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An O2Hb ‘paradox’ in frog blood?
Re,~l~iralion PILvsiolo~y (1978) 35, 119 12;' Co) Elsevier N o r t h - H o l l a n d Biomedical Press
AN O,-Hb 'PARADOX' IN FROG BLOOD? (n-values exceeding 4.0)
G. LYKKEBOE and K. JOHANSEN DepartmeHl o/'Zoophysiolo,Kv, Uuiversit3' o/Aarhu.~, DK-8000 A ttrhus C. Dcttmurk
Abstract. O~-Hb dissociation curves have been determined t'or the anurans. Ram1 It'*tq~oraria and Ratul
catesheialul, and compared with h u m a n blood t\~r the specilic purpose of defining the n-wflue (Hill's cooperativity coefficient) at high O~ saturations. The Psi, values at normal conditions (see fig. 1) for each species were 24.6 m m Hg for h u m a n blood. 37.0 m m Hg for Ram~ lep*y~oraria blood and 53.5 m m Hg for Romt cateshciaml. For h u m a n blood the n-value was 2.7 at saturations from 36",, to 98". levelling off :.tl the highest saturations. For Rana temporaria Hill plots between saturations from 20". to 98",, showed 3 segments. The average n-value increased from 1.6 to 2.4 at about 50",, saturation, increasing again at about 80",, saturation to 7.3. The pattern in Rana c'atexheiatla blood was similar with the n-value changing from 1.6 to 3.1 at about 50"o saturation, averaging 3.5 between 53",, and 89",, saturation. Additionally the Bohr effect in Rana tc'plq~oraria blood more than doubled when compared at 50",, and 90",, saturation. The data show that for R. temporaria blood the tree energy of interaction associated with the binding of O, to Hb is displaced to the upper segment of the dissociation curve. Blood oxygen dissociation curve Bohr effect Cooperativity
Hemoglobin Hill plot n-values
Blood O: affinity in amphibians decreases with increasing importance of pulmonary breathing in overall O~ uptake (McCutcheon and Hall, 1937: Lenfant and Johansen, 1967). A recent study revealed a P~0 value of 60 mm Hg for the very xerophilous tree frog, Chiromautis petersi, at pH 7.6, T 25 C (Johansen et al., 1978) which prompted the question if the lung O: pressure ever was high enough to saturate the blood with O: during passage through the lung. Analysis revealed pulmonary O, pressures up to 130 torr which in equilibrium with blood h7 vitro gave saturations in excess of 950,. A Hill plot, however, revealed the saturation values to grossly Accepted/or publication 4 Juh' 1978 119
120
G. LYKKEBOE A N D K. J O H A N S E N
misalign with the linearity dictated by n-values corresponding to O~ saturations between 30 and 60°o. This suggested a striking increase in the n-value (cooperativity coefficient) at higher O, saturations and stimulated the present investigation of the shape of the O~-dissociation curve in whole blood from 2 species of anuran frogs, Ramt lemporaria and Rana calesbekma. These results are compared with values for human blood obtained using identical experimental procedures and methods.
Material and methods
Human blood was sampled by venipuncture and frog blood was obtained by needle puncture of an aortic or carotid arch. In all cases the blood was drawn into syringes, the dead spaces of which were filled with a heparin solution (50 mg-1 ~). In the case of Rana lemporaria blood samples from several individuals were pooled to yield the needed total volume. Immediately alter sampling the blood from all species were divided into 50 lal aliquots in disposable equilibration tubes (Radiometer D840) and placed in ice water. O, Hb equilibrium curves were constructed based on procedures described in detail earlier (Johansen et al.. 1976). The O, content of 20 gl blood samples equilibrated against gas mixtures of known composition were measured by a Lexocon O, content analyser. A modification of the procedures described earlier consisted in determining the O_~ capacity by equilibration against a very high Po: (i.e. 90"i, Oe) to ensure the highest possible saturation of the hemoglobin. The measured O, contents were corrected for physically dissolved O~ by using the appropriate Bunsen solubility coefficients (Christoforides and Hedley-Whyte, 1969). Particular emphasis was placed on determining the upper portions (70°, < S < 98'~o) of the equilibrium curves as detailed as possible by performing a large number of gas equilibrations in this region of the curves.
Results
Figure 1A shows O~ Hb dissociation curves for human blood and the blood from the anurans Rana temporaria and Rana catesbeiana. Figure 1B delineates the corresponding Hill plots. Each plotted point represents the mean of two determinations. All equilibrations were performed at the body temperatures of the species (in the anurans the acclimation temperature). The Pco2 levels were kept constant for all species. The respective values and the resulting whole blood pH values are shown on figs. 1A and B. The blood 02 binding properties for the 3 species differ in several important respects. Firstly, the O: affinity expressed by the P> value is highest for human blood (24.6 mm Hg) lbllowed by 37.0 mm Hg for Rana ternporaria blood and 53.5 mm Hg for Rana catesheiana.
HIGH n-VALUES IN FROG BLOOD
121
100
80
~" 60
_.9. 40
20
60 80 100 OXYGEN TENSION , m m H g
40
120
1/.0
Fig. IA. Oxygen dissociation curves of whole blood samples f r o m Homo sapiens (A), Rana temporaria (O), and Rana catesbeiana (O), at conditions shown on the figure. Each plotted point represents the mean of two determinations.
