THE JOURNAL OF BIOLOGICAL CHFMSTRY Vol. 2.50, No. 23, Issue of December 10. pp. 9038-9043, 1975

Printed in U.S.A.

Role of Myoglobin in the Oxygen Supply to Red Skeletal Muscle*

(Received for publication, May 9, 1975)

BEATRICE A. WI~ENBERG AND JONATHAN B. WI?TENBERG$

From the Department of Physiology, Albert Einstein College of Medicine, New York, New York 10461

PETER R. B. CALDWELL

From the Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032

The contribution of myoglobin to the oxygen uptake of red skeletal muscle was estimated from the difference in oxygen uptake with and without functional myoglobin. The oxygen uptake of bundles (25 mm long, 0.5 mm mean diameter) of muscle fibers teased from pigeon breast muscle was measured in families of steady states of oxygen pressure from 0 to 250 mm Hg. The oxygen-binding function of myoglobin, in situ in muscle fiber bundles, was abolished by treatment with nitrite or hydroxylamine, which convert oxymyoglobin in situ to high spin ferric myoglobin, or with phenylhydrazine, which converts oxymyo- globin to denatured products, or with 2-hydroxyethylhydrazine, which appears to remove myoglobin from the muscle. The oxygen uptake was again measured. At higher oxygen pressure, where oxygen availability does not limit the respiration of the fiber bundles, oxygen uptake is not affected by any of the four reagents, which is evidence that mitochondrial oxygen uptake is not impaired. At lower oxygen pressure, where oxygen uptake is one-half maximal, the steady state oxygen consumption is roughly halved by abolishing functional myoglobin. Under the steady state conditions studied, the storage function of myoglobin, being static, vanishes and the transport function stands revealed. We conclude from these experiments that myoglobin may transport a significant fraction of the oxygen consumed by muscle mitochondria.

Myoglobin unquestionably serves in some manner to aid the inflow of oxygen into cardiac and red skeletal muscle fibers, so that the demand for oxygen is met instantaneously (1, 2).

Millikan, in his classic study (3), showed that myoglobin in muscle undergoes rapid deoxygenation and oxygenation in response to fluctuations in oxygen supply and demand imposed by intermittent activity. He further showed that myoglobin maintains a steady level of partial oxygenation during sus- tained muscle contraction, implying that myoglobin in this circumstance functions in a very steep gradient of oxygen pressure from the capillary to the most remote mitochondrion (2).

Millikan foresaw that myoglobin might serve the body in three capacities: as an agent in oxygen transport, as an in- tracellular catalyst, or as an oxygen store. If, in fact, myoglobin is free to diffuse within the muscle cell, myoglobin-facilitated

* This work was supported in part by Research Grant BMS72-02491 A02 from the National Science Foundation, by a grant from the Muscular Dystrophy Associations of America, and by Grants HL-02001, HL-17813, and Pulmonary SCOR HL-15088 from the National Heart and Lung Institute.

t Research Career Program Awardee 5 K06 HL 007.33 of the United States Public Health Service, National Heart and Lung Institute.

oxygen diffusion could assure the transport of oxygen (2, 4). Facilitated oxygen diffusion, that is, an oxygen flux brought about by translational diffusion of oxymyoglobin molecules, has been demonstrated in physical model systems (2). A priori we have no assurance that myoglobin serves this function in muscle tissue. In addition, leghemoglobin, the counterpart of myoglobin in plants, serves in the transfer of oxygen from the cytosol to a terniinal oxidase (5). Finally myoglobin acts as a short period oxygen store to buffer fluctuations in the rate of flow of oxygen from the blood to the oxidizing enzymes of the mitochondria, thereby stabilizing the oxygen supply to the beating heart (1, 6-9).

