the fraction of absorbance changes at, A,, and at hsS(~&lo.n.(h,,) and %do.n(hPS)) to that at h,(%do.n (A,)). The fact tha,t the correlation diagram between h,s (Fig. 9(a)) shows a straight line with a 45” angle implies that equation (15) holds at h,s. On the other hand, the correlation diagrams, ~/o~o.~~.(583) vewus ~‘do.r).(h,) and %do.n (588) ?)erslls “/,don(h,), show convex and concave shapes, respectively (Fig. 9(a)). Two series of diagrams (Fig. 9(b) and (c)) measured in bis-Tris buffer and bisTris. inositol hexaphosphate have common characteristics. Namely (1) the correlation diagrams of the fraction of absorbance change at lower and higher wavelengths to that at A,( = 560 nm) deviate to opposite directions, that is. below 584.5 nm the curves deviate upward from the diagonal line and above 584.5 nrn
.’ . . : (.” ,/ ,/
583
I
588
583, 5 88 .
L/’
.i’ ..:... .:’ . . .:’.: (a)
FIG. 9(a).
.. ..-* .A
50;
a
I. ...’
i’
:. ..*
.. . .*
:
,:
507
.I .. :
1 !J 11 i.
590
I:
.:.
..
. ..
:
..
591
..*.
..,.
:
..
:.
:
.:-
.;
,
.. .
,:.
:
..
’
~qT--yiq ..
/ I
:. /
.*-
::
.:
507
....
.:.
:
:
I J
(b)
:I
/
589
. ..’ :. :’
:.
El .J
590
,/
i
:.
..
.*.
..
. .. ..
,:
. .. .. .
--
..9.’ .*’
.,:
:
: .:~
,i’
591
... .*
/
.. .
..: .:
..’ ..
:.
.:.’
.:.
/
: . .~
.:’
:. .*.
a Cc) F1c:. 9(b).
(e)
:
..
:.
OXYGEXATIOS 582
83
BETWEEN
Hb (t AND
. :*
584
’
tc3SUBITNITS
4ti9
’
L 586
t87
(d )
Fit:. 9. (‘orrelation diagrams between (@lo I) (A) or O/,dc.d.(h) (ordinate axis) and %Ao 11.(560) for HbA (abscissa axis). The number in each diagram denotes the measuring warelengt,h. (a) Obtained b) the deoxygenation experiment in potassium phosphate buffer. (b) Obtained by the deoxygenation experiment in bis-Tris buffer. (c) Obtained by the deoxygenation experiment in bis-Tris buffer with added inositol hexaphosphate (IHP). (d) Obtained by the reoxygenation experiment of the same sample as deoxpgenation in his-Tris buffer. Experimental conditions arr shown in Table 2.
downward. (2) The deviations from the diagonal line are larger as the wavelength approaches the isobestic point at 584.5 nm. (3) The correlation diagram between the circular dichroism, %dc.d.(560), and the absorbance, o/,Oo.n.(560), agrees with a diagonal line. The presence of inositol hexaphosphate enhances the above deviations. By comparing Figure 9(d) with Figure 9(a). the above findings are also found to be true in reoxygenation.
5. Discussion The present work again confirms that the oxygen affinity difference between unlike subunits is maintained even in the tetrameric state. In conclusion, the oxygen affinity of /3 subunits is found to be higher than that of (Ysubunits, and the effect of inositol hexaphosphate is to emphasize this tendency. The advantage of the present work is that intact (unmodified) haemoglobins could be used, so that we are freed from any undesirable and unpredictable effects which come from the chemical modification of the protein or the attachment of probes to it. The oxygenation and deoxygenation curves of high data-point density
470
A. NASUDA-KOCYAMA,
H. TA(‘HIKANA
?tNI)
A. W.&I):\
in several spectroscopic dimensions, at a variety of wavelengths, make it possible to provide a perspective view of molecular events produced by the oxygen binding. Slight differences between the spectroscopic properties of the n and the fi subunits can be detected by virtue of the functions of our apparatus. i.e. simultaneous data acquisition and data storing. Precise comparisons of the oxygen equilibrium curves and a detailed examination of their correlations are possible w&h t’his technique.