20
B 98
~6
95
90 .~ 08 80
OZ.
~
70 ~-6O
00
50 z,0
-04
J
30 •
Homo
s~ple~s
370"C
PCO 2 : ~ 2 0
o
g~ono
lemporof,Q
205'C
PCO 2 : IZ, 5 . p H : 7 6 7
•
¢~a~a
¢o~esD~eme2~*O'C
pH;740
20
-08 PC~2:96
p~=755
10
12
14
16
18
20
22
log Po2
Fig. 1B. The Hill plots corresponding to the oxygen dissociation curves shown in fig. 1A. F u r t h e r m o r e , the s h a p e o f the e q u i l i b r i u m curves vary greatly. T h e curve for h u m a n b l o o d w a s d e t e r m i n e d w i t h i n a s a t u r a t i o n r a n g e f r o m 36'!,,; to 98.4'~o. T h e Hill plot (fig. IB) s h o w s an a v e r a g e n - v a l u e (Hill's c o e f f i c i e n t ) o f 2.7, levelling o f f at the highest saturations.
122
el. L Y K K E B O E
A N D K. J O H A N S E N
in sharp contrast the equilibrium curve for Rana tenq~orarkt measured between saturation values of 20"o and 98.Y',, shows an undulating, polyphasic course. Expressed in a Hill plot the curve shows 3 distinct segments. At about 50". saturation the n-value changes from 1.4 to 2.7 and again at about 80",, 0 Z saturation another transition in n-value occurs bringing it to about 11.0. This conspicuously high n-value persists throughout the range of measurements terminating at 98.3'!0 saturation. The marked increase in n-value at higher O. saturations causes the equilibrium curve for Raml temporark/ to actually transect that for human blood in spite of the much lower O, alt]nity of Rana tumporaria blood at 50". saturation. The transect occurs at 96",, saturation corresponding to a Po. of 81 mm Hg. The equilibrium curve for Rana calesheiaml blood shows a pattern between those of human and Ram1 temporarkl blood. At about 5Y',, saturation the n-value changes from 1.6 to 3. l and at the highest saturations a slight levelling off occurs. The average n-value between 5Y',, and 89"o saturation for Rana catushekma was 3.5. Table 1 summarizes the results of a more detailed investigation of R. temporaria blood. A total of 6 0 ~ Hb equilibrium curves covering a range in saturation from 20". to 98,'o have been averaged to give the CO. Bohr effect equations at 50" o, 70°. and 90'!. saturation as well as the mean values of the Hill coefficient, n, corresponding to these three saturation ranges of the equilibrium curves (P~o ranges from 14 to 30 mm Hg resulting in a pH range from 7.8 to 7.4). The Bohr effect is seen to more than double when compared at 50"0 and 90". saturations. The average n-values change from 1.6 at 50". through 2.4 at 70". to 7.3 at 90". saturation. Figure 2 provides the experimental data points showing the change in the CO~ Bohr effect with the degree of saturation. The Bohr lhctor, ~ , as well as the n-value at P~,, accord with results of earlier investigations on amphibian blood (Johansen and Lentimt. 1972). As pointed out in the introduction such low values of O_~ affinity and n predict that blood passing the lung capillaries will never attain complete or near complete saturation. In the case of Rana temporarht with a P5. of 38.0 mm Hg at a pH of 7.55, blood would only be 85",, saturated at a PO~ of 120 to 130 mm Hg. This point is apparent from fig. 4 if the O, equilibrium curve is extended to higher saturations from the n-wdue at P~, (lower broken curve). Meanwhile. our in vitro analyses FABLE
I
CO~ B o h r effect a n d values o f Hill's coefficient, n. tk3r Ramt ",, s a t u r a t i o n
C()~ B o h r effect
tempora;'k; b l o o d at 20.5 C (N - 6) n Mean
Range
50
I o g P ~ . = - 0 . 2 2 . p H + 3.24
1.6
1.4
70 90
log PT. = - 0 . 4 2 • p H + 4.97 IogP~,~= 0.50.pH +5.71
2.4 7.3
1.6 2.9 4.5 10.9
l.g
123 temporar~a
lqana
CO 2 B o h r
effect
•
I o g P~c log Pr0
•
log
o
at
205°C
Pgc
20
100
19
80
a.. t~ o
E 18
.-->_.
•
6o
1? " ~•
o
042
16
40
~5 74
75
76
77
78
pH Fig. 2. CO~ Bohr effect in w h o l e b l o o d from Rana [emporaria. The s y m b o l s O, • a n d • express the r e l a t i o n s h i p between whole b l o o d p H and log Poe at 50, 70 a n d 90'!;. s a t u r a t i o n , respectively. L i n e a r regression a n a l y s i s yields the three different CO 2 Bohr effects.
20 pH
770 755 240
98
95
90 ~ 08 8O ~ Oz.
70 ~--
6O 50
O0
40 30
-04 Rana temporarm
20
205"C
-08
10
12
I4
16
18
20
22
log Po2
Fig. 3. Hill plots lk~r Rana temporario w h o l e b l o o d at three difl'erent pH's. The plots are c o n s t r u c t e d from the data in table 1.