Muscle oxygen demand, Millikan showed, rises instanta- neously with the onset of contraction (3), and oxygen supply may be limited as the contracting muscle constricts the blood vessels. In this extreme circumstance myoglobin acts as an oxygen buffer, supplying oxygen to the mitochondria during contraction and taking on oxygen from the capillary blood during relaxation. On the other hand, in many muscles, fluctuations in supply and demand are strongly damped, and the muscle operates in a family of steady states in which oxygen supply and demand are in instantaneous balance (2). In this circumstance oxygen flows continuously to the mitochondria

9038

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

9039

from outside the muscle fiber, and myoglobin assumes a purely transport function. It is apparent that storage and transport are not necessarily separate functions but are extremes of a continuum, in which storage predominates during changing states of the muscle and transport is dominant in steady states.

Direct evidence that muscle oxygen supply depends on myoglobin is lacking. The present study was designed to address this problem and to answer the question: does myo- globin augment steady state muscle oxygen uptake? We find that it does. Oxygen uptake of muscle fiber bundles was measured in a series of steady states, and the results were expressed graphically as relations between steady state oxygen uptake and ambient oxygen pressure. The oxygen-binding function of myoglobin, in situ in the bundles of muscle fibers, was abolished by chemical treatment, and the contribu- tion of myoglobin toward the oxygen uptake of the muscle fiber bundles was estimated from the difference in the steady state oxygen uptake with and without functional myoglobin.

EXPERIMENTALPROCEDURE

Optical Spectm-Reflectance difference spectra of slabs of pigeon breast muscle were determined using a Cary model 17 recording spectrophotometer equipped with an integrating sphere (Cary cell space total diffuse reflectance accessory).

Bundles of Muscle Fibers-Adult pigeons were decapitated and bled from the neck vessels. Bundles of muscle fibers were teased from the breast muscle by blunt dissection following the natural divisions of the tissue, with care not to stretch the bundles or tear them along their length. A typical fiber bundle is 25 mm long and about 0.5 mm in diameter, and it weighs about 15 mg.

Perwate-This was Krebs’ improved Ringer’s II solution (10) with 40 mM succinate as substrate. The perfusate was equilibrated with known gas mixtures containing 5 ‘/I CO, and varying proportions of 0,and N,.

Chamber for Measurement of Steady State Oxygen Consump- tion-The chamber depicted in Fig. 1 was constructed from a lOO-ml syringe. Vigorous stirring without undue tissue destruction is achieved by the magnetically driven rotating drum. The chamber volume is 40 ml, the flow rate is 1 ml per min, and the assembly is housed in a water bath maintained at 37 + 0.05O. Oxygen pressure in the inlet and outlet streams was measured using a Radiometer (Copenhagen) E 5046/O oxygen electrode and PHM-72 digital acid base analyzer equipped with a PHA 932 p0, module, and recorded continuously using a strip chart recorder

Measurement of Steady State Oxygen Consumption-Tissue, 250 to 800 mg, is placed in the chamber. At the outset, the p0, measured at the outlet electrode declines with tissue oxygen uptake until oxygen consumption is balanced by oxygen entering in the input stream, and a steady state is achieved (half-time to achieve a steady state is about 5 mink The oxygen pressure of the inlet stream is then changed abruptly and the oxygen pressure at the new steady state is noted when it reaches a constant value. As many as 15 step changes in p0, may be made using the same sample of tissue. The time course of a typical experiment is presented in Fig. 2. Oxygen consumption is calculated from the difference in oxygen pressure readings of the two electrodes when the steady state is reached, the flow, and the solubility of oxygen in water at 37”. For this difference to be meaning- ful, the oxygen pressure of the output line must always be greater than zero.

Measurement of Mitochondrial Oxygen Consumption-Mitochon- dria were prepared from pigeon breast muscle by the procedure of Chance and Hagihara (ll), and the oxygen uptake of suspensions (0.2 mg of mitochondrial protein per ml) was measured at 37”.

