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Journal of Applied Physiology
Vol. 82, No. 1, pp. 86-92, January 1997
GAS EXCHANGE, MECHANICS, AND AIRWAYS

Myoglobin oxygen dissociation by multiwavelength spectroscopy

Kenneth A. Schenkman¹, David R. Marble², David H. Burns³, and Eric O. Feigl²

¹ Department of Pediatrics, University of Wisconsin, Madison, Wisconsin 53792; ² Departments of Bioengineering and Physiology, University of Washington, Seattle, Washington 98195; and ³ Department of Chemistry, McGill University, Montreal, Quebec, Canada H3A 2K6

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

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ABSTRACT

Schenkman, Kenneth A., David R. Marble, David H. Burns, and Eric O. Feigl. Myoglobin oxygen dissociation by multiwavelength spectroscopy. J. Appl. Physiol. 82(1): 86-92, 1997.Multiwavelength optical spectroscopy was used to determine the oxygen-binding characteristics for equine myoglobin. Oxygen-binding relationships as a function of oxygen tension were determined for temperatures of 10, 25, 35, 37, and 40°C, at pH 7.0. In addition, dissociation curves were determined at 37°C for pH 6.5, 7.0, and 7.5. Equilibration was achieved with a myoglobin solution, at the desired temperature and pH, and 16 oxygen-nitrogen gas mixtures of known oxygen fraction. Correction for the inevitable presence of metmyoglobin was made by using a three-component least squares analysis and by correcting the end point oxymyoglobin spectra for the presence of metmyoglobin. The PO₂ at which myoglobin is half-saturated with O₂ (P₅₀) was determined to be 2.39 Torr at pH 7.0 and 37°C. The myoglobin dissociation curve was well fit by the Hill equation [saturation = PO₂/(PO₂ + P₅₀)].

equilibration; metmyoglobin; least squares analysis; second derivative

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INTRODUCTION

MYOGLOBIN is an important intracellular O₂-binding protein found in mammalian skeletal and cardiac muscle tissue. It is thought to function both as a reservoir of O₂ and as a transporter of O₂ within muscle cells (17). Myoglobin thus serves as a critical link between the capillary supply of O₂ provided by circulating hemoglobin and the O₂-consuming cytochromes for oxidative phosphorylation in the mitochondrial membrane. The first investigations of the myoglobin O₂-binding relationship date back to Theorell in 1934 (13) and Hill in 1936 (7). In the late 1950s, Antonini and Rossi-Fanelli (12) investigated the effects of temperature and pH on human myoglobin O₂ binding. Recent work on O₂ binding has been performed with other vertebrate hemoglobins and myoglobins (5). As accurate estimates of in vivo intracellular myoglobin saturation become available (6, 11, 14), myoglobin-saturation curves become important for determining intracellular O₂ tension (PO₂). This, in turn, is important in understanding the transfer of O₂ within myocytes and ultimately of myocyte oxidative metabolic regulation.

The early analyses, in which gasometric methods were used, have been replaced by spectrophotometric methods. However, most of the spectrophotometric equilibration experiments have been performed with the use of three or four discrete wavelengths for saturation determinations. Unfortunately, equilibration experiments have been especially difficult to perform at 37°C because of the rapid autooxidation of myoglobin(II) to metmyoglobin(III). Even the widely cited work of Rossi-Fanelli and Antonini (12) was limited at higher temperatures because of rapid autooxidation.

The development of rapid computation has allowed for the analysis of multiwavelength spectra; this analysis has many advantages over the traditional three- or four-wavelength method. The multilinear least squares analysis (3) offers an improved method for analyzing multiple spectra from equilibration systems when all components are known. This method provides a way to account for the presence of multiple species within a given system, thus allowing for correction due to the presence of measurable quantities of metmyoglobin.

The present report is the first analysis of myoglobin-O₂ binding using 150 wavelengths (500-650 nm) while simultaneously measuring and correcting for metmyoglobin contamination that inevitably occurs in vitro. The myoglobin O₂ curves were determined with 16 gas mixtures of known O₂ fraction, plus a zero point obtained with sodium dithionite. Measurements were made at several temperatures (10-40°C) and pH values (6.5-7.5).