124
(~. LYKKEBOE A N D K. J O H A N S E N 100
~" 60 cb
~
40
RQna tempororra
205°C
p H : 755
20
0
* 20
40
60 OXYGEN
. 80
TENSION
.
.
. 1O0
.
.
. 120
. 1z,0
mmHg
Fig. 4. The fully drawn curve represents the oxygen dissociation curve t\~r Rana 1~'mp~Jraria blood corresponding to thc Hill plot at pH 7.55 (fig. 3). The tvxo broken curves rcprescn! extensions of lhe full> dra,a.n cur~,e at n-values 1.6 (lower broken curve) and 2.4 {upper hrokcn curxc).
of Rana temporaria blood showed the blood to be more than 98'}, saturated at PO~ of 120 m m Hg (fig. 1). Figure 4 reveals that this misalignment betweeu predictable and measured O, saturation values is linked to a two step change in the O, equilibrium curve. The fully drawn curve in fig. 4 is the experimentally obtained curve at pH 7.55. The two broken curves represent extensions of the fully drawn curve at n-values 1.6 (lower broken curve) and 2.4 (upper broken curve). The Hill plots at 3 different pH values (fig. 3) are parallel to each other throughout their course indicating that pH exerts no influence on the n-values. The change in distance between the curves at each transition in n-value is expressive of the increase in the Bohr factor as the saturation increases. Whether tile transitions are continuous or not could not be ascertained conclusively since tile equilibrium curves were constructed from point values. The data are, however, suggestive that the transitions in n-value are continuous.
Discussion We have referred to the finding of n-values exceeding 4.0 in Hill plots of O, binding in Rana temporaria blood as a 'paradox" since tile accepted theoretical viewpoint states a maximum of 4.0 for the n-value when a tetramer hemoglobin is the functional unit (Wyman, 1948). While our experimental findings conflict with this view. we can at present offer no well documented alternative molecular mcchanism for our findings. Tile physiological importance oi" bi- or triphasic O, Hb equilibria in anuran blood is, however, clear. As pointed out by Severinghaus, Roughton and Bradley (1972). the technical
H I G H n-VALUES IN F R O G BLOOD
125
problems related to measurement of O, Hb equilibria at very high O, saturations, are considerable and further amplified in the present study because ot" the low O, capacity of frog blood. Whereas the human blood had an O2 capacity of 20.8 vol': o, the two species of anurans studied had 02 capacities of about 9.0 vol"o. Table 1 reflects this difficulty by the large variation in n-value around 90",, saturation. Note, however, that all 6 curves determined showed n-values at 90" o saturation exceeding 4.0. Credence to the data is also given by the close similarity in the Podependence of the n-value for human blood at saturations above 9000 for our data and the ti~r more extensive results reported by Severinghaus et al. (1972). Thus did our data show a decline in n-value for human blood at saturations higher than about 97",, from 2.5 to 1.4, compared to a corresponding change from 2.7 to 1.6 in the results of Severinghaus el al. (1972). The whole blood pH increase with a change in saturation from I00 to 50", at constant Pco, was so small (i.e. ApH less than 0.020) as to not even be considered in fig. 2 relating the Bohr effect to '!,, saturation. We see the following tentative implementations: Maybe the functional hemoglobin unit inside the intact red cell is not only a tetramer, but that tetramer tetramer interactions (linked functions) can be more important than hitherto visualized. Also lbr Rana temporaria blood, the free energy of interaction is displaced to the upper segment of the equilibrium curve. Note in this regard that below 500o saturation, (i.e. up to where the first n-transition occurs) the n-value is only 1.6 suggesting that the probability that a Hb molecule that has bound one O~ molecule binds another O~ molecule is not much greater than for a Hb molecule in the fully deoxygenated state. At about 50oo saturation this situation changes (fig. 4) bringing the average n-value at 70", to 2.4 and to the still higher value of 7.3 at 907;, saturation. This is indicating that most of the apparent total free energy of interaction (Wyman, 1964) is linked to the upper part of the dissociation curve. Or stated differently, transition to the higher affinity oxyconformation predominantly takes place at saturations exceeding 50{Ii,. On this basis we would like to suggest that the concentration of Hb may exert an effect on the cooperativity (n-value). Preliminary data on O: binding in stripped hemolysates at increasing Hb concentration (Rana lemporaria) show an increase in n-wdue from 1.3 to 2.4when tetramer Hb concentration was changed from 0.1 mM to 2.0 raM. In contrast a stripped hemolysate prepared from Rana catesheiana blood showed a n-value of 3.3 when tetramer Hb concentration was 0.3 mM. This n-value is close to the value for Rana catesheiana whole blood (fig. 1B). Since the red cell tetramer Hb concentration for the two frog species is about 4 mM these findings indicate that hemoglobins from Rana teml)orarkt and Rana cateshehma differ in respect to cooperativity dependence on Hb concentration. It should also be noted that some recent studies, particularly on reptilian (MacMahon and Hamer, 1975) and avian whole blood (Lutz et al., 1974: Hirsowitz el al., 1977), have reported n-values slightly exceeding 4.0. These data have either been left without comment by the authors (MacMahon and Hamer, 1975), or