RESULTS

Reactions of Muscle Myoglobin-These were revealed by reflectance difference spectra of reagent-treated minus un- treated slabs of pigeon breast muscle (Fig, 3). These difference spectra show that nitrite and hydroxylamine convert muscle myoglobin to high spin ferric myoglobin. Although purified oxymyoglobin reacts with 2-hydroxyethylhydrazine to give

1

FIG. 1. Chamber for measuring steady state oxygen uptake of tissue fragments.

FIG. 2. Time course of a typical experiment. The oxygen pressure of the input stream, initially 678 mm Hg, was changed stepwise to 480, 374, and 286 mm Hg. Arrows indicate steady states.

predominantly the hydroxyethylhydrazine adduct of ferric myoglobin, 1 muscle fiber bundles incubated in the presence of 3 mM hydroxyethylhydrazine (37”) lose the color of myo- globin and become almost white. Reflectance difference spectra show only myoglobin loss from the muscle slab. Muscle fiber bundles exposed to phenylhydrazine assume the muddy green color of denatured myoglobin.

Reversibility of Effect of Nitrite on Muscle Myoglobin- Bundles of pigeon breast muscle fibers were examined using a microscope fitted with a microspectroscope ocular (Zeiss) or with a Hartridge reversion spectroscope (Beck, London) in place of the ocular. Ferrous (oxy- and deoxy-) myoglobin disappears in a population of fiber bundles incubated with 50 mM sodium nitrite (the broad bands of the product, ferric myoglobin, are not easily seen). When the fiber bundles are rinsed free of nitrite, ferrous myoglobin reappears with a time course of about 20 min at room temperature.

Tissue Preparation and Muscle Fiber Bundle Thickness- Pigeon breast muscle is rich in myoglobin, 0.20 to 0.25 mmol per kg wet weight of tissue. The majority of the fibers contain

‘J. Peisach finds that oxyhemoglobin is reduced by 2-hydroxy- ethylhydrazine to give ferry1 hemoglobin as the initial product (personal communication).

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

9040

myoglobin and are rich in mitochondria (12-15). They are homogeneous in size, are about 30 pm in diameter (14, 16), and could be classified as “red” fibers, with a mitochondrial distribution pattern (14) more typical of “intermediate” fibers (see Ref. 17 for a discussion of muscle fiber types). These red fibers form the core of each fiber bundle, with “white” fibers (about 22% of the total) arrayed at the periphery (14, 16).

The muscle is easily dissected into bundles of fibers. These are irregularly polygonal in cross section. Their shortest dimension in cross section, either when dissected free or in blocks of muscle, was measured with an ocular micrometer and found to range from 300 to 900 Mm with a mean of 500 pm.

Degree of Oxygenation of Myoglobin in Muscle Fiber Bundles-Muscle fiber bundles surrounded by a flowing stream of 5% CO, in wet air were examined at 37” with a microscope fitted with a Zeiss microspectroscope ocular. The absorption bands of oxymyoglobin near 543 and 581 nm were faint, indicating that the myoglobin was substantially deoxygenated. These bands became very intense when tissue oxygen uptake was abolished by cyanide.

Limitation Imposed by Fiber Bundle Size-In an early study with Dr. P. P. J. M. Bindels,* we found that the rate of oxygen uptake depends on the rate of change of ambient p0, and is constant only at constant PO,. For this reason we have made all measurements of oxygen uptake in steady states of con- stant p0 I’

Steady State Oxygen Uptake of Pigeon Breast Muscle- The results of four typical experiments using separated muscle fiber bundles are presented in Fig. 4. Oxygen uptake in- creases monotonically with p0, approaching an apparent plateau near 150 mm Hg, where oxygen uptake becomes nearly independent of oxygen pressure. Half-maximal rate occurs near 30 mm Hg, but substantial uptake is maintained to 5 mm Hg or less.

Caldwell and Wittenberg (18) report that relations similar to those of Fig. 4 obtain for slices of a variety of mammalian tissues.

The oxygen uptake of blocks of tissue, six layers of fiber bundles thick, followed a relation with oxygen pressure (Fig. 4) similar to that of separated fiber bundles, but the oxygen uptake was less.