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METHODS

Myoglobin preparation. One hundred milligrams of commercially obtained lyophilized horse heart myoglobin (Sigma no. M-1882) were dissolved in 1 ml of 50 mM phosphate buffer (NaH₂PO₄ ^· H₂O adjusted to pH 7.0 with NaOH) reacted with excess sodium dithionite (Na₂S₂O₄) to fully reduce the heme to the ferrous state (4) and were quickly separated on a 50-cm G-25 Sephadex column (Sigma no. G-25-150) at 4°C. The pure, reduced myoglobin fraction was collected and diluted in buffer to achieve a final absorbance between 0.4 and 0.5 optical density (OD) at 581 nm. One drop of 0.5% polyoxyethylenesorbitan (Tween; Sigma) and 40 mg/l of gentamicin were added to the solution to decrease bubble formation and to inhibit bacterial contamination, respectively. This solution was then evacuated under low vacuum for 5 min to decrease the dissolved air and was maintained under vacuum on ice until used. All solutions were used within 30 h to minimize conversion to metmyoglobin.

Gas equilibration. The equilibration apparatus consisted of a thin Silastic tubing (0.058-in. ID, 0.077-in. OD, Dow Corning no. 508-006) helically wrapped around a 24-in. long, 7/8-in. hollow-core brass cylinder through which temperature-controlled water was circulated. The Silastic tubing was wrapped 50 times around the core cylinder, with gentle stretching and flattening to increase the surface area-to-volume ratio. The brass cylinder with the Silastic tubing wrapped around it was encased in a 1.5-in.-diameter Plexiglas sleeve. The myoglobin solution was continuously pumped through the Silastic tubing at a rate of ~0.25 ml/min while the desired gas mixture was passed over the tubing in a countercurrent manner through the Plexiglas sleeve at a rate of 1 liter/min.

The entire equilibration apparatus and optical cuvette were contained within a temperature-regulated incubator. A 0.003-in. copper-constantin thermocouple was located within the equilibration apparatus just proximal to the solution exit, and a second thermocouple was placed inside the cuvette. These temperatures were continuously monitored on a multichannel digital thermometer, and the desired temperature was maintained to within 0.1°C by controlling both the incubator and water circulator temperatures.

After sufficient time for equilibration within the apparatus, the solution passed directly into a 70-µl, 1-cm-path-length glass flow-through cuvette (no. 75-G-10, Starna Cells). The solution was initially equilibrated with O₂. Then the apparatus was flushed with utility-grade nitrogen, followed by equilibrations in a stepwise manner with increasing fractions of O₂, using previously calibrated O₂-nitrogen gas mixtures (Airco Industries). The O₂ fraction in the calibrated gases was measured at the supplying company by paramagnetic detection (Siemens paramagnetic O₂ analyzer) for fractions >1%. For O₂ fractions <1%, a trace O₂ analyzer with a B2C type electrochemical fuel cell was used (Teldyne). O₂ fractions were reported with an accuracy of ±2% of the measured fraction.

Spectral acquisition. A custom-built, current-controlled light source provided the illuminating light to the cuvette via a 1/4-in. optical fiber bundle (Edmund Scientific), while a second fiber bundle was used to convey the transmitted light to the spectrophotometer (American Holographic model no. 100S). The light was then dispersed onto a 512-pixel photo diode array (Hammamatsu model no. S3901-512Q) with 1-nm resolution, and the resulting signal was fed via a 16-bit analog-to-digital converter (National Instruments #AT-MIO-16X) to a desktop 486-66 MHz personal computer (AMC). Optical spectra were acquired continuously at a rate of 4 per min during the entire equilibration process. This allowed for assurance that equilibration had taken place. Spectra were first obtained from the myoglobin solution equilibrated with 100% O₂, then with each gas mixture. After acquisition of the entire data set for each experiment, excess Na₂S₂O₄ and potassium ferricyanide [K₃Fe(CN)₆] (2) were added to separate aliquots of the original myoglobin solution and injected into the spectrophotometric cuvette for use as reference deoxymyoglobin and metmyoglobin spectra, respectively.