126
(;. L Y K K E B O E A N D K. J O H A N S E N
dismissed as the result of technical errors (Hirsowitz et a/., 1977), or refuted by
other workers as theoretically impossible (Scheipers el al., 1975). Whereas the molecular basis for the essential finding in this study of n-values exceeding 4.0 in Rana temporarkt whole blood must await further experimental studies and/or theoretical considerations, we have no difficulty in crediting the phenomenon signilicance in improving O, transport by the blood. The wtlue of a low O~ affinity m promoting efficient O_~ unloading from blood in the tissues is often quoted and regarded as adaptive to O, transport Ibr animals in high ambient O~ availability having efficient transfer of O, at their lungs or gills (Bauer, 1974). Among air-breathing vertebrates, amphibians, reptiles and birds show. however, generally much lower P<,, values than COmlalon in maminals. For at least amplfibians and reptiles this is suggestive of submaximal O, loading into the blood in their lungs since the gas to blood O~ gradient in the hmgs of these vertebrates are considerable greater than for avian and mammalian hmgs. P,,, wtlues at arterial pH exceeding 50 mm Hg are often reported for these animals suggesting P,,, values so high as to be of improbable occurrence in puhnonary venous blood. Moreover will continued O, transport from the lung to the tissues in amphibians and most reptiles be threatened by an efficiency loss due to the variable, but often considerable, right to left shunting occurring in the heart and greater systemic outflow vessels of these animals (Johansen, 1978). The changing n-value at increasing O, saturation as reported presently for Raml lemporaria and less pronounced ik)r Ralm calesheiamt promises improved O~ loading and will counter the efficiency loss of right-left shunting in transporting the O~ to the tissues. In anuran amphibians the highest arterial O- saturations are reported l\)r the carotid arches (Haberich, 1965). Oxygen to the CNS will hence be unloaded from the upper segments of the equilibrium curves at high PO,'s and with the possible added advantage of an increased Bohr eff'ect at these higher saturations (fig. 2, table 1). The greatly increasing n-wflue with increasing O~ saturation demonstrated for Ramt ldttq)ordria blood permits the animal to draw on the advantage of a low O_, affinity [\~r efficient O: unloading from Hb while safeguarding the entry of O~ in the gas exchanger and its continued passage through a shunt franc central circulation. References Bauer. C. (19741. On tile respiralory function ofllemoglobin. Rev. Phl'~io/. Biochem. Plmrmaco/. 711:1 31. Christoforides, C. and J. Hcdlcy-\Vhyl.e (1969). Effect of temperature and hemoglobin conccntralion on solubility o1"O, m blood, J. Appl. Phlsio/. 27:592 596. ltaberich, F.,I. II965). Uber die funklionelle Treuning des venOsen und arlerialisierten Blutes im Froschherzen. Pl/iiger,s Archiv 282:76 91. Hirso,aitz. L.A., K. Fell and .I.D. lorrance (1977). Oxygen affinity of avian blood. Rc.v~ir. Physiol. 31:51 62. .Iohansel~, K. and ('. l.enfant (1972). A comparative approach to the adaptability of ()~ fib affinity. Proc. A. Benzon Syrup. Vol. I V . ( ' o p e n h a g e n , Mtmksgaard, pp. 750 780.
H I G H n - V A L U E S IN F R O G BLOOD
127
,lohansen, K.. G. Lykkcboe, R. E. Weber and G. M . O . Maloiy (1976). Respiralory properties of blood in awake and estiwtting lungfish, Protoptcr,s ,mphil~i,.~. Re.v~ir. Ptn'siol. 27:335 345. Johansen, K. (1978). Cardiovascular support of metabolic functions in vertebrates. Ill: Evolution of Respiratory Processes. edited by C. Lenfant and S. C. Wood. New York, M. Decker Inc. (In press). Johansen, K., G. Lykkeboe, S. Kornerup and G . M . O . Maloiy (1978). Oxygen uptake and blood respiratory properties in the tree frog, ('llironl~tnti.~" pc:er.~i. (In preparation). Lenlkmt. C. and K. Johansen (I 967). Respiratory adaptations in selected amphibians. R~'sl;ir. P/n'.~iol. 2: 247 260. Lutz, P.L., I.S. Longmuir and K. Schmidt-Nielsen (1974). Oxygen alfmity of bird blood. Res7~ir. P/n',~iol. 20:325 330. M a c M a h o n , J.A. and A. Hamer (1975). Effects of telnperature and photoperiod on oxygenation and other blood parameters of the sidewinder (('rot~d,.~ cer~rv:c.s') : Adaptive significance. ('o,q~. Bioch~,m. PIn'siol. 51A: 59 69. M cCutchcon, F. H. and F. G. Hall(1937). Hemoglobin in the a mphibia..I. Cell. Co,11~. Ph~'.~iol. 9:191 197. ScheJpers. G., T. Kawashiro and P. Scheid (1975). Oxygcn and carbondioxidc dissociation curxes of duck blood. Respir. Ptn'siol. 24:1 13. Severinghaus. J.W., F . J . W . Roughton and A. F. Bradley 11972). Oxygen dissociation curve analysb, at 98.7 99.6"., saturation. Proc. A. Benzon Syrup. Vol. IV. Copenhagen. Munksgaard. pp. 65 72. W y m a n . J., Jr. (1948). Heme proteins. Ad~,. Prof. Chem. 4 : 4 1 0 531. Wyman. ,1., ,Ir. (1964). Linked functions and reciprocal effects ir~ hemoglobin: A second look. 4d~'. Pro:. Chem. 19:224 286.