The oxygen consumption of muscle fiber bundle prepara- tions ranged from 30 to 60 ~1 of 0, min-‘g-l wet weight. The relation between steady state oxygen uptake and oxygen pressure is independent of the order in which the measure- ments are made (Fig. 4), the rate of stirring, and the pH of the Krebs medium from pH 7.2 to pH 7.8. In the absence of exogenous substrate, oxygen uptake was low and variable. Addition of 40 mM succinate brought oxygen uptake, at high p0, where oxygen is not limiting, to a maximal value. The addition df glucose, 14 mM, separately or together with succinate, did not further increase the rate. Two uncouplers of the mitochondrial electron transport chain, 2,4-dinitro- phenol (800 PM) and carbonyl cyanide m-chlorophenyl hydra- zone (50 FM), were without effect.

Effect of Carbon Monoxide-It was not found possible to block the oxygen-binding function of myoglobin by reaction with carbon monoxide without simultaneously inhibiting cytochrome oxidase.

Lack of Effect of Symmetric Dicarbethoxyhydrazine and

*P. P. J. M. Bindels, unpublished experiments, reported in Ref. 2.

i 1 50 XXI 550 600 650 -3

WAVElENGTH,nm

L 30 a.

FIG. 3. Difference spectra of pigeon breast muscle compared to those of purified myoglobin. A, nitrite-treated muscle minus anaerobic muscle; E, hydroxylamine-treated muscle minus anaerobic muscle; C, high spin ferric myoglobin minus deoxymyoglobin; D, nitrite-treated muscle subsequently exposed to CO minus anaerobic muscle exposed to CO; E, hydroxylamine-treated muscle subsequently exposed to CO minus anaerobic muscle exposed to CO; F, high spin ferric myoglobin minus carbon monoxide myoglobin; G, nitrite-treated muscle sub- sequently exposed to 0.01 M sodium cyanide minus nitrite-treated muscle; H, ferric myoglobin cyanide minus high spin ferric myoglobin.

OXYGEN PRESSURE, mm HQ

FIG. 4. Steady state oxygen uptake of four preparations of pigeon breast muscle fiber bundles and of one preparation of thick slabs of pigeon breast muscle. a, 0, Cl, A, respectively 400, 500,600, and 600 mg of fiber bundles per run. X, slabs of tissue, each about six layers of fiber bundles thick. Two slabs, each 3.0 mm thick, 6 mm wide by 14 mm long, each weighing about 230 mg were used. The sequence of measure- ments is: 0 and A, from low to high ~0~; a, Cl, and x, from high to low po,

of I-Ethyl-P-carboxymethylhydrazine-Neither of these re- agents, which do not react with purified myoglobin, at re- spectively 0.5 mM and 15 mM concentration, changed the steady state oxygen consumption of muscle fragments.

Effect of Reagents Which Abolish Oxygenation of Myo-

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

9041

globin-The oxygen uptake of pigeon breast muscle fiber bundles at low p0, is diminished in the presence of nitrite (Fig. 5), hydroxylamine (Fig. 6), or 2-hydroxyethylhydrazine (Fig. 7). Likewise the oxygen uptake of muscle fiber bundles pretreated with hydroxyethylhydrazine (Fig. 8) or with phenyl- hydrazine (Fig. 9) is diminished at any p0, where oxygen uptake is dependent on oxygen pressure. These effects vanish at high p0, and the oxygen uptake becomes the same in the presence and absence of reagent treatment.

The effect of nitrite is reversible. Fig. 10 presents the results of an experiment in which nitrite-treated muscle fiber bundles were washed free of nitrite and subsequently incubated in Krebs-Ringer solution until the red color and absorption bands of oxymyoglobin (microspectroscope ocular) were restored. The oxygen consumption was restored to the same level as that of a parallel preparation of untreated muscle fiber bundles.

