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Enthalpy of denaturation for human hemoglobin in the oxygenated and deoxygenated state
Thermochimica Acta, 69 (1983) 115--125 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
115
E N T H A L P Y OF D E N A T U R A T I O N FOR H U M A N H E M O G L O B I N IN THE O X Y G E N A T E D AND D E O X Y G E N A T E D STATE
R.G. MULLER,
Institut
K. SCHMID
fur Radiologie der U n i v e r s i t ~ t E r l a n g e n - N H r n b e r g ,
(Key words:
calorimetry,
enthalpy of denaturation,
F.R.G.
human hemoglobin,
thermostability)
Abstract
This paper deals w i t h the e n h a n c e d t h e r m o s t a b i l i t y of h e m o g l o b i n in the d e o x y g e n a t e d
state.
t e m p e r a t u r e of d e n a t u r a t i o n
Calorimetric measurements is increased for D e o x y - H b
show that the by
about
13 K. The specific e n t h a l p y of d e n a t u r a t i o n changes from about 14 J/g for O x y - H b to 32 J/g for Deoxy-Hb. This e x p e r i m e n t a l results may have c o n s e q u e n c e s for the physiological transfer of O x y - H b
to
D e o x y - H b and vice-versa.
of the e x p e r i m e n t a l results on the m o l e c u l a r
The e q u i v a l e n t
level however is not
yet understood.
Introduction
Calorimetric
i n v e s t i g a t i o n of the specific heat c a p a c i t y of
hemoglobin-water-salt mixtures
(12) showed, as a secondary result,
two d i s t i n c t d e n a t u r a t i o n peaks for Hb. O b j e c t of this paper is the v e r i f i c a t i o n and q u a n t i f i c a t i o n of this effect.
Method
The e x p e r i m e n t s were done with an a d i a b a t i c c a l o r i m e t e r and a d i f f e r e n t i a l Uberlingen,
scanning c a l o r i m e t e r
F.R.G.).
0040-6031/83/$03.00
(11)
(DSC 2 Perkin Elmer,
The h e a t i n g rates were
© 1983 Elsevier Science Publishers B.V.
K
10 ~
K
and 10 mi---~
116 r e s p e c t i v e l y over the temperature
interval
from 280 K to about
380 K. The adiabatic c a l o r i m e t e r needs no calibration, for e n t h a l p y nor for temperature. iridium,
n-oktadecane,
neither
The DSC was c a l i b r a t e d with
and cyclohexane.
All Hb samples were p r e p a r e d from fresh human blood. Erythroc s u s p e n s i o n was o b t a i n e d by c e n t r i f u g a t i o n of fresh b l o o d with I/ citrate in addition. E r y t h r o c y t e h e m o l y s a t e was p r e p a r e d by addit of d e s t i l l e d water. The h e m o g l o b i n stock solution was p r o d u c e d following the recipes of d i f f e r e n t authors
(2,6,7,9). The stock
s o l u t i o n was c o m p l e t e l y free of salt.
The transfer of Hb into the deoxy state was o b t a i n e d by bubbl the sample with CO 2 under a CO 2 atmosphere~
Results
Ampoule sealed samples of erythrocyte s u s p e n s i o n showed in the adiabatic c a l o r i m e t e r quite d i f f e r e n t areas of d e n a t u r a t i o n (fig.
la, Ib,
Ic). The only d i f f e r e n c e thereby was the time d e l a
between sealing and m e a s u r e m e n t
(la:
12 h; Ib: 24 h;
DSC i n v e s t i g a t i o n showed that the early m e a s u r e m e n t done w i t h O x y - H b and the late one with Deoxy-Hb
Ic:40:) (fig.
(fig.
Ic) .
The
la) wa
117
E .< nO m
HUMAN
.000
£_9 W Q UJ __I ZD 0
ER¥
12ol
I
1 0,0
°750
2 m
.500
0_ < o [..< w "ira u
I
J
• w
Q_ u)
10
20
30
e.O
50
IN
DEGo
TEMPERATURE
fi@.
la: ~'specific
heat capacity"
GO
70
@0
90
CEI.SIUS
of human erythrocytes
in
completely oxygenated state. Region a: heat production of the living cells b: melting of the membrane c: denaturation
of Oxy-Hb
d: denaturation
of Deoxy-Hb.