AN O,-Hb 'PARADOX' IN FROG BLOOD? (n-values exceeding 4.0)
G. LYKKEBOE and K. JOHANSEN DepartmeHl o/'Zoophysiolo,Kv, Uuiversit3' o/Aarhu.~, DK-8000 A ttrhus C. Dcttmurk
Abstract. O~-Hb dissociation curves have been determined t'or the anurans. Ram1 It'*tq~oraria and Ratul
catesheialul, and compared with h u m a n blood t\~r the specilic purpose of defining the n-wflue (Hill's cooperativity coefficient) at high O~ saturations. The Psi, values at normal conditions (see fig. 1) for each species were 24.6 m m Hg for h u m a n blood. 37.0 m m Hg for Ram~ lep*y~oraria blood and 53.5 m m Hg for Romt cateshciaml. For h u m a n blood the n-value was 2.7 at saturations from 36",, to 98". levelling off :.tl the highest saturations. For Rana temporaria Hill plots between saturations from 20". to 98",, showed 3 segments. The average n-value increased from 1.6 to 2.4 at about 50",, saturation, increasing again at about 80",, saturation to 7.3. The pattern in Rana c'atexheiatla blood was similar with the n-value changing from 1.6 to 3.1 at about 50"o saturation, averaging 3.5 between 53",, and 89",, saturation. Additionally the Bohr effect in Rana tc'plq~oraria blood more than doubled when compared at 50",, and 90",, saturation. The data show that for R. temporaria blood the tree energy of interaction associated with the binding of O, to Hb is displaced to the upper segment of the dissociation curve. Blood oxygen dissociation curve Bohr effect Cooperativity
Hemoglobin Hill plot n-values
Blood O: affinity in amphibians decreases with increasing importance of pulmonary breathing in overall O~ uptake (McCutcheon and Hall, 1937: Lenfant and Johansen, 1967). A recent study revealed a P~0 value of 60 mm Hg for the very xerophilous tree frog, Chiromautis petersi, at pH 7.6, T 25 C (Johansen et al., 1978) which prompted the question if the lung O: pressure ever was high enough to saturate the blood with O: during passage through the lung. Analysis revealed pulmonary O, pressures up to 130 torr which in equilibrium with blood h7 vitro gave saturations in excess of 950,. A Hill plot, however, revealed the saturation values to grossly Accepted/or publication 4 Juh' 1978 119
120
G. LYKKEBOE A N D K. J O H A N S E N
misalign with the linearity dictated by n-values corresponding to O~ saturations between 30 and 60°o. This suggested a striking increase in the n-value (cooperativity coefficient) at higher O, saturations and stimulated the present investigation of the shape of the O~-dissociation curve in whole blood from 2 species of anuran frogs, Ramt lemporaria and Rana calesbekma. These results are compared with values for human blood obtained using identical experimental procedures and methods.
Material and methods
Human blood was sampled by venipuncture and frog blood was obtained by needle puncture of an aortic or carotid arch. In all cases the blood was drawn into syringes, the dead spaces of which were filled with a heparin solution (50 mg-1 ~). In the case of Rana lemporaria blood samples from several individuals were pooled to yield the needed total volume. Immediately alter sampling the blood from all species were divided into 50 lal aliquots in disposable equilibration tubes (Radiometer D840) and placed in ice water. O, Hb equilibrium curves were constructed based on procedures described in detail earlier (Johansen et al.. 1976). The O, content of 20 gl blood samples equilibrated against gas mixtures of known composition were measured by a Lexocon O, content analyser. A modification of the procedures described earlier consisted in determining the O_~ capacity by equilibration against a very high Po: (i.e. 90"i, Oe) to ensure the highest possible saturation of the hemoglobin. The measured O, contents were corrected for physically dissolved O~ by using the appropriate Bunsen solubility coefficients (Christoforides and Hedley-Whyte, 1969). Particular emphasis was placed on determining the upper portions (70°, < S < 98'~o) of the equilibrium curves as detailed as possible by performing a large number of gas equilibrations in this region of the curves.
Results
Figure 1A shows O~ Hb dissociation curves for human blood and the blood from the anurans Rana temporaria and Rana catesbeiana. Figure 1B delineates the corresponding Hill plots. Each plotted point represents the mean of two determinations. All equilibrations were performed at the body temperatures of the species (in the anurans the acclimation temperature). The Pco2 levels were kept constant for all species. The respective values and the resulting whole blood pH values are shown on figs. 1A and B. The blood 02 binding properties for the 3 species differ in several important respects. Firstly, the O: affinity expressed by the P> value is highest for human blood (24.6 mm Hg) lbllowed by 37.0 mm Hg for Rana ternporaria blood and 53.5 mm Hg for Rana catesheiana.
HIGH n-VALUES IN FROG BLOOD
121
100
80
~" 60
_.9. 40
20
60 80 100 OXYGEN TENSION , m m H g
40
120
1/.0
Fig. IA. Oxygen dissociation curves of whole blood samples f r o m Homo sapiens (A), Rana temporaria (O), and Rana catesbeiana (O), at conditions shown on the figure. Each plotted point represents the mean of two determinations.
20
B 98
~6
95
90 .~ 08 80
OZ.
