Virtual cooperativity in myoglobin oxygen saturation curve in skeletal muscle in vivo

Background Myoglobin (Mb) is the simplest monomeric hemoprotein and its physicochemical properties including reversible oxygen (O2)binding in aqueous solution are well known. Unexpectedly, however, its physiological role in intact muscle has not yet been established in spite of the fact that the role of the more complex tetrameric hemoprotein, hemoglobin (Hb), in red cells is well established. Here, I report my new findings on an overlooked property of skeletal Mb. Methods I directly observed the oxygenation of Mb in perfused rat skeletal muscle under various states of tissue respiration. A computer-controlled rapid scanning spectrophotometer was used to measure the oxygenation of Mb in the transmission mode. The light beam was focused on the thigh (quadriceps) through a 5-mm-diameter light guide. The transmitted light was conducted to the spectrophotometer through another 5-mm-diameter light guide. Visible difference spectra in the range of 500–650 nm were recorded when O2 uptake in the hindlimb muscle reached a constant value after every stepwise change in the O2 concentration of the buffer. Results The O2 dissociation curve (ODC) of Mb, when the effluent buffer O2 pressure was used as the abscissa, was of a sigmoid shape under normal and increased respiratory conditions whereas it was of rectangular hyperbolic shape under a suppressed respiratory condition. The dissociation curve was shifted toward the right and became more sigmoid with an increase in tissue respiration activity. These observations indicate that an increase in O2 demand in tissues makes the O2 saturation of Mb more sensitive to O2 pressure change in the capillaries and enhances the Mb-mediated O2 transfer from Hb to cytochrome oxidase (Cyt. aa3), especially under heavy O2 demands. Conclusion The virtual cooperativity and O2 demand-dependent shifts of the ODC may provide a basis for explaining why Mb has been preserved as monomer during molecular evolution.


Background
Mb is a monomeric hemoprotein with a molecular weight of 17 kDa, carrying a single oxygen (O 2 )-binding site per molecule. It is located near the contractile elements and cell membranes in the red skeletal and cardiac muscles of vertebrates [1]. Previously, Millikan [2,3] proposed the following three possible physiological functions for Mb: (a) an O 2 store during temporary deficits in O 2 supply, (b) an intracellular O 2 transport agent and (c) an intracellular catalyst. Among them, the first function has traditionally been accepted. In the muscles of a beating heart and exercising skeletal muscles, Mb acts as a short-term O 2 store (i.e., an O 2 buffer), tiding the muscles over from one contraction to the next. The rich Mb content in skeletal muscles in aquatic mammals is considered to provide a longterm O 2 store during diving. However, this role of Mb, at least in human, is not significant because its oxygen storage capacity is so low that the total oxygen bound to Mb is exhausted within ca. 5.5 s after being cut off from the O 2 supply [4]. The second function, called "facilitated O 2 -diffusion by Mb", was based on findings in in vitro experiments [5,6]. The conditions required for this facilitated diffusion to occur are [7]: (a) existence of deoxygenated Mb in a certain fraction (or certain low intracellular partial pressure of O 2 ), (b) existence of a spatial gradient of oxygenated Mb concentration as a driving force for translational diffusion of Mb, and (c) sufficient mobility of the oxygenated Mb to permit diffusion. Although this mechanism has been widely accepted, several discrepancies remain unresolved [8][9][10][11][12]. As for the third function, Doeller and Wittenberg [13] proposed the occurrence of Mbmediated oxidative phosphorylation in heart myocytes under aerobic conditions. However, Mb concentration is not closely related to the oxidative capacity of muscles, that is, the concentration is higher in skeletal muscles (~0.5 mmole/kg wet wt.) than in heart muscles (~0.25 mmole/kg wet wt.) [7].