(8)
The broken line shows the Cp of the thermally denaturated sample.
118
ERY NAT.TVE
4,,000 < rr"
f
I
/
I O,O m (-9 LLI C]
3.t50
d
LLI J
(13 -3
3.500
2~
< b< m I
(3_ U~
3°250-
........
10 , .........
20 , ......
3,0
40 , . . . . . . . .
, . . . . . . . . . . .
50 60 , . . . . . . . . . . . . . . . . . . .
"~0 , ..............
TEMPERATURE IN CELSIUS
fig.
Ib: The same as fig.
la only half oxygenated.
E <
3°75oj
10
30
0
50
60
10
80
90
TEMPERATURE IN DEG° CELSIUS
fig.
Ic: The same as fig.
la nearly
fully deoxygenated.
80
,,,,
119
Differential scanning calorimetry is not so precise as the adiabatic one but it was used to get results in a shorter time. The relation between the specific heat capacity of the samples and the denaturation enthalpy is a little bit unfavourable (fig. 2). Therefore it was sometimes difficult to define the baseline and the physical quantities as enthalpy and temperature of denaturation (fig. 3).
fig. 2: Complete DSC thermogram of
fig. 3: DSC thermogram of an
an ery sample, where the relation of c and denaturation is to be P seen.
oxygenated ery sample. The best estimate of baseline and the parallels for half value width and temperature of the maximum are shown. The denaturation temperature T is the intersect tg of the tangent of the ascending branch with the baseline.
120
The and
plotted
the a r e a s
Digital Oxy-Hb The
and
Deoxy-Hb
reason
were
of d e n a t u r a t i o n
Equipment).
is t h a t
(Wl, w 2) and be
thermograms
the
separated
have the
were
with
calculated
different
unknown
by c o m p u t e r
by
quantities,
(PDP 11/
of d e n a t u r a t i o n iterative
the w e i g h t
of d e n a t u r a t i o n
stic)
f(
computatJ
fractions
(Ah I, Ah2), c o u l d
not
I)
Ah = w I Ah I + W 2 Ah 2
with
Ah:
specific
w I , w2:
weight
Ahl,Ah2:
enthalpy
fraction
the
equations
iterative (2)
and
computation
specific
enthalpy
equation
of d e n a t u r a t i (
Deoxy-Hb
(I) was
respectivel~
transformed
into
(3)
Ah I = Ah
[z 1 + Y l
(1 - Z l ) [ I
(3)
Ah 2 = Ah
[z 2 + Y2
(I - z2) ]
Yl
of O x y - H b ,
respectively
(2)
where
of d e n a t u r a t i (
Deoxy-Hb
of O x y - H b ,
For
an u l t r a s o u n d
enthalpies
to be c a l c u l a t e d
enthalpies
(equ.
(I)
The
digitized
with
Ah I z I = Ah 2 ,
and
Yl
a n d Y2 are
of d e n a t u r a t i o n ,
=
Ah 2 z 2 = Ah I
w I Ah I Ah '
experimental
while
z I = z2-I
Y2 =
w 2 Ah 2 Ah
values
calculated
is the p a r a m e t e r
from of
the
iterat
121 The following table shows a survey of the experimental results.
Table 1: experimental results
sample type
AhI J-g
Ah2 -1
J * g
Ttg,l -1
OC
Ttg,2 OC
14.2 (0.5)
32.5 (1.3)
78.9 (0.5)
92.6 (0.5)
16.2
33.0 (3.2)
76.4
89.8 (0.5)
_--
27.1 (4.3)
___
90.4 (0.4)
15.4 (1.0)
35.6 (2.2)
79.8 (0.7)
93.1 (0.2)
20.0 (4.1)
45.6 (9.5)
79.5 (0.5)
88.3
14.4 (0.5)
32.8 (1.6)
74.1 (0.3)
91.4 (0.2)
14.7 (2.4)
33.6 (5.6)
76.1 (0.5)
85.1 (0.5)
13.6 (0.4)
31.0 (1.0)
74.7 (0.4)
91.3 (0.4)
13.4 (1.0)
30.7 (2.2)
72.7 (0.5)
88.0 (0.4)
Sample type (1):
fresh erythrocyte suspension; (2) as (I)
after 48 h sealed storage: (3) erythrocyte suspension under co2: (4) erythrocyte hemolysate ;(5) as (4) under C02; (6) Hb-solution:(7) as (6) under C02; (8) Hb-H20-NaCl mixture;
(9) as (8) under C02.