Previous spectroscopic studies with modilied haemoglobins have provided conclusions similar to those of the present study (Brunori et ~1.. 1970: (:ra~, k Gibson, 1971: Gibson, 1973; Imai et al., 1980: Maeda et a,Z.. 1972: Makino & Sugita. 1978; Nagai, 1977: Yamamoto Pr Yonetani. 1975). except, for the Mn-Fe tiybrid haemoglobin (Waterman &, Y onetani, 1970) and the a(Meso)2/?(Proto)2 hybrid haemoglobin (Yamamoto 8~ Yonetani. 1975). Imai et al. (1980) observed the oxygenation of k and fi subunits separately under, conditions similar to ours. They estimated the degrees of oxygenation of the subunits in an iron haemoglobin. YE and Y,, by referring to the microscopic oxygen equilibrium constants of Co-Fe hybrid haemoglobin. Using their numerical values, we calculated oxygen equilibrium curves and correlation diagrams of intact iron haemoglobin with 0.1 M-potassium phosphate buffer (pH7.4) at 15” as plotted in Figure 10. The results show curves reasonably similar to ours. A study on spin-labelled haemoglobin can provide detailed information about. the haem and residues which are functionally important (Asakura & Lau. 1978: Huestis & Raftery, 1972a,h.1973; Ogawa Kr MacConnell, 1967: Ogawa et nl.. 196s: Ogata 8: MacConnell. 1971,1972a,b). However. the effect of spin-labellinp
log PO2
FIG. IO. (a) Oxygen equilibrium CU~W reproduced from the data of Imai rt nl. (IWO). (~-. (..........) yei,: (-. -. -) y,. (b) (y? orrelation diagram of I’, and Ye to Y from the data of lmai (1980).
) 1’.
rt trl.
OXYGENATIOX
BETWEEN
Hb 1 AND
p SUBUNITS
471
modification on haemoglobin function is still not quite clear. It is necessary, therefore, to be assured how functionally equivalent the modified isolated chains are to the intact isolated chains. In the study on differently spin-labelled (SL) haemoglobins, CX(SL)~/~~and a’fl(SL)‘, the degrees of oxygenation obtained from the signal change of the probe, Y, and Y,, do not compensate for each other, so that their mean values are not equal to the fractional oxygen saturation of whole molecule, Y (Asakura & Lau, 1978). Nuclear magnetic resonance is a powerful method which can investigate several conformational aspects of haemoglobin simultaneously without modifying the haem groups. However, previous studies by nuclear magnetic resonance have not provided a unified conclusion (Huang & Redfield, 1976; Johnson & Ho, 1974; Lindstrom & Ho, 1972). The reasons for this lack of agreement are the difficulties in assigning the resonance peaks (Davis et aE.. 1971,1976; Huang & Redfield, 1976), and in estimations of the degree of oxygenation, which is obtained from a separate measurement of the optical absorbance change in the visible region. In the kinetic study, kinetic pathways from one state to another are not necessarily equal to the pathways of the fluctuation between the states in equilibrium. As a matter offact, the equilibrium constant evaluated by the rates of association and dissociation of oxygen is much different from that obtained in the oxygen equilibrium experiments (Gibson, 1973). Qualitatively, our result agrees well with that of Imai et al. (1980), whereas quantitatively the separation of our two experimental curves (Figs 7 and 9(a)) is narrower than theirs (Fig. 10). The values of PsO in the present study are a little higher than their values. These slight disagreements might be due to the higher temperatures in our experiments (Imai, 1979; Imai & Yonetani, 1975a,b). (b) Problems
in the present study on which further
discussion
is required
We must point out the problems that underlie the assumptions and the experimental conditions used in the present argument. The former concern the effect of the quaternary structural change and the quaternary structure formation. and the latter the effect of the heterotropic allosteric effector such as inositol hexaphosphate. The quaternary structure formation and the quaternary structural change are manifested indirectly by the spectral change o which reflects the association of subunits (Ueda et al., 1970; Philo et al., 1981) and the haem-haem interaction. However, the additivity equations, equations (2) and (4), take only the former factor into account. Therefore, if the haem-haem interaction exists, o.~.,(h, Y,) and So.n.,(X, Y,) should be affected by Y, and vice versa, so that the additivity no longer stands. An additional cross term of Y, and Ys should be involved in the equations in a strict sense. However, the fact that absorbance spectra retain their isobestic points throughout the oxygenation process (Enoki & Tyuma, 1964 ; and the present work) suggests that the major haemoglobin populations are the fully oxygenated and deoxygenated species, whereas partially oxygenated intermediates are minorities. Thus, the additivity assumed in equation (2) may be a reasonable assumption. D.Q
472
A SASL’DA-KOUYAMA.