Thus, the physiological roles of Mb have not yet been established. Recently, alternative functions of (d) O 2 sensing and (e) nitric oxide scavenging were proposed [14]. Another recent paper [15] seemed to have totally scrambled the past long-term disputes about the physiological significance of Mb. It was shown using gene-knockout technology that mice without Mb are fertile, exhibit normal exercise capacity, and have a normal ventilatory response to low O 2 levels, suggesting that Mb is not essential for apparently normal cardiovascular and musculoskeletal function in a terrestrial, homoiothermic mammal. However, it has been reported that the disruption of Mb results in the activation of multiple compensatory mechanisms such as increases in Hb concentration, hematocrit, coronary flow, coronary reserve, and capillary density [16]. Further, a Mb-like hemoprotein, neuroglobin, has been found in the vertebrate brain [17] contrary to the long-held belief that Mb is restricted to vertebrate cardiomyocytes and oxidative skeletal myofibers. These studies imply that further investigations are required to reveal the physiological role of Mb in intact organs.
In contrast to Mb, which shows a rectangular hyperbolic ODC, the vertebrate Hb, a tetramer carrying four O 2 binding sites, shows a sigmoid ODC that is described in terms of a four-step cooperative O 2 binding. It is widely accepted that the sigmoid ODC enables Hb to transport O 2 with high efficiency: it is nearly fully saturated with O 2 in the lungs and it unloads O 2 sensitively depending on decreases in the partial pressure of oxygen (PO 2 ) in peripheral tissues. Here, no convincing explanation has been given for the question: does the hyperbolic ODC of Mb have any physiological adequacy or reasonability? The Bohr effect of Hb (pH dependence of O 2 affinity) has physiological significance, in that it enhances O 2 unloading from Hb in the capillaries where pH tends to decrease and in that it increases the solubility of CO 2 as bicarbonate in the venous blood through deoxygenation-induced uptake of protons by Hb. In contrast, Mb lacks the Bohr effect and it had long been believed that Mb was a totally non-allosteric protein, although recently lactate, a metabolic product, was found to cause a right-shift of the ODC for horse and sperm whale Mbs [18].
It is well established that the O 2 affinity of Mb is higher than that of Hb but lower than that of Cyt. aa 3 , as known from the relative positions of the ODCs for Mb and Hb and the oxidation curve for Cyt. aa 3 (Fig. 1). This fact led one to the idea that Mb acts as an intracellular O 2 transfer agent from Hb (vascular space) to Cyt. aa3 (mitochondria). Here, one must not overlook an important fact. The three curves in Fig. 1 are drawn with the same PO 2 scale. Therefore, they give O 2 saturation (Y) or the degree of oxidation for the individual proteins when dissolved in the same solution and are in equilibrium with oxygen at the given PO 2 value. However, in vivo, they sense different PO 2 values due to the presence of a PO 2 gradient along the path from the inside of red cells to the mitochondria in myocytes. Thus, the relative positions of the three curves in Fig. 1 must be considered with this precaution, and direct in vivo observations of Y or the degree of oxidation for these three individual proteins are required to get insight into their ensemble functional roles. Recently, using 1 H nuclear magnetic resonance spectroscopy, Mole et al. [19] and Richardson et al. [20] directly observed Y Oxygen dissociation curves (ODCs) for Hb (whole blood) and Mb and oxidation curve for Cyt Figure 1 Oxygen dissociation curves (ODCs) for Hb (whole blood) and Mb and oxidation curve for Cyt. aa 3 (at 37°C). PO 2 , partial pressure of oxygen in mmHg. Data from Imai [36].
for Mb in human skeletal muscles under exercise of different intensities or during breathing of air with different O 2 contents. In these studies, Mb was used as an indicator of intracellular PO 2 , and no attention was paid to the relation between Mb saturation and capillary PO 2 .