The values in parenthesis are the single standard deviations of five measurements at most.
Discussion
The transfer of Oxy-Hb into Deoxy-Hb is accompanied with an enhanced thermostability. The temperature as well as the enthalpy of denaturation are increased. Inter- and intramolecular interaction may be the reason for a change
122
of t h e r m o s t a b i l i t y .
Azari
(I) already s h o w e d in 1958 that Conalbu
and T r a n s f e r r i n became more stable against p r o t e o l y s i s and denatu by b i n d i n g
iron ions. D o n o v a n et al.
by d i f f e r e n t i a l molecules
scanning calorimetry.
(4) v e r i f i e d this finding
19
The binding of m u c h larger
to proteins may induce also a change of t h e r m o s t a b i l i t y
D o n o v a n et al.
(3) described an increased t h e r m o s t a b i l i t y
for a
t r y p s i n - i n h i b i t o r complex. Thereby only the d e n a t u r a t i o n t e m p e r a t u was i n c r e a s e d while the enthalpy r e m a i n e d unchanged. There was also Donovan et al.
(5), who p u b l i s h e d a t h r e e f o l d
increase in d e n a t u r a t i o n enthalpy for the system A v i d i n - B i o t i n , which can't be e x p l a i n e d by the enthalpy of binding. Two effects my be c o n s i d e r e d as responsible
for the t h e r m o s t a b
of D e o x y - H b :
I) H e m o g l o b i n is s t a b i l i z e d by 2 , 3 - D i p h o s p h o g l y c e r a t e
(2,3-DPG
in the d e o x y g e n a t e d state. The e n t h a l p y of b i n d i n g 2,3-DPG to D e o x y - H b however amounts only to 7 k c a l / M o l or to about 0.43 J/g of hemoglobin.
This is to small to explain an e f f e
of 19 J/g. M o r e o v e r there could not be seen any s i g n i f i c a n t d i f f e r e n c e b e t w e e n the salt- and 2,3-DPG-free samples and the other samples
(table I, sample type 6,7,8,9).
2) B e c a u s e of the increased number of h y d r o p h o b i c
interactions
b e t w e e n w a t e r and d e n a t u r e d Hb its specific h e a t c a p a c i t y i~ increased
for a ACp of about 0.33 J g -I k-1
(12). An enhanc~
d e n a t u r a t i o n t e m p e r a t u r e with the amount of AT therefore sh~ give an i n c r e a s e d d e n a t u r a t i o n e n t h a l p y with
AAh = AT • Aep.
123 For Deoxy-Hb, AAh = I3 K - 0.33 J g
-1
= 4.3 J/g, nearly
is one fourth the experimental effect.
The surplus denaturation enthalpy of Deoxy-Hb of about 14 to 15 J/g or 235 kcal/Mol of tetramer is not yet understood. Probably an alteration of ternary structure by the transition from Oxy- to Deoxy-Hb should be considered too supplementary to the well known change of quaternary structure (IO, 13). This should be done unaffected by the very established results of X-ray studies, which suggest only minimal ternary alteration in the neighbourhood of the
porphyrin ring.
From the calorimetric point of view an alteration of the ternary structure of the 01-and B-subunits doesn't seem unlikely, because the known alterations and the additional four salt-bridges of Deoxy-Hb (13) can't be responsible for the surplus denaturation enthalpy.
Calorimetric investigation of isolated a- and B-chains and myoglobin as well should give a little more insight in this problem.