~
70 ~-6O
00
50 z,0
-04
J
30 •
Homo
s~ple~s
370"C
PCO 2 : ~ 2 0
o
g~ono
lemporof,Q
205'C
PCO 2 : IZ, 5 . p H : 7 6 7
•
¢~a~a
¢o~esD~eme2~*O'C
pH;740
20
-08 PC~2:96
p~=755
10
12
14
16
18
20
22
log Po2
Fig. 1B. The Hill plots corresponding to the oxygen dissociation curves shown in fig. 1A. F u r t h e r m o r e , the s h a p e o f the e q u i l i b r i u m curves vary greatly. T h e curve for h u m a n b l o o d w a s d e t e r m i n e d w i t h i n a s a t u r a t i o n r a n g e f r o m 36'!,,; to 98.4'~o. T h e Hill plot (fig. IB) s h o w s an a v e r a g e n - v a l u e (Hill's c o e f f i c i e n t ) o f 2.7, levelling o f f at the highest saturations.
122
el. L Y K K E B O E
A N D K. J O H A N S E N
in sharp contrast the equilibrium curve for Rana tenq~orarkt measured between saturation values of 20"o and 98.Y',, shows an undulating, polyphasic course. Expressed in a Hill plot the curve shows 3 distinct segments. At about 50". saturation the n-value changes from 1.4 to 2.7 and again at about 80",, 0 Z saturation another transition in n-value occurs bringing it to about 11.0. This conspicuously high n-value persists throughout the range of measurements terminating at 98.3'!0 saturation. The marked increase in n-value at higher O. saturations causes the equilibrium curve for Raml temporark/ to actually transect that for human blood in spite of the much lower O, alt]nity of Rana tumporaria blood at 50". saturation. The transect occurs at 96",, saturation corresponding to a Po. of 81 mm Hg. The equilibrium curve for Rana calesheiaml blood shows a pattern between those of human and Ram1 temporarkl blood. At about 5Y',, saturation the n-value changes from 1.6 to 3. l and at the highest saturations a slight levelling off occurs. The average n-value between 5Y',, and 89"o saturation for Rana catushekma was 3.5. Table 1 summarizes the results of a more detailed investigation of R. temporaria blood. A total of 6 0 ~ Hb equilibrium curves covering a range in saturation from 20". to 98,'o have been averaged to give the CO. Bohr effect equations at 50" o, 70°. and 90'!. saturation as well as the mean values of the Hill coefficient, n, corresponding to these three saturation ranges of the equilibrium curves (P~o ranges from 14 to 30 mm Hg resulting in a pH range from 7.8 to 7.4). The Bohr effect is seen to more than double when compared at 50"0 and 90". saturations. The average n-values change from 1.6 at 50". through 2.4 at 70". to 7.3 at 90". saturation. Figure 2 provides the experimental data points showing the change in the CO~ Bohr effect with the degree of saturation. The Bohr lhctor, ~ , as well as the n-value at P~,, accord with results of earlier investigations on amphibian blood (Johansen and Lentimt. 1972). As pointed out in the introduction such low values of O_~ affinity and n predict that blood passing the lung capillaries will never attain complete or near complete saturation. In the case of Rana temporarht with a P5. of 38.0 mm Hg at a pH of 7.55, blood would only be 85",, saturated at a PO~ of 120 to 130 mm Hg. This point is apparent from fig. 4 if the O, equilibrium curve is extended to higher saturations from the n-wdue at P~, (lower broken curve). Meanwhile. our in vitro analyses FABLE
I
CO~ B o h r effect a n d values o f Hill's coefficient, n. tk3r Ramt ",, s a t u r a t i o n
C()~ B o h r effect
tempora;'k; b l o o d at 20.5 C (N - 6) n Mean
Range
50
I o g P ~ . = - 0 . 2 2 . p H + 3.24
1.6
1.4
70 90
log PT. = - 0 . 4 2 • p H + 4.97 IogP~,~= 0.50.pH +5.71
2.4 7.3
1.6 2.9 4.5 10.9
l.g
123 temporar~a
lqana
CO 2 B o h r
effect
•
I o g P~c log Pr0
•
log
o
at
205°C
Pgc
20
100
19
80
a.. t~ o
E 18
.-->_.
•
6o
1? " ~•
o
042
16
40
~5 74
75
76
77
78
pH Fig. 2. CO~ Bohr effect in w h o l e b l o o d from Rana [emporaria. The s y m b o l s O, • a n d • express the r e l a t i o n s h i p between whole b l o o d p H and log Poe at 50, 70 a n d 90'!;. s a t u r a t i o n , respectively. L i n e a r regression a n a l y s i s yields the three different CO 2 Bohr effects.
20 pH
770 755 240
98
95
90 ~ 08 8O ~ Oz.
70 ~--
6O 50
O0
40 30
-04 Rana temporarm
20
205"C
-08
10
12
I4
16
18
20
22
log Po2
Fig. 3. Hill plots lk~r Rana temporario w h o l e b l o o d at three difl'erent pH's. The plots are c o n s t r u c t e d from the data in table 1.
124
(~. LYKKEBOE A N D K. J O H A N S E N 100
~" 60 cb
~
40
RQna tempororra
205°C
p H : 755
20
0
* 20
40
60 OXYGEN
. 80
TENSION
.