In the present study, we directly measured Y for Mb in isolated rat hindlimb muscles, perfused with a Hb-free medium, under vigorous changes in respiration conditions. We plotted the Y values as a function of buffer PO 2 and found that the apparent ODC thus plotted for skeletal muscle Mb was rectangular hyperbolic under a suppressed metabolic activity condition but it became sigmoid under enhanced metabolic activity conditions, realizing virtually cooperative O 2 binding by monomeric Mb.

Muscle perfusion
All experimental procedures were performed according to the institutional guidelines for animal care and use of the Committee for Animal Care of Osaka University and the Japanese Physiological Society. Male Wistar rats (250 to 300 g body weight, N = 12) fed on a commercial diet were used. Rats were anesthetized with sodium pentobarbital (30 mg/kg body wt., intraperitoneal injection). Prepara-tion of the isolated rat hindlimb and the perfusion apparatus were described previously [21,22]. Surgery was modified from those of Ruderman et al. [23] and Shiota et al. [24]. After a midline abdominal incision, the superficial epigastric vessels were ligated. The abdominal wall was then incised from the pubic symphysis to the xiphoid process. The spermary, testis, and inferior mesenteric arteries and veins were ligated, and the spermaries, the testises, and part of the descending colon were excised, together with contiguous adipose tissue. The caudal artery and internal iliac artery and vein were also ligated. Ligature were placed around the neck of the bladder, the coagulating gland and the prostate gland. While carefully Apparent ODCs for Mb in perfused muscle at various steady-state O 2 uptake levels Figure 3 Apparent ODCs for Mb in perfused muscle at various steady-state O 2 uptake levels. PO 2 is the same as in Fig. 2. A, ODCs as O 2 saturation (Y) plotted against log PO 2 . The lines were calculated from the Hill equation (see below). Each ODC was obtained from three animals (three muscle preparations). Symbols (mean ± SD) express observed points and their meaning is the same as in Fig. 2. The lines without symbols are the ODCs for Mb in non-respiring muscle [21]. B, Apparent ODCs as expressed by the Hill plot which is based on the linearized Hill equation [25]: log {Y/(1 -Y)} = n (log PO 2 -log P Y50 ). The slope of the plot (n) was constant and expressed as n app in the present paper. The n app and intercept values obtained from the Hill plots are listed in Table 2.
Steady-state O 2 uptake rate (V) of perfused rat hindlimb muscles as a function of effluent buffer PO 2 Figure 2 Steady-state O 2 uptake rate (V) of perfused rat hindlimb muscles as a function of effluent buffer PO 2 . The rat hindlimb was perfused with Krebs-bicarbonate buffers containing no additive (•) as control, 0.4 mM of KCN (❍) for suppression of muscle respiration, and 5 µM (᭝) or 10 µM (▲) 2,4-dinitrophenol for stimulation of muscle respiration. Symbols express observed points. Each plot is the mean of experiments using three animals, and the errors for each data point are less than the size of symbols. The solid lines were calculated using a rectangular hyperbolic curve: V = V max (PO 2 / P V50 )/{1 + (PO 2 /P V50 )}. The values of P V50 and V max are given in Table 1 which also includes the maximal values of influent and effluent PO 2 .