124 References
Azari, P.R., Feeney, R.E.: Resistance of Water Complexes of Conalbumin to Proteolysis and to thermal Denaturation. J. Biol. Chem. 232 (1958) 293-302 Berger, R.L.: Human Isoionic Hemoglobin: Preparation and Kinetic Properties. Univ. San Diego La Jolla, Anal. Let., 6 (2), (1973) 125-138 Donovan, J.W., Beardslee, R.A.: Protein-Protein Association. J. Biol. Chem., 250 (1975) 1966-1971 Donovan, J.W., Ross, K.D.: Iron Binding to Conalbumin. Calorimetric Evidence for two Distinct Species with one Bound Iron Atom. Fed. Proc. 32 (1973) 540 Donovan, J.W., Ross, K.D.: Increase in the Stability of Avidin Produced by Binding of Biotin. A Differential Scanning Calorimetric Study of Denaturation by Heat. Biochemistry 12 (1973) 512-517 Drabkin, D.L., Austin, J-H.: Spectrometric Studies V. A Technique for the Analysis of Undiluted Blood and Concentrated Hemoglobin Solutions. J. Biol. Chem. 112 (1935-1936) 105 Hinson, J.A., McMeekin, T.L.: A Rapid Method for Preparance Crystallin Human Haemoglobin and the Separation of Crystallin Haemoglobin A in Quantity. Univ. South Carolina, Columbia, march 6 (1969); Biochem. and Biophys. Res. Comm. Vol. 35, No. 1 (1969) Jackson, W.M., Kostyla, J., Nordin, J.H., Brandts, J.F.: Calorimetric Study of Protein Transitions in Human Erythrocyte Ghosts. Biochemistry 12 (1973)
3662-3667
125
Menges, G.: Der Aktivit~tskoeffizient yon KCI in hochkonzentrierten H~mog l o b i n l 6 s u n g e n und in Erythrozyten. Dissertation, Universit~t Erlangen-NHrnberg (1973) Miurhead, H., Cox, J.M., Mazzarella, L., Perutz, M.F.: Structure and Function of Haemoglobin. III. A Three-dimensional Fourier Synthesis of Human Deoxyhaemoglobin at 5.5 ~ Resolution. J. Mol. Biol. 28 (1967)
MUller,
117-156
R., Hasl, G., Pauly, H.:
Design and performance of a precise adiabatic scanning calorimet6 for the measurement of the heat capacity of small samples in the temperature range between 283 and 353 K. J. Chem. Thermodynamics 10 (1978)
591-601
MUller, R., Hasl, G., Pauly, H.: Determination of the Specific Heat Capacity of Hemoglobin and Methemoglobin-Water Mixtures Using an Adiabatic Calorimeter. Thermochimica Acta 22 (1978) 339-345
Perutz, M.F.: Stereochemistry of Cooperative Effects in Haemoglobin. Nature 228
(1970
726-739
115
E N T H A L P Y OF D E N A T U R A T I O N FOR H U M A N H E M O G L O B I N IN THE O X Y G E N A T E D AND D E O X Y G E N A T E D STATE
R.G. MULLER,
Institut
K. SCHMID
fur Radiologie der U n i v e r s i t ~ t E r l a n g e n - N H r n b e r g ,
(Key words:
calorimetry,
enthalpy of denaturation,
F.R.G.
human hemoglobin,
thermostability)
Abstract
This paper deals w i t h the e n h a n c e d t h e r m o s t a b i l i t y of h e m o g l o b i n in the d e o x y g e n a t e d
state.
t e m p e r a t u r e of d e n a t u r a t i o n
Calorimetric measurements is increased for D e o x y - H b
show that the by
about
13 K. The specific e n t h a l p y of d e n a t u r a t i o n changes from about 14 J/g for O x y - H b to 32 J/g for Deoxy-Hb. This e x p e r i m e n t a l results may have c o n s e q u e n c e s for the physiological transfer of O x y - H b
to
D e o x y - H b and vice-versa.
of the e x p e r i m e n t a l results on the m o l e c u l a r
The e q u i v a l e n t
level however is not
yet understood.
Introduction
Calorimetric
i n v e s t i g a t i o n of the specific heat c a p a c i t y of
hemoglobin-water-salt mixtures
(12) showed, as a secondary result,
two d i s t i n c t d e n a t u r a t i o n peaks for Hb. O b j e c t of this paper is the v e r i f i c a t i o n and q u a n t i f i c a t i o n of this effect.
Method
The e x p e r i m e n t s were done with an a d i a b a t i c c a l o r i m e t e r and a d i f f e r e n t i a l Uberlingen,
scanning c a l o r i m e t e r
F.R.G.).