H TA1’HIKASA
.1X1) .. . 1VAl1.4
As for the second problem, it has been pointed out that’ the allosteric effecters. such as organic phosphates and Cl -. influence the absorbance spect)ra of oxyhaemoglobin (Adams & Shuster, 1974: Imaizumi et n/.. 1978). According to these reports. the shape of the effector-induced difference spectra does not, depend upon the kind of effecters, while the magnitude of absorption decreases in the order of inositol hexaphosphate, 2,3-diphosphoglycerate and (‘I- (Imaizumi et al.. 1978). Unfortunately, one of the peaks of effector-induced difference spectra is close to t,hp isobestic point of oxy-minus-deoxp difference spectra. i.c>. 584.5 nm. However. in the following discussions, it will be shown t,hat t~his evidence does not affect our conclusion. In our experiments. 0.1 M-KU was always added. so that. if oxygenation produces the dissociation of Cl - which affects the optical absorption. the following result will be obtained. According t’o t,he report of Imaizumi et al. (1978). 560 nm and 585 nm are the wavelengths corresponding to a peak and a zero-point, in the difference spectrum, respectively. Therefore. a series of correlation diagrams between bhe absorbance changes at A,( = 560 nm) and those at, any wavelength in the ricinitjy of 585 nm should give similar profiles. However. correlation diagrams of the absorbance changes below and above 5X4.5 nm ex hihit. opposite features as shown in Figure 9(b) and (d). We therefore conclude tha.t thcs difference of o(- and &sensitivity exceeds the effect. of (‘1 - The binding sites for ( ‘1. are not considered to k~r in one particular type of subunits (Bonaventura et tsl.. 1976: Chiancone et al., 1974,1975,1976: Nigen & Manning. 1975: 0’J)onnell et (I/ 1979; Perutz et ~1.. 1980: Van Reek et al.. 1979). On the other hand, the effect of inositol hexaphosphate may not be ignored since it’ has a significant influence on oxygen binding affinity (Tynrna) et (II., 1973) and on the oxy spectra (Adams & Shuster. 1974: Imaizumi et crl.. 1978). The presence of 0.1 M-(“1 - reduces t,he effect of inositol hexaphosphat,e on t,he oxy spectrum. but it is still significant, (Imaizurni et 01.. 197X). To examine the effect of these ions. let us compare the correlation diagrams 01’ HbA with added 2 mr\l-inositol hexaphosphate and 0.1 LX-(“~-. As shown in Figure 9(c). there are large deviations from the diagonal line in bhe correlation diagrams between o;ldo.~).(p,) and ()&!o I) (A,) : especiall\- in t)he diagram between 584 nm and 560 nm. it, appears that p subunits are ’ fully oxygenated a,t half saturation of whole molecules. On t,he other hand. the deviations from the diagonal line in the correlation diagram between A,, and 560 nm (Fig. 9(c)) are smaller tha,rr those between A,, and 560 nm. These results may indicate the dissociation of inositol hexaphosphate bound to the p’P/32 Interface (&none & Perutz. 1971). In any case. the presence of inositol hexaphosphate emphasizes t.he difference in the binding affinity (Gray c(r Gibson, 1971 ). and this supports our oxygen interpretation.