removing the skin covering the lower half of the animal, the vessels that supply the subcutaneous region were ligated. Then, the inferior epigastric, iliolumbar and renal arteries and veins were ligated as well as the coeliac axis and portal vein. Further, a ligature was also placed around the tail. A hemoglobin-free Krebs-bicarbonate buffer (NaCl, 115 mM; KCl, 5.9 mM; MgCl 2 , 1.2 mM; NaH 2 PO 4 , 1.2 mM; Na 2 SO 4 , 1.2 mM; NaHCO 3 , 25 mM; CaCl 2 , 2.5 mM; glucose, 10 mM; pH 7.4) containing 4% (w/v) polyvinylpyrrolidone (PVP-40T; average M.W., 40,000; Sigma) was perfused from the abdominal aorta in the flow-through mode at a constant flow rate of 1.0 ml/min/ g muscle. Perfusate and muscle temperature were maintained at 25 ± 0.5°C. The effluent was collected from the inferior vena cava in order to measure the O 2 uptake rate. PO 2 in the influent and the effluent buffers was monitored with an oxygen electrode. The rate of O 2 uptake was calculated from the flow rate and the difference in O 2 concentration between the influent and the effluent buffers. Before each measurement, the rat hindlimb was perfused with the buffer equilibrated with 95% O 2 + 5% CO 2 for 30 min. Then, the O 2 concentration in the perfusate was decreased stepwise by mixing a buffer equilibrated with 95% O 2 + 5% CO 2 and another equilibrated with 95% N 2 + 5% CO 2 , and the measurement was started. As required, potassium cyanide or 2,4-dinitrophenol was infused to modify the O 2 uptake rate of the perfused muscle. During each measurement of about 60 min, the perfusion pressure remained nearly constant at 73-78 mmHg. All chemicals used were of analytical reagent grade.

Spectrophotometric measurement of myoglobin oxygenation
A computer-controlled rapid scanning spectrophotometer (USP-501, Unisoku, Osaka, Japan) was used to measure the oxygenation of Mb in the transmission mode [21,22]. The light beam was focused on the thigh (quadriceps) through a 5-mm-diameter light guide. The transmitted light was conducted to the spectrophotometer through another 5-mm-diameter light guide. Visible difference spectra in the range of 500-650 nm were recorded when O 2 uptake in the hindlimb muscle reached a constant value after every stepwise change in the O 2 concentration of the buffer.

Analysis of data
Changes in the O 2 uptake rate were analyzed using a rectangular hyperbolic curve equation: V = V max (PO 2 /P V50 )/ {1 + (PO 2 /P V50 )}. Here, the maximal rate of O 2 uptake (V max ) and effluent buffer PO 2 at half maximal O 2 uptake (P V50 ) were obtained from the slope (1/V max ) and the ordinate intercept (P V50 /V max ) of the Hanes-Woolf plot (effluent PO 2 /V vs. effluent PO 2 ). Changes in oxygen saturation of Mb (Y) were analyzed using the Hill equation [25], Y = PO 2 n /(PO 2 n + P Y50 n ), where P Y50 is PO 2 at half saturation of Mb (Y 50 ) and n is the Hill coefficient. In the original Hill equation, n was treated as a constant. This equation expressed the ODC of Mb well but not the ODC of Hb because the Hill plot for Hb deviated from a straight line at both extremes. To make the Hill plot applicable to Hb, Wyman [26] extended the equation by linearizing it in the form: log {Y/(1 -Y)} = n (log PO 2 -log P Y50 ) where n was treated as a variable. This extension allowed cooperativity measured by n to vary depending on Y. Figure 2 shows the steady-state O 2 uptake rate (V) of a perfused muscle. The respiration rate of the muscle was varied by controlling mitochondrial respiration activity by about 7.5-fold (compare the V max values described below) from a suppressed state with an inhibitor (KCN) of mitochondrial respiration to enhanced states with two levels of an uncoupler (2,4-dinitrophenol) of mitochondrial respiration. Three preparations of muscle were used for the experiments in each mitochondrial activity state. The actual PO 2 values of the influent and effluent buffers at the maximal O 2 inflow rate are listed in Table 1. Changes in the value of V were well expressed by a rectangular hyperbolic curve as a function of effluent buffer PO 2 (Fig.  2). Table 1 also gives the estimated V max and P V50 obtained from these data as described in Materials and Methods. V max and P V50 became larger by approximately 7.5-fold and 2-fold, respectively, for the maximal increase in respiration activity. With elevation of respiration activity, the critical PO 2 , at which O 2 uptake of perfused hindlimb muscle starts to decrease, increased to higher values. This indicates that, under higher respiration activity, O 2 supply a, 2,4-dinitorophenol; b, Maximal value of steady-state O 2 uptake rate (V) at infinite influent PO 2 (in µmol/min/g muscle); c, effluent PO 2 at V = half V max (in mmHg). Values of V max and P V50 were obtained from solid lines shown in Figure 2.