0040-6031/83/$03.00
(11)
(DSC 2 Perkin Elmer,
The h e a t i n g rates were
© 1983 Elsevier Science Publishers B.V.
K
10 ~
K
and 10 mi---~
116 r e s p e c t i v e l y over the temperature
interval
from 280 K to about
380 K. The adiabatic c a l o r i m e t e r needs no calibration, for e n t h a l p y nor for temperature. iridium,
n-oktadecane,
neither
The DSC was c a l i b r a t e d with
and cyclohexane.
All Hb samples were p r e p a r e d from fresh human blood. Erythroc s u s p e n s i o n was o b t a i n e d by c e n t r i f u g a t i o n of fresh b l o o d with I/ citrate in addition. E r y t h r o c y t e h e m o l y s a t e was p r e p a r e d by addit of d e s t i l l e d water. The h e m o g l o b i n stock solution was p r o d u c e d following the recipes of d i f f e r e n t authors
(2,6,7,9). The stock
s o l u t i o n was c o m p l e t e l y free of salt.
The transfer of Hb into the deoxy state was o b t a i n e d by bubbl the sample with CO 2 under a CO 2 atmosphere~
Results
Ampoule sealed samples of erythrocyte s u s p e n s i o n showed in the adiabatic c a l o r i m e t e r quite d i f f e r e n t areas of d e n a t u r a t i o n (fig.
la, Ib,
Ic). The only d i f f e r e n c e thereby was the time d e l a
between sealing and m e a s u r e m e n t
(la:
12 h; Ib: 24 h;
DSC i n v e s t i g a t i o n showed that the early m e a s u r e m e n t done w i t h O x y - H b and the late one with Deoxy-Hb
Ic:40:) (fig.
(fig.
Ic) .
The
la) wa
117
E .< nO m
HUMAN
.000
£_9 W Q UJ __I ZD 0
ER¥
12ol
I
1 0,0
°750
2 m
.500
0_ < o [..< w "ira u
I
J
• w
Q_ u)
10
20
30
e.O
50
IN
DEGo
TEMPERATURE
fi@.
la: ~'specific
heat capacity"
GO
70
@0
90
CEI.SIUS
of human erythrocytes
in
completely oxygenated state. Region a: heat production of the living cells b: melting of the membrane c: denaturation
of Oxy-Hb
d: denaturation
of Deoxy-Hb.
(8)
The broken line shows the Cp of the thermally denaturated sample.
118
ERY NAT.TVE
4,,000 < rr"
f
I
/
I O,O m (-9 LLI C]
3.t50
d
LLI J
(13 -3
3.500
2~
< b< m I
(3_ U~
3°250-
........
10 , .........
20 , ......
3,0
40 , . . . . . . . .
, . . . . . . . . . . .
50 60 , . . . . . . . . . . . . . . . . . . .
"~0 , ..............
TEMPERATURE IN CELSIUS
fig.
Ib: The same as fig.
la only half oxygenated.
E <
3°75oj
10
30
0
50
60
10
80
90
TEMPERATURE IN DEG° CELSIUS
fig.
Ic: The same as fig.
la nearly
fully deoxygenated.
80
,,,,
119
Differential scanning calorimetry is not so precise as the adiabatic one but it was used to get results in a shorter time. The relation between the specific heat capacity of the samples and the denaturation enthalpy is a little bit unfavourable (fig. 2). Therefore it was sometimes difficult to define the baseline and the physical quantities as enthalpy and temperature of denaturation (fig. 3).
fig. 2: Complete DSC thermogram of
fig. 3: DSC thermogram of an
an ery sample, where the relation of c and denaturation is to be P seen.
oxygenated ery sample. The best estimate of baseline and the parallels for half value width and temperature of the maximum are shown. The denaturation temperature T is the intersect tg of the tangent of the ascending branch with the baseline.