The effect of hydroxylamine on steady state oxygen uptake was found negligible at 0.1 mM hydroxylamine, it was sub- stantial at 0.25 mM, and it reached a plateau of maximal magnitude near 0.5 to 1.0 mM.

Effects of Reagents on Oxygen Uptake of Isolated Mito- chondria-Nitrite (25 mM) inhibited the oxygen uptake of a mitochondrial preparation about 20%, at all p0, from 5 to 110 mm Hg. This inhibition vanishes in the presence of 135 FM myoglobin, which becomes ferric and may serve to trap inhibitory products formed from nitrite. 9 2-Hydroxy- ethylhydrazine (5 mM) is without effect on mitochondrial oxygen uptake either in the presence or absence of ADP. Hydroxylamine (0.3 mM) inhibits mitochondrial oxygen up- take about 30 to 50%, independent of p0, from 10 to 200 mm Hg. The effect of phenylhydrazine could not be tested be- cause of nonenzymatic air oxidation of phenylhydrazine in neutral solution.

Effect of Cyanide-Cyanide is without effect on the reversi- ble oxygenation of myoglobin. The concentration of cyanide used (125 PM) is about one-third that required to give 95% inhibition of cytochrome oxidase in a Keilin-Hartree prepara- tion (19). Cyanide inhibits oxygen uptake of muscle fiber bundles at all oxygen pressures (Fig. 11).

Effect of Antimycin A-This inhibitor of mitochondrial electron transport does not affect the reversible oxygenation of myoglobin. Antimycin A inhibits the oxygen uptake of muscle fiber bundles at all oxygen pressures (Fig. 11). The concentration of antimycin A used in this experiment was 30 /rg per ml but 0.6 pg per ml is sufficient.

DISCUSSION

Our intent in undertaking these experiments was to study the oxygen uptake of muscle under steady state conditions of oxygen supply and consumption where the storage func- tion of myoglobin, by being static, vanishes, and the kinetic function of myoglobin in oxygen transport stands revealed. For this sufficient reason all rates of oxygen uptake were measured in steady states of constant ambient oxygen pres- sure. In addition, we report that steady state measurement is a requirement set by the long diffusion path for oxygen in the relatively large tissue fragments used. The apparent rate of oxygen uptake of the muscle fiber bundles depends strongly on the rate of change of oxygen pressure, and is constant only at unchanging ambient oxygen pressure. *

Pigeon breast muscle was chosen because it is rich in myo- globin and because it can readily be dissected into bundles

3 H. Adler. personal communication.

of fibers with minimum damage to the integrity of the tissue architecture. At oxygen pressures greater than 150 mm Hg, the oxygen uptake of pigeon breast muscle fiber bundles is inde- pendent of oxygen pressure (Figs. 4, 6, 7B, and 9). In this range, the oxygen uptake of these muscle fiber bundles is comparable to that of resting cat heart papillary muscles of about the same size (20), and is about one-third to one-half that of skeletal and cardiac muscles doing steady state work in situ with oxygen supplied from the capillaries (2, 21). Abolition of myoglobin function through the action of the reagents, which remove the oxygen-binding function of myo- globin, had no effect on muscle oxygen uptake in this range of oxygen pressure.

In the range of lower oxygen pressure, steady state oxygen uptake is dependent on oxygen pressure, rising steeply and monotonically from zero at zero oxygen pressure toward an apparent plateau at 150 mm Hg. The effect of myoglobin which we can demonstrate occurs in this range of oxygen pressure. This effect, the major finding of this study, is that abolition of the oxygen-binding function of myoglobin de- creases the steady state oxygen uptake of muscle at any oxygen pressure within this range (Figs. 5 to 7, 9, and 10). Near the oxygen pressure for half-maximal oxygen uptake, treatment of the tissue with nitrite, hydroxylamine, or hy- droxyethylhydrazine (Figs. 5, 7A, 10) roughly halves the rate of oxygen uptake. To restore the original rate of oxygen uptake it is necessary to double the ambient oxygen pressure.