Oxygen uptake by perfused muscle in different respiration states
to the perfused muscle was limited even at very high influent PO 2 (~700 mmHg). This situation occurred because the flow rate of the perfusate and the capillary PO 2 were controlled independently of the respiration activity state so that the PO 2 gradient between the perfusate and the mitochondria became larger at higher respiration states. Figure 3A shows ODCs for Mb in the perfused muscle.

Relationship between effluent buffer PO 2 and Mb oxygenation in perfused muscle
Here, Y is plotted against effluent buffer PO 2 . These ODCs are apparent in the sense that the PO 2 is not the value at the location where Mb is working. The curve was shifted to the right and became steeper as muscle respiration activity was enhanced. These oxygenation data were further expressed by means of the Hill plot (log [Y/(1-Y)] vs. log PO 2 ), yielding linear plots (Fig. 3B). The effluent buffer PO 2 at half saturation (P Y50 ) and the slope of the Hill plot (the Hill coefficient, n) obtained from these plots are listed in Table 2, where n is expressed as n app (apparent n). The P Y50 value became larger with an increase in muscle respiration activity. The log P Y50 value was nearly linearly related to the log P V50 value (not shown). The n app value also increased from 1.10 in the suppressed respiration activity state to 1.85 in the 7.5-fold enhanced respiration activity state.
Since this virtual cooperativity is of particular interest, its relation to O 2 uptake rate was further examined. Figure 4 shows the dependence of n app on V at the half O 2 saturation point of Mb (V Y50 ). The n app value asymptotically increased from unity for the non-respiring state to 2.23 at infinite V Y50 . These results indicate that the apparent ODC of Mb in the perfused muscle is transformed from a hyperbolic curve to a sigmoid curve depending on the magnitude of tissue respiration. Effect of the Hill coefficient (n) on ratio of substrate (or ligand) concentrations necessary to change enzyme activity from 90% to 10% of maximal can be expressed with a parameter, R (= 81 1/n ) [27]. Here, the O 2 transport efficiency (EO 2 ) was estimated as ratio of the parameter at n app = 1 to that at a given value of n app (Fig. 4 inset). Figure 5 shows the effect of muscle respiration on the O 2 gradient between effluent and the perfused tissue. Assuming the effluent buffer PO 2 approximates the capillary PO 2 , the calculated O 2 gradient from capillary to cytoplasmic space (∆PO 2 ) is plotted against V Y50 . Here, the P 50 value of Mb in the perfused muscle was 2.3 mmHg [21]. ∆PO 2 increased with the increase in V Y50 . This result indicates the presence of a large O 2 diffusion barrier between capillary lumen and cytoplasmic space.

Discussion
In the present study, by using computer-controlled rapid scanning fiber-optic spectrophotometry, I directly measured Y for Mb in isolated rat hindlimb muscles under extensive changes in respiration rate caused by mitochondrial activity control or perfusate PO 2 control. It is assumed that capillary PO 2 may be approximated by effluent PO 2 in the present experiment, and I plotted the Y values as a function of effluent buffer PO 2 . Thereby, I expected that this treatment enabled a meaningful comparison of the ODCs for Mb and Hb. I found that thus plotted apparent ODC for skeletal muscle Mb was hyperbolic under a suppressed metabolic activity condition whereas it became sigmoid under enhanced metabolic activity conditions, realizing virtually cooperative oxygenation of the monomeric Mb.
