The physiological significance of the position and shape of the oxygen equilibrium curve (OEC) of horse hemoglobin (Hb) is considered from the viewpoint of oxygen (O2) transport efficiency and the effectiveness of the Bohr effect. In horse fetal and maternal bloods, their physiological O2 affinities are nearly optimized with respect to the effectiveness of the Bohr shift occurring at the O2 release site, when it is measured by the change in O2 saturation per unit change in P50. With relatively low cooperativity (n=2.69) of horse Hb under physiological conditions, the effectiveness of the Bohr shift for fetal blood at O2 uptake site and maternal blood at O2 release site is high. These facts imply that the position and the cooperativity of horse Hb OEC are optimized to receive maximal benefit from the double Bohr shift. Before exercise, the position of the OEC for adult mares is nearly optimized for the effectiveness of the Bohr shift occurring at the O2 release site, whereas, at maximal exercise, the position of the OEC tends to become advantageous for O2 transport efficiency.
An increase in partial pressure of CO2 (PCO2) or a decrease in pH lowers the O2 affinity of Hb. Thus, they shift the OEC to the right, causing release of additional O2 from Hb to the tissues. This shift caused by concomitant PCO2 change and pH change is called the “classical Bohr effect” (Bohr et al., 1904), while the shift of OEC only by pH change called the “Bohr effect”. In the case of fetal blood, the Bohr shift is considered to be of particular importance. The diffusion of CO2 from fetal blood into maternal blood increases pH of fetal blood and shifts the fetal OEC to the left, the simultaneous decrease in pH of maternal blood shifts its OEC to the right (MaCarthy, 1943). This phenomenon is called the “double Bohr effect”. It has been considered that the enhanced difference in P50 between fetal and maternal bloods by the double Bohr effect promotes the mother-fetus transfer of O2. P50 is partial pressure of O2 at half saturation.
The magnitude of the Bohr effect is quantitatively measured by the change in log P50 per unit change in pH (dlogP50/dpH), which is called the “Bohr coefficient”. The additional O2 released from Hb by the Bohr shift without any change in venous PO2 is dependent on not only the magnitude of the Bohr coefficient but also the steepness of the OEC. In our previous study, we found that the position of the OEC of human adult Hb at rest is optimal for the effectiveness of the Bohr shift occurring at O2 release sites (venous PO2(PvO2)=40 torr) (Kobayashi et al., 1996, Itoh et al., 2001).
The cooperativity of O2 binding is expressed in terms of the highest slope of the Hill plot (log(S/(1–S)) vs. log P plot), n (Hill, 1910). The functional significance of the sigmoid shape of the OEC has been explained by efficient O2 transport: the higher the cooperativity, the more O2 is transported to the tissues. However, mammalian tetrameric Hb usually has relatively low cooperativity (n value of approximately 2.8). The physiological significance of this low cooperativity has not been explained so far. Recently, using the human OEC data, which was described by Adair's stepwise O2 binding equation, it was reported that the relatively low cooperativity of the OEC of human tetrameric Hbs is designed to receive a maximal benefit from the double Bohr shift (Zhang et al., 2003). In this study, the above-mentioned approach was applied to confirm the functional significance of the relatively low cooperativity of other mammalian tetrameric Hbs.
In each many species of mammalian species, Hb of the fetal blood differs structurally from that found in the adult blood. The subunit structure of human adult Hb is α2β2, and that of fetal Hb is α2γ2. The amino acid sequences of the β-chain of adult Hb and the γ-chain of fetal Hb differ at 31 of 146 residues (Schroeder et al., 1963). In contrast, the Hbs of the fetal and adult horse bloods have been shown to be structurally identical with each other (Stockell et al., 1961; Comline and Silver, 1974). However, fetal blood has higher O2 affinity than maternal blood because of the low concentration of 2,3-diphosphoglycerate (2,3-DPG) in fetal red blood cells compared to that in maternal red blood cells (Bunn and Kitchen, 1973). There are literature values for not only the Bohr coefficient of horse blood (dlogP50/dpH=−0.47, Bunn and Kitchen, 1973; Fenger et al., 2000), but also PO2 for the arterial and venous blood and P50 for both fetal and maternal bloods (Comline and Silver, 1974; Comline and Silver, 1975). In addition, the OEC of horse adult blood, for which the four Adair constants (see below) were determined, is also available in the literature (Clerbaux et al., 1993). To our knowledge, horse fetal blood OEC data, for which the four Adair constants have been determined, are not available in the literature. Then, we measured OECs of horse Hb at various 2,3-DPG concentrations and found that cooperativity is not sensitive to 2,3-DPG concentration. It is known that cooperativity is also insensitive to pH. These facts give the basis for generating an OEC for either adult or fetal Hb under given 2,3-DPG and pH conditions from the Adair constants for horse adult Hb. Using these calculated OECs, the physiological significance of the position and the cooperativity of horse Hb can theoretically be examined from the viewpoint of O2 transport efficiency and the effectiveness of the Bohr shift.