(c) Analysis generalized
of the wavelength,
concerted-transition
dependence
of correlatim
model and an assumption
diagrattrs
according
tv thr
about do u.,(h. Y. AS)
The generalized concerted-transition (GCT) model, which is an extended Monod. Wyman & Changeux (MWC’) model (Monod et al.. 1965). was proposed by Oga.ta &MacConnell (1971). They took the oxygen affinit,y difference between z and /3
OXYGENATION
BETWEEN
Hb a AND
/3 SUBUNITS
473
subunits into account on the assumption that the oxygen binding equilibrium constants of each subunit at the relaxed (R) state, KE and Ki, are equal to the experimentally measured affinities of isolated a and /3 chains. Their conclusion is that the affinity for oxygen of a subunits is higher than that of /I subunits for the tense (T) state, i.e. Klf > Kg. Let us try to interpret the wavelength dependence of correlation diagrams, F,,,,(A, Y.AY) versus F,,,,(h,, Y,AY), in the vicinity of hi,,( = 584.5 nm) for the condition that the absorbance change caused by the subunit association is proportional to Y, i.e. in accordance with equation (15). From equations (12) and (15). F,,,,(h. Y. AY) is written as: -AO.D.,(A) +Ao.D$(h) Ay Ao.Da(h)+Ao.I~.~(A)+Ao.D.Q(h) .
F,,,,(h, Y.AY) = Y+ The first derivative
(22)
of F,,,,(h, Y, AY) with respect to h is obtained as: AY + Ao.np(h) + Ao.D&}~
$&,,(A Y,AY)= {Am.,(h)
- [~Ao.D.&)
X
+ [~Ao.D.,(/\)
+
+
Ao.I,.~@)] & Ao.r).,(h)
do.~.~(A)]
$
+ [Ao.r).,(h) - Amp(h)] At A = ho,, by substituting
Ao.D.&~)
.
$ Ao.r,&)
(23)
= 0 into equation (23), we have:
Ao.Ix,@~,)
&Y ; F, 1)(A Y>A Yhs, =
1 {do D.p(ha,)+ Ao.I~.&,)}~ X
-
[~Ao.D.~(/\~,)
+do.~.~(h,J]
$
x Ao.r~,(h)),~~+ Ao.Ix,(~,,)
d - Ao.D.,(+d z Ao.~)&)l,~.
$Ao.D.,(X)I,.
I
> 0. where the relations, as illustrated -Ao.~.,&~)
(24’)
in Figure 1, among the absorbance changes: < Ao.~~.~(h,,) < 0 < Ao.~x~(h~J.
(25)
and their derivatives : & Ao.rxQ(h) < 0. & Ao.~.(h) + $ Ao.D.#)
i: $
Ao.r,.,(/\) < $ Ao.D.#)
< 0.
A/Js< h < Ls
(26)
474
il. ?;ASIlI)~-~C)I;Y~ill.~.
are used. Similarly.