Spectral analysis. Analysis of the resulting spectra was based on a multilinear least squares method (3), using the wavelength range of 500-650 nm. The least squares analysis method is based on the assumption that a given spectrum from a limited component system is a linear combination of the individual spectral components. Linear regression of a given spectrum against the known single component spectra yields the fractional contribution of each component to the given spectrum. Because myoglobin undergoes rapid autooxidation to metmyoglobin at higher temperatures, a three-component least squares analysis was used, with pure oxymyoglobin, deoxymyoglobin, and metmyoglobin spectra used as the individual components. Figure 1 shows the visible spectra of these three pure components. To avoid baseline shifts affecting the analysis, the spectra were preprocessed by taking the second derivative with respect to wavelength (8), using a second difference method with a three-point smoothing average. Fractional saturation was defined as the ratio of oxymyoglobin/(oxymyoglobin + deoxymyoglobin). In a similar manner, the percentage metmyoglobin for each of the spectrum was determined as metmyoglobin/(metmyoglobin + oxymyoglobin + deoxymyoglobin).

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Fig. 1. Spectra of oxy-, deoxy-, and metmyoglobin. Oxymyoglobin - and -peaks are quite distinct from single deoxymyoglobin peak in visible spectral region. Smaller, but distinct, metmyoglobin peak at 628 nm can be seen. OD, optical density.
[View Larger Version of this Image (19K GIF file)]

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End point determinations. Accurate determination of the three end point spectra was critical to the analysis, and thus new end points were determined for each spectral data set. Metmyoglobin spectra were obtained by the addition of excess K₃Fe(CN)₆ to the original solution. Complete oxidation occurs in minutes. Similarly, deoxymyoglobin was produced by the addition of Na₂S₂O₄ to the original solution. Care was taken to acquire these spectra at the same temperature as the data set. The presence of modest excess amounts of either the K₃Fe(CN)₆ or Na₂S₂O₄ were shown not to affect the optical spectra.

The third end point needed for the analysis, oxymyoglobin, proved to be the most difficult to obtain. A pure 100% oxygenated myoglobin solution theoretically occurs only in a 100% O₂ environment at an infinite atmospheric pressure [see determination of PO₂ at which myoglobin is half-saturated with oxygen (P₅₀) below]. For practical reasons, it is assumed that myoglobin is sufficiently oxygenated in a solution that has been equilibrated with 100% O₂ at atmospheric pressure. However, due to the rapid autooxidation of myoglobin to metmyoglobin, especially at higher temperatures, it is difficult to obtain a pure oxymyoglobin spectrum. This difficulty was circumvented by correcting the resultant spectra for the presence of metmyoglobin.

Metmyoglobin correction of the oxymyoglobin end point. As described above, both the metmyoglobin and deoxymyoglobin end points were prepared chemically with reactions that go to completion; thus, their spectra represent essentially pure components. However, due to the rapid autooxidation of oxymyoglobin to metmyoglobin, it is difficult to prepare a sample of pure oxymyoglobin that has been equilibrated with 100% O₂. Because oxidation of myoglobin to metmyoglobin is inhibited by low temperature, the best spectrum of pure oxymyoglobin obtained from these experiments occurred after equilibration with 100% O₂ at 10°C. This spectrum was used as a reference calibration spectrum to develop a method to correct for metmyoglobin at higher temperatures.

Metmyoglobin has a relatively broad but distinct absorption band at ~628 nm, as shown in Fig. 1. Thus, the presence of metmyoglobin can be observed as the presence of increased absorption around this wavelength. An approach to quantifying the metmyoglobin contamination might be based on a visual comparison of the oxymyoglobin with the metmyoglobin spectra and then subtraction of the metmyoglobin spectrum from the oxymyoglobin spectrum, as shown in Fig. 2. However, determination of the optimal amount of correction remains ambiguous. A quantitative determination can be made by using the second-derivative spectra.