Horse blood sample was purchased from Nippon biological material center. Hemoglobin was stripped of organic phosphates according to the method of Condo et al. (1992), and its OECs were measured with an automatic oxygenation apparatus developed by Imai and Yonetani (1977) and Imai (1981) at an Hb concentration of 60 μM on a heme basis and 25°C. Oxygen saturation of Hb was calculated from the change in absorbance at 576 nm measured with a Shimadzu spectrophotometer (model UV 2000). The concentration of O2 in the sample cell was decreased by replacing air with pure N2 gas and its change was monitored with a Clark-type O2 electrode. The buffer solution used for OEC measurements was 0.05 M Tris-HCl (pH 7.4). Methemoglobin (Met-Hb) formed by auto-oxidation was reduced using an enzymatic reducing system as described by Hayashi et al. (1973). The Met-Hb concentration at the end of OEC measurement as determined as described by Evelyn et al. (1938), did not exceed 5% of total Hb. The 2,3-DPG concentrations were measured by the enzymatic procedure of Ericson and Verdier (1972). The experimentally obtained OEC data were analyzed by curve fitting method described by Imai (1981) to estimate the Adair constants. Using these Adair constants, P50 and n values were calculated.
The Adair equation (Adair, 1925) is given by
The literature Ai values for horse adult whole blood at pH 7.4, PCO2 of 40 torr and 37°C are: A1=3.103. 10−2 torr−1, A2=8.451. 10−4 torr−2, A3=1.447. 10−5 torr−3 and A4=3.961. 10−6 torr−4 (Clerbaux et al., 1993). The values of P50 and n are 23.8 torr and 2.69, respectively. Using Adair constants, theoretical OECs with various P50 values were generated by multiplying each PO2 value by a common factor. In this multiplication, the position of the OEC was shifted freely without changing the shape.
In order to investigate the effect of cooperativity on the effectiveness of the Bohr shift, Hill's empirical equation (Hill, 1910) and OEC data of horse Hb solutions measured under various experimental conditions (Imai, 1983) were used.
The O2 transport efficiency and the effectiveness of the Bohr shift of horse fetal and maternal bloods at rest were calculated using the following literature values of blood PO2 and P50 (Comline and Silver, 1974). Fetal umbilical venous PO2(arterialized fetal blood), fetal umbilical arterial PO2 (“venous blood” coming from the fetus to the placenta), and physiological P50 under normal conditions were assumed to be 49, 33, and 27 torr, respectively. In the placental circulation, “arterial blood” and “venous blood” meant the blood flowing through the umbilical vein and the umbilical artery, respectively. Maternal arterial PO2, uterine venous PO2 and physiological P50 values were assumed to be 95, 50 and 31 torr, respectively.
The O2 transport efficiency and the effectiveness of the Bohr shift during incremental exercise of horse adult were calculated using the PO2, P50 and n values reported by Fenger et al. (2000). The arterial PO2 (PaO2), venous PO2 (PvO2), P50 and n values before exercise were 106, 38, 22.9 torr and 2.649, respectively, and these at maximal exercise were 86, 20, 31.8 torr and 2.707, respectively.
All computations were performed on a personal computer (model PC-9821 AP2; Nippon Electric Co., Tokyo) using MS-FORTRAN.
RESULTS AND DISCUSSIN
Effect of 2,3-DPG on cooperativity of horse Hb
Fig. 1 shows the effect of 2,3-DPG concentration on the O2 affinity and cooperativity of horse Hb. These data demonstrate a strong influence of 2,3-DPG on the O2 affinity, but little effect on cooperativity. Hill's coefficient (n) value was nearly constant. Therefore, in this study, the OEC with an n value of 2.69 of adult blood measured under standard conditions pH 7.4, PCO2 40 mmHg, 37°C (Clerbaux et al., 1993) was used to generate OECs for horse fetal and maternal bloods under various conditions.