120
The and
plotted
the a r e a s
Digital Oxy-Hb The
and
Deoxy-Hb
reason
were
of d e n a t u r a t i o n
Equipment).
is t h a t
(Wl, w 2) and be
thermograms
the
separated
have the
were
with
calculated
different
unknown
by c o m p u t e r
by
quantities,
(PDP 11/
of d e n a t u r a t i o n iterative
the w e i g h t
of d e n a t u r a t i o n
stic)
f(
computatJ
fractions
(Ah I, Ah2), c o u l d
not
I)
Ah = w I Ah I + W 2 Ah 2
with
Ah:
specific
w I , w2:
weight
Ahl,Ah2:
enthalpy
fraction
the
equations
iterative (2)
and
computation
specific
enthalpy
equation
of d e n a t u r a t i (
Deoxy-Hb
(I) was
respectivel~
transformed
into
(3)
Ah I = Ah
[z 1 + Y l
(1 - Z l ) [ I
(3)
Ah 2 = Ah
[z 2 + Y2
(I - z2) ]
Yl
of O x y - H b ,
respectively
(2)
where
of d e n a t u r a t i (
Deoxy-Hb
of O x y - H b ,
For
an u l t r a s o u n d
enthalpies
to be c a l c u l a t e d
enthalpies
(equ.
(I)
The
digitized
with
Ah I z I = Ah 2 ,
and
Yl
a n d Y2 are
of d e n a t u r a t i o n ,
=
Ah 2 z 2 = Ah I
w I Ah I Ah '
experimental
while
z I = z2-I
Y2 =
w 2 Ah 2 Ah
values
calculated
is the p a r a m e t e r
from of
the
iterat
121 The following table shows a survey of the experimental results.
Table 1: experimental results
sample type
AhI J-g
Ah2 -1
J * g
Ttg,l -1
OC
Ttg,2 OC
14.2 (0.5)
32.5 (1.3)
78.9 (0.5)
92.6 (0.5)
16.2
33.0 (3.2)
76.4
89.8 (0.5)
_--
27.1 (4.3)
___
90.4 (0.4)
15.4 (1.0)
35.6 (2.2)
79.8 (0.7)
93.1 (0.2)
20.0 (4.1)
45.6 (9.5)
79.5 (0.5)
88.3
14.4 (0.5)
32.8 (1.6)
74.1 (0.3)
91.4 (0.2)
14.7 (2.4)
33.6 (5.6)
76.1 (0.5)
85.1 (0.5)
13.6 (0.4)
31.0 (1.0)
74.7 (0.4)
91.3 (0.4)
13.4 (1.0)
30.7 (2.2)
72.7 (0.5)
88.0 (0.4)
Sample type (1):
fresh erythrocyte suspension; (2) as (I)
after 48 h sealed storage: (3) erythrocyte suspension under co2: (4) erythrocyte hemolysate ;(5) as (4) under C02; (6) Hb-solution:(7) as (6) under C02; (8) Hb-H20-NaCl mixture;
(9) as (8) under C02.
The values in parenthesis are the single standard deviations of five measurements at most.
Discussion
The transfer of Oxy-Hb into Deoxy-Hb is accompanied with an enhanced thermostability. The temperature as well as the enthalpy of denaturation are increased. Inter- and intramolecular interaction may be the reason for a change
122
of t h e r m o s t a b i l i t y .
Azari
(I) already s h o w e d in 1958 that Conalbu
and T r a n s f e r r i n became more stable against p r o t e o l y s i s and denatu by b i n d i n g
iron ions. D o n o v a n et al.
by d i f f e r e n t i a l molecules
scanning calorimetry.
(4) v e r i f i e d this finding
19
The binding of m u c h larger
to proteins may induce also a change of t h e r m o s t a b i l i t y
D o n o v a n et al.
(3) described an increased t h e r m o s t a b i l i t y
for a
t r y p s i n - i n h i b i t o r complex. Thereby only the d e n a t u r a t i o n t e m p e r a t u was i n c r e a s e d while the enthalpy r e m a i n e d unchanged. There was also Donovan et al.
(5), who p u b l i s h e d a t h r e e f o l d
increase in d e n a t u r a t i o n enthalpy for the system A v i d i n - B i o t i n , which can't be e x p l a i n e d by the enthalpy of binding. Two effects my be c o n s i d e r e d as responsible
for the t h e r m o s t a b
of D e o x y - H b :
I) H e m o g l o b i n is s t a b i l i z e d by 2 , 3 - D i p h o s p h o g l y c e r a t e
(2,3-DPG
in the d e o x y g e n a t e d state. The e n t h a l p y of b i n d i n g 2,3-DPG to D e o x y - H b however amounts only to 7 k c a l / M o l or to about 0.43 J/g of hemoglobin.