The simplest interpretation of these data is that myo- globin serves to transport oxygen to the mitochondria. In the range of lower oxygen pressure, this transport contributes a major fraction of the total oxygen uptake, the balance being the simple diffusive flow of dissolved oxygen.

We have used four reagents to abolish the oxygen-binding function of myoglobin. Although the mechanism of the initial attack of each of these reagents on oxymyoglobin may be the same, the products differ. Two reagents, nitrite and hydroxylamine, convert oxymyoglobin, both in situ in muscle (Fig. 3) and when isolated to high spin ferric myoglobin. One, hydroxyethylhydrazine, converts isolated oxymyoglobin to a mixture of products and attacks myoglobin in situ with apparent loss of the heme group so that at the end of an experiment the muscle fragments appear nearly white. The last reagent, phenylhydrazine, irreversibly denatures myo- globin, either purified or in situ, to a green product.

All of the myoglobin-reactive reagents have the potential to affect other heme proteins as well. The most convincing evidence that they exert their main effect on myoglobin function is the aforementioned fact that the effect of all the myoglobin-reactive reagents vanishes at high oxygen pressure, showing that the rate of mitochondrial oxygen uptake, per se, has not been affected. Further evidence for the integrity of mitochondrial function in reagent-treated muscle fiber bundles is the reversibility of the effect of nitrite (Fig. 10). When the tissue myoglobin returns to the ferrous, oxy- and deoxy-, state through the presumed action of tissue enzymes, the rate of oxygen uptake is restored to its original large value. Finally, we note that the pattern of inhibition obtained with the known inhibitors of mitochondrial oxygen uptake, cyanide and antimycin A (Fig. ll), is entirely different from that found with the myoglobin-reactive reagents. Cyanide and antimycin A inhibit the oxygen uptake of muscle fiber bundles at all oxygen pressures up to the highest examined.

Less direct evidence in support of the conclusion that

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

9042

ir’ OXYGEN PRESSURE, mm HQ

FIG. 5. Effect of nitrite on the steady state oxygen uptake of pigeon breast muscle fiber bundles. 0, untreated tissue. 0, the same in the presence of 25 rnM sodium nitrite.

OXIGEN PRESSURE. mm Hp

FIG. 7. Effect of 2-hydroxyethylhydrazine on the steady state oxygen uptake of pigeon breast muscle fiber bundles; experiments in which 2-hydroxyethylhydrazine was injected into the chamber. Open symbols, untreated fiber bundles. Solid symbols, the same in the presence of 3 mM 2-hydroxyethylhydrazine. Different symbols repre- sent separate experiments. A, an experiment chosen to demon- strate the usual magnitude of the effect. B, an experiment chosen to demonstrate the plateau of oxygen uptake at high oxygen pressure.

40

30

20 I

0

.

. 0

-::~i

.

20 40 60 SO 120 140

OXYGEN PRESSURE, mm Hg

FIG. 9. Effect of phenylhydrazine on steady state oxygen uptake of pigeon breast muscle fiber bundles. Open symbols, untreated fiber bundles. Solid symbols, fiber bundles which were preincubated for 30 min at 4” in a solution of phenylhydrazine, 5 mM, in Krebs solu- tion, rinsed, and subsequently transferred to phenylhydrazine-free Krebs solution in the measuring chamber. Different symbols represent separate experiments.

v 1 1 I 1 1 I 1 I I 3 20 40 60 80 100 I20 140 160 lS(

OXYGEN PRESSURE, mm Hg

FIG. 6. Effect of hydroxylamine on the steady state oxygen up- take of pigeon breast muscle fiber bundles. 0 and Cl, untreated fiber bundles; 0, the same in the presence of 0.25 rnM hydroxylamine. Different symbols represent separate experiments.