It is generally accepted that cooperative O 2 binding by Hb is advantageous for efficient O 2 transfer from the alveolar gas to red cells and from red cells to peripheral tissues. Based on the Hill equation, Graby and Meldon [28] showed that an n value (here, n is a constant) of 1.5 to 2.0 is more favourable for minimizing the change in blood flow under resting conditions than the normal n value of 2.5 to 3.0, whereas an n value as large as 3 is beneficial for a large amount of O 2 extraction under vigorous exercise. Kobayashi et al. [29] showed that, under resting conditions, O 2 release from Hb becomes most sensitive to PO 2 change at Y = 38% where cooperativity measured by n (here, n is a variable of PO 2 ) is not maximal, whereas it becomes less sensitive at the mixed venous blood PO 2 where Y is around 70% and cooperativity is nearly maximal. These reports indicate that, under resting conditions, the blood reserves an O 2 transport capacity to meet possible increases in O 2 demand, e.g. under exercise conditions, and the sigmoid character of ODC becomes more important under such conditions. This situation is real- ized by maintaining Y at a rather high level (70%) below which the Y value drops sharply upon PO 2 decrease within the very steep middle portion of ODC.
The present study has clearly shown that the apparent ODC for Mb in intact skeletal muscle is sigmoid, the n app value being 1.46 under the control condition (Table 2) and 2.23 under the maximal respiratory condition (Fig.  4). These n app values greater than unity imply that the muscle Mb binds O 2 in a virtually cooperative manner with variation of effluent buffer PO 2 . This phenomenon implies that the sensitivity of Y for Mb to vessel PO 2 change becomes higher for increased O 2 demands than for normal O 2 demand. In addition to this effect, the rightward shift of the ODC upon increases in oxygen demand will undoubtedly enhance O 2 unloading from Mb. These effects will facilitate Mb-mediated O 2 transfer from Hb to Cyt. aa 3 , especially for heavy O 2 demands. Based on the Hill equation, the O 2 transport efficiency of Mb in the perfused muscle is estimated to increase ca. 4-fold under the control condition and ca. 11-fold under maximally respiring condition (Fig. 4 inset).
The Mbs isolated from body wall or radular muscle of a limited number of annelidan and molluscan species are dimers and show some cooperativity in oxygen binding (1 < n < 2) but no Bohr effect [30]. The physiological signif-icance of these dimeric Mbs is unknown. As shown in the present study, the ODC of monomeric Mb can exhibit virtual cooperativity and O 2 demand-dependent shifts. The virtually cooperativity and O 2 demand-dependent shifts of Mb oxygenation in vivo are probably common features at least for vertebrate Mbs, and this may provide a basis for explaining why the vertebrate Mb has been preserved as a monomer during molecular evolution.
The virtual cooperativity in Mb oxygenation observed in the present study is explained in terms of the PO 2 gradient along the O 2 diffusion path. If the tissue O 2 demand was null, then the PO 2 gradient would be absent and the apparent ODC for Mb would be identical with the real ODC for Mb in solution. At a steady state with a certain level of O 2 demand a PO 2 gradient develops across red cell membrane, blood plasma, capillary wall, sarcolemma and sarcoplasm, making the PO 2 sensed by Mb lower than the capillary PO 2 . Then, the apparent ODC will be shifted toward the right because a capillary PO 2 value higher than the PO 2 value sensed by Mb is needed to maintain the same Y value as that which occurs in the absence of a PO 2 gradient. When the tissue O 2 demand is kept constant, the ratio of capillary PO 2 to sarcoplasm PO 2 will become larger at low capillary PO 2 than at high capillary PO 2 . This will cause a more extensive rightward shift of the apparent ODC in the low saturation range than in the high saturation range, making the curve steeper than the real one. Increase in tissue oxygen demand will enhance this mechanism and make the curve more right-shifted and sigmoid. All the apparent ODCs observed in the present study are shifted toward the right compared to the real one measured for Mb in solution (Fig. 3).