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Fig. 2. Correction for metmyoglobin in oxymyoglobin spectrum. Incremental wavelength-by-wavelength subtraction of the metmyoglobin spectrum from oxymyoglobin results in a corrected oxymyoglobin when all the metmyoglobin in original spectrum has been subtracted. Small amount of absorbance in the 620- to 650-nm region in original oxymyoglobin spectrum was caused by 7.3% contamination from metmyoglobin. Corrected spectrum (center) has no absorption due to metmyoglobin. Overcorrection due to subtraction of 14.6% metmyoglobin is shown for illustration, as well. A negative absorption band is seen in 620- to 650-nm region. The 7.3% metmyoglobin contamination is difficult to detect by using raw spectra.
[View Larger Version of this Image (23K GIF file)]

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The second-derivative spectrum of metmyoglobin has a zero crossing at 651 nm, where the second-derivative spectrum of oxymyoglobin is virtually flat (slope = 1.183 × 10⁷ OD/nm²), as shown in Fig. 3. The slope of the second-derivative metmyoglobin spectrum is quite steep compared with the second-derivative oxymyoglobin spectrum at this point (slope = 3.329 × 10⁵ OD/nm²). For this reason, as the amount of metmyoglobin contamination of an oxymyoglobin spectrum increases, the second-derivative spectrum has an increasingly positive slope at ~651 nm. Subtracting small amounts of the second-derivative metmyoglobin spectrum from the oxymyoglobin spectrum results in new spectra with progressively less positive slopes around that point.

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Fig. 3. Second-derivative spectra of reference oxymyoglobin and metmyoglobin for the spectral region from 610 to 700 nm, at 10°C and pH 7.0. The zero crossing of the metmyoglobin spectrum at 651 nm can be seen. The oxymyoglobin second-derivative spectrum is virtually flat in this region.
[View Larger Version of this Image (14K GIF file)]

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A grid-search incremental subtraction was performed by an iterative wavelength-by-wavelength subtraction of small amounts (0.1% increments) of the metmyoglobin second-derivative spectrum from the measured oxymyoglobin second-derivative spectrum, using the equation

(1)

where corrMb_j() is the corrected myoglobin spectrum, after j iterations in tenths of percent, for each wavelength (). Division by (1  0.001j) is used to normalize the spectrum to the original OD, after subtracting 0.001 jmetMb. The optimum correction for metmyoglobin was found when the slope of the second-derivative spectrum at ~651 nm is minimized, assuming that the true second-derivative oxymyoglobin slope is zero, as discussed below.

As a test of the assumption that the oxymyoglobin third derivative is zero at 651 nm, the algorithm was reapplied in a circular manner to the calibration oxymyoglobin sample (10° and 100% O₂) which yielded a metmyoglobin contamination of 0.4%. This amount of metmyoglobin contamination seems plausible for the sample obtained under the best experimental conditions. The actual measured slope of this calibration spectrum at 651 nm (1.183 × 10⁷ OD/nm²) might be used as a standard slope on which to base the metmyoglobin correction for the other equilibration experiments. However, the magnitude of this slope is dependent on the myoglobin concentration of the original sample. Although the myoglobin concentration for the various experiments was similar, it certainly was not constant. Note that end point spectra were obtained for each individual experiment, so that saturation determinations within an experimental data set were all made based on samples from the same solution at exactly the same concentration. Thus, for the practical reason of avoiding errors caused by different myoglobin concentrations in the individual experiments, and the observation that the third derivative of the oxymyoglobin spectrum is very close to zero at 10°C, the metmyoglobin correction method assumed that the third derivative of a pure oxymyoglobin spectrum is zero at 651 nm. It should be noted that this metmyoglobin correction is only applied to the oxymyoglobin end point for any given equilibration experiment. Saturation values for all intermediate points are determined by a three-component least squares analysis that includes oxymyoglobin, deoxymyoglobin, and metmyoglobin.