OXYGEN PRESSURE (mmHg1

FIG. 8. Effect of 2-hydroxyethylhydrazine. Two experiments in which fiber bundles were pretreated with 2-hydroxyethylhydrazine until the color of myoglobin had disappeared. Open symbols, untreated fiber bundles. Solid symbols, fiber bundles which had been incubated at 37” for 90 min in Krebs solution containing 100 rnM 2-hydroxy- ethylhydrazine, rinsed for a total of 20 min at 37” in four changes of reagent-free Krebs solution, and subsequently transferred to reagent- free Krebs solution in the measuring chamber. Different symbols represent separate experiments.

OXYGEN PRESSURE.mm Hg

FIG. 10. Reversibility of the effect of nitrite on the steady state oxygen uptake of pigeon breast muscle fiber bundles. 0, fiber bundles in the presence of .W mM sodium nitrite. The sequence of measure- ments is from high to low PO,. 0, the same fiber bundles after replacing the perfusing medium with nitrite-free Krebs solution and incubating for 1 hour at 37”. The sequence of measurements is from low to high p0 ,.

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

OXYGEN PRESSURE mm Hg

FIG. 11. Effect of cyanide and ot’ antimycin A on the steady state oxygen uptake of’ pigeon breast muscle f’iber bundles. Open symbols, untreated fiber bundles. 0, the same in the presence of 125 pM potassium cyanide. The sequence of measurements in the experiment with cyanide is indicated by the numbers of the data points. n , fiber bundles in the presence of antimycin A, 3Org per ml. Different symbols represent separate experiments.

the myoglobin-reactive reagents do not largely affect the mitochondria is of several kinds. Two hydrazine derivatives, symmetric dicarbethoxyhydrazine and 1 -ethyl - 2 - carboxy - methylhydrazine, which do not react with oxymyoglobin, are without effect on the oxygen uptake of muscle fiber bun- dles. The effect of hydroxylamine increases with concentra- tion up to a saturating level at about 1 mM. This is con- sonant with a progressive effect on myoglobin function, and not consistent with an effect on mitochondrial function, which would be expected to increase with reagent concentration until oxygen consumption was abolished. Finally, 2-hydroxy- ethylhydrazine was without effect on the oxygen uptake of isolated mitochondria, and the known inhibitory effect of nitrite on isolated mitochondria (22, 23) disappears in the presence of myoglobin.

The question arises whether the results found with muscle fiber bundles fit what might be predicted for simultaneous diffusion and chemical reaction in a cylinder consuming oxygen at a uniform rate. None of the several mathematical treatments (e.g. Refs. 24 to 28) adequately describe the data of the present study. While there is approximate agreement for the results obtained with muscle fiber bundles of small diameter, the predictions fail in the case of the thick slabs of pigeon breast muscle which consume oxygen at a much greater than predicted rate. For this reason we do not believe it appropriate to use our findings for relatively thick muscle fiber bundles as a basis for quantitative predictions such as those made by Wyman (29, 30) and Murray (31) for the events in actual muscle where the diffusion path is 20-fold less.

We conclude from our experiments that myoglobin acts to enhance the inflow of oxygen into red muscle fibers. Our finding that myoglobin in the muscle fiber bundles is only partially oxygenated implies a steep gradient of myoglobin oxygenation in the tissue. Indeed, such a gradient is a pre-

9043

condition for any carrier-mediated oxygen transport (2, 31, 32), and gradients of oxygenation have been demonstrated in work- ing cardiac and skeletal muscle (3, 33-36). Thus myoglobin, in enhancing the oxygen flow, operates within steep gradi- ents of oxygen pressure and partial oxygenation.

Acknowledgments-Dr. P. P. J. M. Bindels participated in the early stages of this work and discovered that steady state conditions were required for measurement of oxygen uptake in this system. It is a pleasure to acknowledge the skilled assistance of Miss Lourdes Fernandez. We thank Dr. A. N. Stokes for discussion of mathematical aspects of this work.