-+,,(A;
Y.LlY)I,\
H. 7'.4('HIH.AS.4
ASI) .A \S'Ai,A
at h = A,, (00 I),~(,&) = 0) we have: 1
=-----
{do r).(h,,)+do.l).o(/\a,)j2 X
x
- & dO.l~.,(A)l,,~-tl;:Ali
do I).&,) i I) ,xlA,~+ 1;; A() I) Q(h)/,;\
I
I
+ IdO.l).,(h,,)-dO.I) (,(h,,)l (II, Do I) Q(h)j,,~ > 0. where the relations
among
(27) the absorbance
do.n.,(h,,)
changes:
< do I) Q(h,,) < 0,
(2X)
and their derivatives, i.e. equation (26): are used. From equations (24) and (27). the effect of the subunit association on the wavelength dependence of F,, ,) (A. Y. d Y) ‘I:ers?rs Y is found to be to produce a ~OIP convex curve below A,,,, and a less concave curve above A,,,,. respectivelg. than t,hose obtained when the effect of subunit association is neglected or happens to be zero. Since F,, ,, (A. Y. A Y) can be substituted by Y because of the perfect correlation at h,s, 560 nm and 576 nm, the above feature is also the case in the wavelengt,h dependence of the correlation diagrams. F,,,,(h. 1.. AY) WI’SUS F,,,,(h,. I’. A Y). A quantitative agreement between predicted values by the U(‘T model and values obtained by the experiments is examined as follows. First. do.u.o(h,, = 583) is assumed to be zero. because AC, 1).,(583) is nearly zero NS coinc-ides shown in Figure 1, and because the isobestic point of HbM Milwaukee with that of isolated Y chains: t,he latter fact, indicates t,he absence of an> contribution by n subunits to Ao.t).o(583). Therefore. F,,,,(583. I’. A Y) can br equated to YP. By comparing the correlation diagram of Y,, WTSIIS Y. which is obtained by the GCT model, with that of F,) ,)(5X3. I’. d Y) versus F,, ,)(560. Y. d k’ j. the ratio of the oxygen-binding equilibrium constants of n and /3 subunits in the ‘I’ state, Ki/Kz. can be estimated to he between 2.5 and 13 (the most probable value is 51). By using the most probable value. the ratio of ilo o o(588) and do.1).,(588) WII also be estimated to be l/2 from the deviation of ii’,, ,, (588, k’. A I’) from Y = 12 at P,*. Secondly, ilo.~)&) a.nd Ao.I).&) are obtained by assuming a linearity of t.hc absorbance change against the wavelength between 583 nm and 5X8 nm. Thirdly. do.~).o(A) is obtained by eyuation (11) by using do I) ,:,,(A) in Figure 6. These valur>s are summarized in the upper four rows of Table 4. By substituting the values of’ ~o.I).,(/\). Ao.I).~(/\) and o.IL~(X) into eyuation (22). t,he degrees of deviation of F ,,,, (A. Y. d Y) from Y = l/2 at P,, at various h values bet,ween 5X3 nm and 588 nm are estimated and summarized in the fifth row of Table 1. The degrees of the deviation of E’,, ,) (A. Y. A Y). which are estimated above. agree fairly well with those obtained by the experiments (c.ompare the lower two rows of Table 4). For other values of Ki/Kz. 2.5 and 13. the deviations of F,, l) (A. Y, A Y) are
OXY(:EX;ATION
RETWEES
Hb \ AXD
/l SUBTSITS
475
T..\HI,E 4 dependence of Ao.n .(A), Ao.II.~(/\), Ao.u.,(h), Ao.n.,Jh) F,,,, (A, Y, AY) from Y = l/Z at P,j,,
Wavelength
Wawlength
(nm)
5u3t
5x4
0f)00
- emu
5x4.5
5x5
and deviations
586
- 0~042 - 0~056 - 0084
,587
5uut
- 0.1 12
- @I10
:Lh I) I (0 I) ) :A(1 I)0
0.1 15
:do I) Q
0400
a() ‘) T2Q2 Ikiation of P,, ,) predicted (T/o)
0.1 15 651
12*3
-17+2
-7*1
-.5*1
-4*1
Drviation
6+1
1X&2
-lo+2
-7+1
-j*1
-4&l
of F,, ,, experiment
t The vnlurs arc obtained taxperimental values.