Water vapor pressure. PO₂ was determined for each of the dry gas mixtures from the known fraction of O₂ in the cylinder and the atmospheric pressure at the time of the experiment. However, the equilibration took place at the Silastic-liquid interface. The effective water vapor pressure in the Silastic membrane was estimated by a series of experiments. Serial absorption spectra were obtained from a myoglobin solution that was equilibrated alternately with dry stock gas and with the same gas mixture fully humidified at the same temperature (37°C). Theoretically, if the equilibration occurred at full saturation, the humidification of the gas should result in no difference in the fractional saturation measured. Alternatively, if the equilibration took place with a completely dry gas, a change in fractional saturation should be measured that would correspond to the theoretical change in PO₂ caused by the presence of water vapor. This change in saturation should be predictable, based on the slope of the dissociation curve for that temperature at that PO₂.

The measured change in saturation in switching from dry to wet gas was approximately one-half (50 ± 10%) that predicted, demonstrating that the equilibration took place in the presence of approximately half-saturated water vapor. Therefore, calculation of PO₂ was determined by the equation

(2)

where PB is the measured barometric pressure, PH₂O is the water vapor pressure at that temperature, and FO_{2 gas} is the O₂ fraction of the dry gas mixture. This water vapor correction equals 0.08 Torr in the P₅₀ value at 37°C.

Verification of equilibration. Equilibration of the myoglobin solution with the gas mixture is determined by the relative flow rates of gas across the outside of the Silastic tubing and the flow of the solution inside the Silastic tubing. To assure that sufficient time was allowed for complete equilibration, the effect of changes in the flow rate of the solution with constant gas flow was investigated. A series of experiments was performed in which the flow rate of the gas was maintained at the usual 1 l/min, while the myoglobin solution flow rate was halved from the study flow rate of 0.25 to 0.13 ml/min. The myoglobin solution entering the equilibrium apparatus was in room air, and the gas used for these experiments contained 4,080 parts per million O₂ in nitrogen, to provide a PO₂ near the P₅₀ of myoglobin at 37°C. No decrease in myoglobin saturation was observed when the solution flow rate was halved, indicating that equilibration was essentially complete under the study conditions.

Determination of P₅₀. The reaction of myoglobin with O₂ can be described by the equilibrium equation

(3)

where Mb is deoxymyoglobin, MbO₂ is oxymyoglobin, and k and k are the equilibrium rate constants for the forward and reverse reactions, respectively. The P₅₀ for myoglobin is defined by the rate constants as

(4)

and can be determined by the Hill equation, which in the general form is

(5)

where, in the present case, y is the fractional O₂ saturation of myoglobin and x is the PO₂. Thus y_min = 0.0 and y_max = 1.0. H is an empirical index of the heme-heme interaction (1), which for myoglobin is assumed to be 1.0, because myoglobin exists as a monomer and thus exhibits no cooperativity for O₂ binding.

Under these conditions (y_min = 0.0, y_max = 1.0, and H = 1.0), the Hill equation reduces to

(6)

and

(7)

However,

(8)

is also true, demonstrating that the equation is undefined when y = 1.0. This means that the end point obtained with 100% O₂ cannot be assumed to give a fractional myoglobin saturation of 1.0 but rather a fractional saturation very close to 1.0, as given by Eq. 6. For the case of a barometric pressure of 760 mmHg and a temperature of 37°C where the water vapor pressure is 47 Torr (16), combining Eqs. 2 and 6 gives However, this presupposes a value of x₅₀ which is found by fitting Eq. 6 to the data. Therefore, x₅₀ was estimated, and the fitting procedure was iterated to obtain the fractional saturation with 100% O₂ and the x₅₀. Individual Hill fitting to Eq. 6 (SigmaPlot, Jandel) was performed with three iterations for each data set, and the resulting five P₅₀ were averaged for each condition. The maximal change in P₅₀ between the second and third iteration for any experiment was 0.001 Torr.