.
. 1O0
.
.
. 120
. 1z,0
mmHg
Fig. 4. The fully drawn curve represents the oxygen dissociation curve t\~r Rana 1~'mp~Jraria blood corresponding to thc Hill plot at pH 7.55 (fig. 3). The tvxo broken curves rcprescn! extensions of lhe full> dra,a.n cur~,e at n-values 1.6 (lower broken curve) and 2.4 {upper hrokcn curxc).
of Rana temporaria blood showed the blood to be more than 98'}, saturated at PO~ of 120 m m Hg (fig. 1). Figure 4 reveals that this misalignment betweeu predictable and measured O, saturation values is linked to a two step change in the O, equilibrium curve. The fully drawn curve in fig. 4 is the experimentally obtained curve at pH 7.55. The two broken curves represent extensions of the fully drawn curve at n-values 1.6 (lower broken curve) and 2.4 (upper broken curve). The Hill plots at 3 different pH values (fig. 3) are parallel to each other throughout their course indicating that pH exerts no influence on the n-values. The change in distance between the curves at each transition in n-value is expressive of the increase in the Bohr factor as the saturation increases. Whether tile transitions are continuous or not could not be ascertained conclusively since tile equilibrium curves were constructed from point values. The data are, however, suggestive that the transitions in n-value are continuous.
Discussion We have referred to the finding of n-values exceeding 4.0 in Hill plots of O, binding in Rana temporaria blood as a 'paradox" since tile accepted theoretical viewpoint states a maximum of 4.0 for the n-value when a tetramer hemoglobin is the functional unit (Wyman, 1948). While our experimental findings conflict with this view. we can at present offer no well documented alternative molecular mcchanism for our findings. Tile physiological importance oi" bi- or triphasic O, Hb equilibria in anuran blood is, however, clear. As pointed out by Severinghaus, Roughton and Bradley (1972). the technical
H I G H n-VALUES IN F R O G BLOOD
125
problems related to measurement of O, Hb equilibria at very high O, saturations, are considerable and further amplified in the present study because ot" the low O, capacity of frog blood. Whereas the human blood had an O2 capacity of 20.8 vol': o, the two species of anurans studied had 02 capacities of about 9.0 vol"o. Table 1 reflects this difficulty by the large variation in n-value around 90",, saturation. Note, however, that all 6 curves determined showed n-values at 90" o saturation exceeding 4.0. Credence to the data is also given by the close similarity in the Podependence of the n-value for human blood at saturations above 9000 for our data and the ti~r more extensive results reported by Severinghaus et al. (1972). Thus did our data show a decline in n-value for human blood at saturations higher than about 97",, from 2.5 to 1.4, compared to a corresponding change from 2.7 to 1.6 in the results of Severinghaus el al. (1972). The whole blood pH increase with a change in saturation from I00 to 50", at constant Pco, was so small (i.e. ApH less than 0.020) as to not even be considered in fig. 2 relating the Bohr effect to '!,, saturation. We see the following tentative implementations: Maybe the functional hemoglobin unit inside the intact red cell is not only a tetramer, but that tetramer tetramer interactions (linked functions) can be more important than hitherto visualized. Also lbr Rana temporaria blood, the free energy of interaction is displaced to the upper segment of the equilibrium curve. Note in this regard that below 500o saturation, (i.e. up to where the first n-transition occurs) the n-value is only 1.6 suggesting that the probability that a Hb molecule that has bound one O~ molecule binds another O~ molecule is not much greater than for a Hb molecule in the fully deoxygenated state. At about 50oo saturation this situation changes (fig. 4) bringing the average n-value at 70", to 2.4 and to the still higher value of 7.3 at 907;, saturation. This is indicating that most of the apparent total free energy of interaction (Wyman, 1964) is linked to the upper part of the dissociation curve. Or stated differently, transition to the higher affinity oxyconformation predominantly takes place at saturations exceeding 50{Ii,. On this basis we would like to suggest that the concentration of Hb may exert an effect on the cooperativity (n-value). Preliminary data on O: binding in stripped hemolysates at increasing Hb concentration (Rana lemporaria) show an increase in n-wdue from 1.3 to 2.4when tetramer Hb concentration was changed from 0.1 mM to 2.0 raM. In contrast a stripped hemolysate prepared from Rana catesheiana blood showed a n-value of 3.3 when tetramer Hb concentration was 0.3 mM. This n-value is close to the value for Rana catesheiana whole blood (fig. 1B). Since the red cell tetramer Hb concentration for the two frog species is about 4 mM these findings indicate that hemoglobins from Rana teml)orarkt and Rana cateshehma differ in respect to cooperativity dependence on Hb concentration. It should also be noted that some recent studies, particularly on reptilian (MacMahon and Hamer, 1975) and avian whole blood (Lutz et al., 1974: Hirsowitz el al., 1977), have reported n-values slightly exceeding 4.0. These data have either been left without comment by the authors (MacMahon and Hamer, 1975), or