by setting
0~092 - 0004 wotio
the deviations
04~81
oa59
0946
- oG39
- 0.058
- oG67
owe
of F,,,,
of
0023
0900
- 047 1
- 0.070
- O-045 - 0~105 - 0.160
- 0210
to br equal to the corresponding
within the precision of the predicted values estimated for the most probable value, Ki/Kz = 51. Therefore, the conclusion of higher affinity of ,9 subunits than n subunits. which has been stated above, is true even when the effect of the quaternary structural formation of the tetrameric form is taken into account. We thank Dr T. Iizuka, Dr K. Imai, Dr K. Nagai, Dr H. Morimoto. Dr T. Shiga and Prof. M. Kotani for useful advice and discussions. We also thank Dr K. Nagai and Dr M. Yamazaki for their gift of M-type abnormal haemoglobins. REFERENCES Adams. 11. L. Xr Shuster, J. M. (1974). Riochem. Hiophys. Rrs. ~‘om,mun. 58. 525-531. Amone, A. & Perutz, M. F. (1974). lVuture (London), 249, 34-36. Asakura. T. & Lau, P. W. (1978). Proc. AVat. Acad. Sci.. I’.S.A. 75. 546%5465. Benesch, R., Benesch, R. E. 8r Yu, C”. I. (1968). Proc. Xut. Acccd. Sci., l:.S.A 59, 526-532 Bonaventura, J., Bonaventura. C., Sullovan, B.. Ferruzzi. G.. McC’urdg, Y. R.. Fox. ,J. & Moo-Penn. W. F. (1976). J. Biol. (‘hem. 251, 7563-7571. Brunori. AMM.,Antonini, G. 8 Wyman. J. (1970). J. Mol. Riol. 49, 461-471. (‘hiancone, E., Norne, J. E., Bonaventura, J., Bonaventura, (‘. Hr For&n. S. (1974). Riochim. Bioph.ys. ilcta, 336, 403-406. (‘hiancone, E.. Some, J. E., For&n. S., Bonaventura, .I.. Brunori, M.. Antonini. A. Ki Wyman. J. (1975). Eur. J. Biochem. 55, 385395. (‘hianconr. E.. Borne. J. E., For&n. S.. Mansouri. A. & Winterhalter. K. H. (1976). FF:BS Letters, 63. 309-312. Davis. D. G.. Lidstrom, J. R.. Baldrassare. J. ?J.. (‘hararhe. S.. Jones. R. T. & Ho, (‘. (1971). J. Mol. Hiol. 60. 101~111. l)ax-is. D. G.. Lidstrom, ,J. R., Baldrassare. J. ,J. Kr Winterhalter. K. H. (1976). l”fC:BA’ Letters, 63, 309-312. Drabkin, D. L. (1946). J. l?iol. (‘hem. 164, 703-723. Enoki. Jr. 8 Tyuma, I. (1964). Jap. J. Physiol. 14, 280-293. Gibson. Q. H. (1973). Proc. Xat. Acad. Sci.. 1T.S.A. 70, 1-4. Gray. R. D. XT Gibson. Q. H. (1971). J. Riol. Chem. 246, 5176-5178. Hayashi, A., Suzuki. T. & Shin, M. (1973). Riochim. Biophys. Acta. 207, 18-29. Huang, T. 8: Redfield, A. G. (1976). J. Biol. Chem. 251, 7114-7119. Huestis, W. H. 8r Raftery, M. A. (1972a). Biochem. Biophys. Res. Commun. 49, 1358-1365 Hwstis, VI;. H. Rr Raftery, M. A. (1972h). Biochemistry. 11, 1648-1654.
37G
A. N.~Sl’i)A-liO~:~AMA.
H. ‘l’A(‘HIB.?iNA
ANl) :I. tVAlj.4
Huestis. W. H. K- Rafter?. 11. A. (1973). Biochrrnist~y. 12. 6:?31m %3S. Imai, K. (1979). J. Mol. Riol. 133, 233-247. Imai. K. H: Yonetani, T. (1975a). .I. Rio/. (‘hum. 250. 224i--2231. Imai. K. Kr Yonetani. T. (1975h). J. Hiol. Chem. 250. 7(339-7098. Imai, K.. Ikeda-Saito, hf., Tamamoto. H. K: Yonetani. T. (19X0). J. Mol. /