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RESULTS

Temperature effects. Figure 4 shows the saturations as a function of time determined from an equilibration experiment. Equilibration experiments were repeated five times for each of the five temperatures investigated (10, 25, 35, 37, and 40°C at pH 7.0). Figure 5 shows the raw data along with the averaged Hill-fit curve. The significant left shift in P₅₀ with decreasing temperature is easily seen in this representation. Also, it can readily be appreciated that curve fitting to the Hill equation closely matches the experimental data. The steepness of these curves at lower PO₂ contrasts sharply with the sigmoid-shaped curve of the hemoglobin O₂ dissociation curve. The relationship between P₅₀ and temperature (T) can be described by the equation

(9)

which was determined by a best fit of the data, using the curve-fitting software in Matlab (The Math Works). Figure 6 demonstrates the exponential relationship of the P₅₀ with T, and the experimental results are given in Table 1.

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Fig. 4. Record from equilibration experiment. A 3-component least squares analysis using oxy-, deoxy-, and metmyoglobin determines the fractional saturation of myoglobin as oxymyoglobin/(oxymyoglobin + deoxymyoglobin). Calculated myoglobin saturation values are shown as a function of time (solid line). Equilibration is started with 100% O₂, followed by flushing of the chamber with N₂. Stepwise increase in saturation corresponds to change from 1 gas mixture to the next. Fraction of metmyoglobin (broken line) is also determined by least squares as metmyoglobin/(oxymyoglobin + deoxymyoglobin + metmyoglobin).
[View Larger Version of this Image (16K GIF file)]

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Fig. 5. Myoglobin-O₂ dissociation curves as a function of temperature at pH 7.0. Calculated saturations for each experiment are shown, with Hill-fit dissociation curve from averaged values for PO₂ at which myoglobin is half-saturated with oxygen (P₅₀) superimposed. Each dissociation experiment was repeated 5 times. Hill equation is seen to fit data quite well. A significant left shift is seen, as temperature decreases from 40 to 10°C. As described in the text, maximum theoretical fractional saturation was determined by Hill equation but approached 100% even at atmospheric pressure and 37°C. In addition to the zero point with sodium dithionite (Na₂S₂O₄) and the 14 gas mixtures shown, saturations were obtained with room air and 100% O₂.
[View Larger Version of this Image (21K GIF file)]

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Fig. 6. Relationship of myoglobin P₅₀ with temperature at pH 7.0. Averaged values of P₅₀ for temperatures are shown, along with the best-fit exponential equation. Error bars are not shown because they fall within symbols.
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Table 1. Effect of temperature on myoglobin P50 at pH 7.0  Temp, °C P50, Torr 40.0 3.11 ± 0.04  37.0 2.39 ± 0.06  35.0 2.03 ± 0.02  25.0 0.78 ± 0.02  10.0 0.17 ± 0.01 Values for P50 are means ± SD; n = 5 experiments in each case. Temp, temperature; P50, PO2 at which myoglobin is half-saturated with oxygen.

pH effects. Equilibrium experiments were performed at 37°C for pH 6.5, 7.0, and 7.5. With the use of different concentrations of NaOH, the phosphate buffer was made in the same manner for each set of experiments. To assure that the change in P₅₀ with change in pH was not due to variation in ionic strength, equilibration experiments were also performed at a constant pH of 7.0 with phosphate buffers of 25 and 50 mM. No difference in P₅₀ was observed between these ionic strengths. Although the difference is small, there is a demonstrable linear relationship between P₅₀ and pH, as shown in Fig. 7. This relationship can be described by the equation

(10)

These results are tabulated in Table 2. The Pearson correlation coefficient for the linear regression including all 15 equilibration experiments (5 at each pH) is 0.832 with P < 0.001. 

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Fig. 7. Relationship of myoglobin P₅₀ with pH at 37°C. Averaged P₅₀ ± SE for each pH are shown, along with the best-fit linear equation. As shown, Bohr effect due to pH for myoglobin is quite small.
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Table 2. Effect of pH on myoglobin P50 at 37°C pH P50, Torr 6.5 2.46 ± 0.04  7.0 2.39 ± 0.06  7.5 2.32 ± 0.02 Values for P50 are means ± SD; n = 5 experiments.