This is to small to explain an e f f e
of 19 J/g. M o r e o v e r there could not be seen any s i g n i f i c a n t d i f f e r e n c e b e t w e e n the salt- and 2,3-DPG-free samples and the other samples
(table I, sample type 6,7,8,9).
2) B e c a u s e of the increased number of h y d r o p h o b i c
interactions
b e t w e e n w a t e r and d e n a t u r e d Hb its specific h e a t c a p a c i t y i~ increased
for a ACp of about 0.33 J g -I k-1
(12). An enhanc~
d e n a t u r a t i o n t e m p e r a t u r e with the amount of AT therefore sh~ give an i n c r e a s e d d e n a t u r a t i o n e n t h a l p y with
AAh = AT • Aep.
123 For Deoxy-Hb, AAh = I3 K - 0.33 J g
-1
= 4.3 J/g, nearly
is one fourth the experimental effect.
The surplus denaturation enthalpy of Deoxy-Hb of about 14 to 15 J/g or 235 kcal/Mol of tetramer is not yet understood. Probably an alteration of ternary structure by the transition from Oxy- to Deoxy-Hb should be considered too supplementary to the well known change of quaternary structure (IO, 13). This should be done unaffected by the very established results of X-ray studies, which suggest only minimal ternary alteration in the neighbourhood of the
porphyrin ring.
From the calorimetric point of view an alteration of the ternary structure of the 01-and B-subunits doesn't seem unlikely, because the known alterations and the additional four salt-bridges of Deoxy-Hb (13) can't be responsible for the surplus denaturation enthalpy.
Calorimetric investigation of isolated a- and B-chains and myoglobin as well should give a little more insight in this problem.
124 References
Azari, P.R., Feeney, R.E.: Resistance of Water Complexes of Conalbumin to Proteolysis and to thermal Denaturation. J. Biol. Chem. 232 (1958) 293-302 Berger, R.L.: Human Isoionic Hemoglobin: Preparation and Kinetic Properties. Univ. San Diego La Jolla, Anal. Let., 6 (2), (1973) 125-138 Donovan, J.W., Beardslee, R.A.: Protein-Protein Association. J. Biol. Chem., 250 (1975) 1966-1971 Donovan, J.W., Ross, K.D.: Iron Binding to Conalbumin. Calorimetric Evidence for two Distinct Species with one Bound Iron Atom. Fed. Proc. 32 (1973) 540 Donovan, J.W., Ross, K.D.: Increase in the Stability of Avidin Produced by Binding of Biotin. A Differential Scanning Calorimetric Study of Denaturation by Heat. Biochemistry 12 (1973) 512-517 Drabkin, D.L., Austin, J-H.: Spectrometric Studies V. A Technique for the Analysis of Undiluted Blood and Concentrated Hemoglobin Solutions. J. Biol. Chem. 112 (1935-1936) 105 Hinson, J.A., McMeekin, T.L.: A Rapid Method for Preparance Crystallin Human Haemoglobin and the Separation of Crystallin Haemoglobin A in Quantity. Univ. South Carolina, Columbia, march 6 (1969); Biochem. and Biophys. Res. Comm. Vol. 35, No. 1 (1969) Jackson, W.M., Kostyla, J., Nordin, J.H., Brandts, J.F.: Calorimetric Study of Protein Transitions in Human Erythrocyte Ghosts. Biochemistry 12 (1973)
3662-3667
125
Menges, G.: Der Aktivit~tskoeffizient yon KCI in hochkonzentrierten H~mog l o b i n l 6 s u n g e n und in Erythrozyten. Dissertation, Universit~t Erlangen-NHrnberg (1973) Miurhead, H., Cox, J.M., Mazzarella, L., Perutz, M.F.: Structure and Function of Haemoglobin. III. A Three-dimensional Fourier Synthesis of Human Deoxyhaemoglobin at 5.5 ~ Resolution. J. Mol. Biol. 28 (1967)
MUller,
117-156
R., Hasl, G., Pauly, H.:
Design and performance of a precise adiabatic scanning calorimet6 for the measurement of the heat capacity of small samples in the temperature range between 283 and 353 K. J. Chem. Thermodynamics 10 (1978)
591-601
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726-739