Calculation of O2 transport by fetal and maternal blood
Fig. 2 illustrates an example calculation of the O2 transport by horse fetal and maternal bloods and the contribution of the Bohr shift occurring at the O2 uptake and release sites.
The additional O2 released from fetal blood due to the Bohr shift was estimated from the decrease in O2 saturation at PO2 of 33 torr, ΔS(33A–33B). In the placenta, the additional O2 uptake by fetal blood due to the Bohr shift was estimated from the increase in O2 saturation at PO2 of 49 torr, ΔS(49A–49B).
In maternal blood, the additional O2 released at PO2of 50 torr due to the Bohr shift was represented by ΔS(50C–50D), and the additional O2 uptake in the lungs caused by the Bohr shift at PO2of 95 torr was represented by ΔS(95C–95D).
The amount of O2 transported to the tissues was estimated from the arterio-venous difference in O2 saturation ΔS(PaO2–PvO2).
O2 transport efficiency of fetal and maternal blood
The slope of the OEC (dS/dP=S') was quantified to represent the O2 transport efficiency of Hb (Kobayashi et al., 1994), and this measure was used to compare the fetal and maternal bloods. The S' vs. P plot of fetal blood exhibited a steep slope under physiological O2 environment (at PO2 ranging from 33 to 49 torr) (Fig. 3A), indicating high O2 transport efficiency. In contrast, the slope of the OEC of maternal blood was rather flat under physiological O2 environment (at PO2 ranging from 50 to 95 torr), showing low O2 transport efficiency (Fig. 3B).
Relation to the optimal P50 for O2 transport efficiency
In order to explain the significance of the position of the OEC of fetal blood, the arterio-venous difference in O2 saturation (ΔS(PaO2–PvO2)) was calculated as a function of P50. Fig. 4A shows the relationship between P50 and the fetal “venous blood” O2 saturation (S(33)), “arterial blood” O2 saturation (S(49)) and arterio-venous difference in O2 saturation (ΔS(49–33)). The ΔS(49–33) vs. P50 plot had one maximum value. The P50 that gave the highest O2 transport efficiency was called the “optimal P50” for O2 transport. There are slight difference in the physiological P50 of horse blood and the optimal P50 values. The amount of O2 transported at physiological P50 was slightly lower than that of the maximum value at optimal P50 value. In human fetal Hb, it is well known that the physiological P50 of human fetal Hb is close to the optimal P50 (Itoh et al., 2001; Sold, 1982; Willford et al., 1982). Therefore, the O2 tranasport efficiency of horse fetal blood is not so high as that of human fetal blood.
In maternal blood, the amount of O2 transported at physiological P50 (31 torr) was roughly one-half that of theoretically obtained maximum value (Fig. 4B). This indicates that maternal venous blood is a large O2 reservoir and a rightward shift of the OEC enhances O2 transport as observed at hard exercise.
Effectiveness of Bohr shift in fetal and maternal blood
The effectiveness of the Bohr shift at various P50 was estimated from the change in O2 saturation per unit change in P50, i.e. the slope of the S(PO2) vs. P50 plot. The slope (dS(PO2)/dP50) was usually a negative value because S(PO2) was decreased with an increase in P50. Therefore, in this study, the magnitude of the effectiveness of the Bohr shift was expressed as –dS(PO2)/dP50.
Fig. 5A shows the effectiveness of the Bohr shift at the O2 uptake and release sites (at PO2s of 49 and 33 torr, respectively) for fetal blood. The theoretical highest effectiveness of the Bohr shift at O2 release site was observed at P50 of 23 torr. Clearly the physiological P50 value is close to the optimal P50 value for the effectiveness of the Bohr shift. At O2 uptake site, there are a slight difference in the physiological P50 and optimal P50 values. This implies that the position of the OEC of fetal blood is nearly optimal with respect to the effectiveness of the Bohr shift at O2 release site.