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DISCUSSION

Previously, accurate determinations of myoglobin O₂ saturation relationships have been plagued by the rapid autooxidation of oxymyoglobin to metmyoglobin. Perhaps, in part, for this reason, much of the early work was performed at room temperature because this reaction rate is slow at room temperature. The results presented above account for the metmyoglobin problem and allowed for determination of accurate myoglobin O₂ dissociation curves at physiologically relevant temperatures. The three-component least squares analysis allows for a spectrum-by-spectrum correction for the presence of metmyoglobin. This is particularly important, since the production of metmyoglobin is, in part, dependent on the PO₂ (15). Equally important is the recognition of the contamination of the end point oxymyoglobin spectrum by even small amounts of metmyoglobin. The method used to determine the amount of metmyoglobin present in a given sample can be used to demonstrate that several percent metmyoglobin can easily be hidden in what otherwise appears to be a pure oxymyoglobin spectrum. Although previous investigators mention the problem of metmyoglobin production, there are no reports of attempts to quantify or correct for this problem.

The present results compare favorably with the data available in the literature. Rossi-Fanelli and Antonini (12) reported a P₅₀ of 0.7 Torr for horse myoglobin at 20°C. Theorell (13) reported a P₅₀ value of 0.65 Torr at 20°C. Other mammalian species have been reported to have P₅₀ at 20°C ranging from 0.46 Torr in sheep muscle to 1.3 Torr in rat skeletal muscle (1). Human myoglobin P₅₀ has been reported to be quite similar to that of the horse, with values ranging from 0.65 to 0.72 Torr at 20°C (12). Recent investigations using kinetic methods have determined a P₅₀ of 0.48 and 0.59 Torr for cat and mouse myoglobin at 20°C, respectively (5). The present work yields a P₅₀ of 0.46 Torr at 20°C, as calculated from Eq. 9. It should be noted that in all of these investigations, the only data reported at temperatures >25°C are from Antonini and Brunori (1), where the oxymyoglobin-dissociation curves for human myoglobin were determined by six data points at 35°C and two data points at 40°C. For these temperatures, Antonini and Brunori report a P₅₀ of 2.09 and 3.02 Torr, whereas the results from the current work (shown in Table 1) yield values of 2.03 and 3.11 Torr for 35 and 40°C, respectively.

The present results describe the behavior of myoglobin with an emphasis on the physiologically relevant range that will allow an improved understanding of the role of myoglobin within the cell. The regulation of O₂ flux within the cell is intricately related to the rate of oxidative phosphorylation in the mitochondria, but it remains to be determined what aspects of O₂ flux are rate limiting and what constitute all of the regulatory mechanisms behind oxidative phosphorylation. New magnetic resonance spectroscopy methods for interrogation of living muscles are emerging that can give in vivo estimates of myoglobin O₂ saturation in working muscle (9, 10). Thus the development of a more precise understanding of the relationship of the myoglobin-O₂ dissociation curve with changes in pH and temperature can lead to more accurate measurements of in vivo intracellular PO₂.

In conclusion, the present results provide a comprehensive description of the myoglobin-O₂ dissociation relationship for equine myoglobin. In particular, this report focuses on the physiologically relevant conditions of pH and temperature, including low temperatures that may be encountered in hypothermic clinical settings. Multiwavelength optical spectroscopy was used to correct for the inevitable presence of metmyoglobin contamination during in vitro measurements.

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ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-49228 and R01-HL-49822 and by American Heart Association Grant 94-WA-104.

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FOOTNOTES

Address for reprint requests: K. A. Schenkman, Dept. of Pediatrics, Univ. of Wisconsin, 600 Highland Ave., Madison, WI 53792-4108 (E-mail: kaschenk{at}facstaff.wisc.edu).

Received 13 March 1996; accepted in final form 21 August 1996.

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