126
(;. L Y K K E B O E A N D K. J O H A N S E N
dismissed as the result of technical errors (Hirsowitz et a/., 1977), or refuted by
other workers as theoretically impossible (Scheipers el al., 1975). Whereas the molecular basis for the essential finding in this study of n-values exceeding 4.0 in Rana temporarkt whole blood must await further experimental studies and/or theoretical considerations, we have no difficulty in crediting the phenomenon signilicance in improving O, transport by the blood. The wtlue of a low O~ affinity m promoting efficient O_~ unloading from blood in the tissues is often quoted and regarded as adaptive to O, transport Ibr animals in high ambient O~ availability having efficient transfer of O, at their lungs or gills (Bauer, 1974). Among air-breathing vertebrates, amphibians, reptiles and birds show. however, generally much lower P<,, values than COmlalon in maminals. For at least amplfibians and reptiles this is suggestive of submaximal O, loading into the blood in their lungs since the gas to blood O~ gradient in the hmgs of these vertebrates are considerable greater than for avian and mammalian hmgs. P,,, wtlues at arterial pH exceeding 50 mm Hg are often reported for these animals suggesting P,,, values so high as to be of improbable occurrence in puhnonary venous blood. Moreover will continued O, transport from the lung to the tissues in amphibians and most reptiles be threatened by an efficiency loss due to the variable, but often considerable, right to left shunting occurring in the heart and greater systemic outflow vessels of these animals (Johansen, 1978). The changing n-value at increasing O, saturation as reported presently for Raml lemporaria and less pronounced ik)r Ralm calesheiamt promises improved O~ loading and will counter the efficiency loss of right-left shunting in transporting the O~ to the tissues. In anuran amphibians the highest arterial O- saturations are reported l\)r the carotid arches (Haberich, 1965). Oxygen to the CNS will hence be unloaded from the upper segments of the equilibrium curves at high PO,'s and with the possible added advantage of an increased Bohr eff'ect at these higher saturations (fig. 2, table 1). The greatly increasing n-wflue with increasing O~ saturation demonstrated for Ramt ldttq)ordria blood permits the animal to draw on the advantage of a low O_, affinity [\~r efficient O: unloading from Hb while safeguarding the entry of O~ in the gas exchanger and its continued passage through a shunt franc central circulation. References Bauer. C. (19741. On tile respiralory function ofllemoglobin. Rev. Phl'~io/. Biochem. Plmrmaco/. 711:1 31. Christoforides, C. and J. Hcdlcy-\Vhyl.e (1969). Effect of temperature and hemoglobin conccntralion on solubility o1"O, m blood, J. Appl. Phlsio/. 27:592 596. ltaberich, F.,I. II965). Uber die funklionelle Treuning des venOsen und arlerialisierten Blutes im Froschherzen. Pl/iiger,s Archiv 282:76 91. Hirso,aitz. L.A., K. Fell and .I.D. lorrance (1977). Oxygen affinity of avian blood. Rc.v~ir. Physiol. 31:51 62. .Iohansel~, K. and ('. l.enfant (1972). A comparative approach to the adaptability of ()~ fib affinity. Proc. A. Benzon Syrup. Vol. I V . ( ' o p e n h a g e n , Mtmksgaard, pp. 750 780.
H I G H n - V A L U E S IN F R O G BLOOD
127
,lohansen, K.. G. Lykkcboe, R. E. Weber and G. M . O . Maloiy (1976). Respiralory properties of blood in awake and estiwtting lungfish, Protoptcr,s ,mphil~i,.~. Re.v~ir. Ptn'siol. 27:335 345. Johansen, K. (1978). Cardiovascular support of metabolic functions in vertebrates. Ill: Evolution of Respiratory Processes. edited by C. Lenfant and S. C. Wood. New York, M. Decker Inc. (In press). Johansen, K., G. Lykkeboe, S. Kornerup and G . M . O . Maloiy (1978). Oxygen uptake and blood respiratory properties in the tree frog, ('llironl~tnti.~" pc:er.~i. (In preparation). Lenlkmt. C. and K. Johansen (I 967). Respiratory adaptations in selected amphibians. R~'sl;ir. P/n'.~iol. 2: 247 260. Lutz, P.L., I.S. Longmuir and K. Schmidt-Nielsen (1974). Oxygen alfmity of bird blood. Res7~ir. P/n',~iol. 20:325 330. M a c M a h o n , J.A. and A. Hamer (1975). Effects of telnperature and photoperiod on oxygenation and other blood parameters of the sidewinder (('rot~d,.~ cer~rv:c.s') : Adaptive significance. ('o,q~. Bioch~,m. PIn'siol. 51A: 59 69. M cCutchcon, F. H. and F. G. Hall(1937). Hemoglobin in the a mphibia..I. Cell. Co,11~. Ph~'.~iol. 9:191 197. ScheJpers. G., T. Kawashiro and P. Scheid (1975). Oxygcn and carbondioxidc dissociation curxes of duck blood. Respir. Ptn'siol. 24:1 13. Severinghaus. J.W., F . J . W . Roughton and A. F. Bradley 11972). Oxygen dissociation curve analysb, at 98.7 99.6"., saturation. Proc. A. Benzon Syrup. Vol. IV. Copenhagen. Munksgaard. pp. 65 72. W y m a n . J., Jr. (1948). Heme proteins. Ad~,. Prof. Chem. 4 : 4 1 0 531. Wyman. ,1., ,Ir. (1964). Linked functions and reciprocal effects ir~ hemoglobin: A second look. 4d~'. Pro:. Chem. 19:224 286.