In fetal blood, the highest value at the O2 release site (PvO2=50 torr) was observed at P50 of 38 torr (Fig. 5B). This optimal P50 value is somewhat different from the physiological P50 value (31 torr). In contrast, at O2 uptake site there was a large difference between the theoretical optimal P50 value (72 torr) and the physiological P50 value. Therefore, the position of the OEC of maternal blood is nearly optimal to maximize the effectiveness of the Bohr shift in venous blood. The effectiveness of the Bohr shift at O2 uptake site of fetal blood is almost equal to that of maternal blood at O2 release site with physiological P50. This phenomenon is similar to that observed in human bloods (Zhang et al., 2003)
Influence of cooperativity on effectiveness of the Bohr shift in fetal and maternal blood
Using physiological PaO2, PvO2 and P50 values, the influence of cooperativity on the effectiveness of the Bohr shift was investigated for a wide range of n values.
In fetal blood, the effectiveness of the Bohr shift at the O2 release site (dS(33)/dP50) was increased with an increase in n value (Fig. 6A), and reached the highest value at approximately n=7 (this point is out of range in Fig. 6A). Conversely, at the O2 uptake site of fetal “arterial” blood, the highest effectiveness of the Bohr shift (dS(49)/dP50) was observed at n=2.6 (Fig. 6A).
In maternal blood, the highest value at the O2 release site (dS(50)/dP50) was observed at n=3.3, and that at the O2 uptake site (dS(95)/dP50) was observed at n=1.5 (Fig. 6B).
Correlation between effectiveness of the Bohr shift of fetal “arterial” blood and that of maternal uterine venous blood
The effectiveness of the Bohr shift at physiological P50 was compared between at the O2 uptake site (fetal “arterial” blood) and that at O2 release site (maternal uterine venous blood) to consider the gas exchange across the placental membrane. As shown in Fig. 7, the two dS(PO2)/dP50 values at PO2 values of 49 and 50 torr were nearly equal at n values below 3.0. The highest values were observed at an n values ranged from 2.5 to 3.0. The n value of horse Hb under standard conditions was reported to be 2.69 (Clerbaux et al., 1993). If there was large difference in effectiveness of the Bohr shift of both bloods, the increase or decrease in CO2 and H+ concentration in the fetal blood could not be prevented. The results obtained in this study seem to show that relatively low cooperativity is adequate for gas exchange across the placental membrane.
2,3-DPG has little effect on the Bohr coefficient of horse blood (Pellegrini et al., 1996) and this seems to suggest that fetal blood have the same Bohr coefficient as that of maternal blood. From these results, it is concluded that the physiological P50 values of horse fetal and maternal bloods are appropriate for the effectiveness of the Bohr shift occurring at the O2 uptake and release sites. The relatively low cooperativity of fetal and maternal bloods is well suited for the efficient coupling of O2 and H+ transport through the placental membrane. These theoretically obtained results confirm the results on human Hbs reported in our previous paper (Zhang et al., 2003)
O2 transport efficiency and effectiveness of the Bohr shift before exercise and at maximal exercise
Using literature values of in vivo O2 environment and P50 during incremental exercise in horse (Fenger et al., 2000), we calculated O2 transport efficiency at O2 release site and the effectiveness of the Bohr shift before exercise and at maximal exercise. The fetal umbilical arterial PO2 and physiological P50 are different to the literature values used in Fig. 2 (Comline and Silver, 1974). The O2 consumption rate at maximal exercise was approximately 20 times higher than that before exercise. Fig. 8 shows the relationship between the O2 transport efficiency and the effectiveness of the Bohr shift at various P50 values with n fixed at physiological values. Before exercise, the physiological P50 (22.9 torr) was nearly optimal for the effectiveness of the Bohr shift. The O2 transport efficiency is about 2/5 that of the maximal value (Fig. 8A). In contrast, at maximal exercise, the physiological P50 (31.8 torr) tended to be advantageous for O2 transport efficiency (Fig. 8B). The effectiveness of the Bohr shift was low, approximately 3/4 of that before exercise. The decrease in O2 affinity and the effectiveness of the Bohr shift at maximal exercise indicate both a decrease in pH and an increase in PCO2 must occurr. In fact, PvCO2 and pH values changed from 49.9 torr and 7.43 before exercise to 82.9 torr and 7.15 at maximal exercise (Fenger et al., 2000).
The P50 of the OEC before exercise, which is lower than PvO2, is nearly optimized with respect to the effectiveness of the Bohr shift. On the other hand, at maximal exercise, the physiological P50, which is higher than PvO2 but lower than PaO2, tends to be advantageous for O2 transport efficiency. These trends are basically similar to those of human Hb (Itoh et al., 2001)