At present, detailed explanations for this cooperative mechanism is difficult. However, it could be argued that heterogeneous oxygenation in tissue [31] and in single myocytes [32] might be responsible in part for the shift and the shape change of the Mb ODC, and might also enhance intercellular O 2 transfer, i.e., re-distribution of O 2 among adjacent myocytes, although we adopted high and constant flow rate perfusion conditions (i.e., about 50 times higher than normal blood flow) and, thus, the perfused vessels of muscle were always passively dilated.
Unfortunately, it is not practical to use a Hb solution or a red cell suspension as the perfusate in our experiments because the absorption spectra for Hb and Mb are too similar and independent observations of Mb oxygenation are not feasible, especially when the concentration of Hb is much higher than that of Mb. To overcome the problem that the O 2 solubility of the buffer is much smaller than that of a Hb solution or a red cell suspension two strategies were employed: one was to make the PO 2 of the influent buffer as high as that of water vapor-saturated O 2 (ca. Relationship between n app and steady-state O 2 uptake rate at Y = 50% (V Y50 ) Figure 4 Relationship between n app and steady-state O 2 uptake rate at Y = 50% (V Y50 ). The V Y50 values were obtained from the hyperbolic curves in Fig. 2. The symbols (mean ± SD) are as in Fig. 2. The solid line, which was calculated from the equation: n app = 1 + 1.23*V Y50 /(0.193 + V Y50 ), simulates the observed dependence. The maximal value of n app at infinite V Y50 is 2.23, which was obtained using the Hanes-Woolf plot, V Y50 /(n app -1) vs. V Y50 . The inset figure shows the relationship between n app and O 2 transport efficiency (EO 2 ) (see Text).
700 mmHg) and the other was to use a high flow rate for the buffer, which was about 50 times higher than that of normal blood flow. As a result, the inflow of O 2 was about 5 times larger than that of the tissue O 2 consumption at the control metabolic rate. The large O 2 diffusion barrier (see Fig. 5) and the high PO 2 of the influent buffer (and consequently, the high capillary PO 2 ) are an additional (and probably, major) cause for the rightward shift of the apparent ODC of Mb. The apparent ODCs of Mb in the control and enhanced respiration activity states (Fig. 3A) are right-shifted compared to the whole blood ODC (Fig.  1). One may suppose that Mb cannot work when the capillary PO 2 is in the physiological range (40 to100 mmHg) because its O 2 saturation is too low to function, as judged from Fig. 3A. However, the actual apparent ODCs for Mb in muscles with blood circulation will be shifted much more toward the left compared to those shown in Fig. 3A, and Mb can be saturated with O 2 to practical levels. The important point is that the difference in in vivo O 2 saturation between Hb and Mb is not so large as that expected from the ODCs in Fig. 1. In fact, Y for Mb in working muscles is less than around 50% [19,20,[31][32][33]. Red blood cell (RBC) in perfusion buffer appears to exert considerable effects on intracellular oxygenation in the beating heart [34], probably due to the facilitated O 2 transfer by RBC motion within capillary lumen [35]. Therefore, the virtually cooperative oxygenation of Mb might be only demonstrated in organs perfused with RBC-free medium. However, it is well known that the blood flow in the capillary bed is not constant and frequently only plasma flow is observed. In this case, the virtually cooperative oxygenation of Mb may play a significant role for O 2 transfer from capillary to mitochondria.
In summary, I found that the ODC for Mb in intact skeletal muscle is sigmoid and right-shifted. This virtually cooperative O 2 binding by Mb and the right-shift of ODC become more marked as tissue respiration activity is increased. Hence, increase in O 2 demand in tissues makes the O 2 saturation of Mb more sensitive to capillary PO 2 change and enhances Mb-mediated O 2 transfer from red cell to motochondria. The virtual cooperativity and O 2 demand-dependent shifts of ODC may give a basis for explaining why Mb has been preserved as a monomer during molecular evolution. Preservation of a monomeric structure may be required to retain multi-functional role of Mb in vivo.