Millikan, G. A. (1939) Physiol. Reu. 19, 503-523 Wittenberg, J. B. (1970) Physiof. Reo. 50.559-636 Millikan, G. A. (1937) Proc. R. Sot. Lond. Ser. B 123.218-241 Riveros-Moreno, V. & Wittenberg, J. B. (1972) J. Biol. Chem.

247,895-901 Appleby, C. A., Bereersen, F. J., Macnicol. P. K.. Turner, G. L..

-Wit&berg, B. A: & Wittenberg, J. B. (1975) in Proceedings International Symposium on Nitrogen Fixation (Newton. W. E. & Nyman, C.-J., eds), Washington State University Press, Pullman, Wash.

6.

7. 8.

Barcroft, J. (1934) Features in the Architecture of Physiological Function, pp. l-368, Cambridge University Press, Cambridge.

Hill, R. (1936) Proc. R. Sot. Lond. Ser. E 120,472-483 Chance, B., Tamura, M., Oshino, M. & Salkowitz, I. (1974)

Fed. Proc. 33,425 9.

10. 11.

Kessler, M. (1974) Microuasc. Res. 8, 283-290 Krebs, H. A. (1950) Biochim. Biophys. Acta 4,249-269 Chance, B. & Hagihara, B. (1962) J. Biol. Chem. 237, X40-

3545 12. George, J. C. & Berger, A. J. (1966) Auian Myology, Academic

Press, London 13. George, J. C. & Talesara, C. L. (1961) J. Cell. Comp. Physiol.

58,253-260 14. 15. 16.

Ashhurst, D. E. (1969) Tissue Cell 1.485-496 James, N. T. (1972) Comp. Biochem. Physiol. 41B, 457-460 George, J. C. & Naik, R. M. (1959) Biol. Bull. (Woods Hole)

116,239-247 17. Gauthier, G. F. (1974) in Advances in Muscle Biology (Pearson,

A. M., ed), Vol. 2, Michigan State University. East Lansing, in press

18. Caldwell, P. R. B. & Wittenberg, B. A. (1974) Am. J. Med. 57, 447-452

19. 20.

Slater, E. C. (1967) Methods Enzymol. 10,48-57 Cranefield, P. F. & Greenspan, K. (1960) J. Gen. Physiol. 44,

235-249 21. Blinks, J. R. & Koch-Weser, J. (1963) Pharmacol. Reu. 15,

531-599 22. Walters, C. L. & Taylor, A. McM. (1965) Biochim. Biophys. Acta

96, 522-524 23. Cheah, K. S. (1974) int. J. Biochem. 5.349-352 24. Warburg, 0. (1914) Ergeb. Physiol. 14,255-333 25. Krogh, A. (1919) J. Physiol. 52,409-415 26. Hill, A. V. (1929) Proc. R. Sot. B 104.39-96 27. Hill. A. V. (1965) in Trails and Trials in Physiology, Chap. 6,

pp. 208-241, Williams & Wilkins Company. Baltimore 28.

29. 30.

31. 32.

Landis, E. M. & Pappenheimer. J. i. (i963) in Handbook of Physiology. Circulation, Sect. 2, Vol. 2, pp. 961-1034, Ameri- can Physiology Society, Washington, D.C.

Wyman, J. (1966) J. Biol. Chem. 241, 115-121 Wyman, J. (1972) in Structure and Function of Oxidation-

keduction Enzymes (Akeson, A. & Ehrenberg, A., eds), pp. 113-138, Pergamon Press, Oxford

Murray, J. D. (1974) J. Theor. Biol. 47, 115-126 Murray, J. D. & Wyman, J. (1971) J. Biol. Chem. 246,\5903-

5906

REFERENCES

33. Chance, B., Mayevskg, A., Goodwin, C. & Mela, L. (1974) Micro- uasc. Res. 8.276-282

34. JFbsis, F. F. & Stainsby, W. N. (1968) Respir. Physiol. 4,292-300 35. JSbsis, F. F. (1972) Fed. I+oc. 31, 1404-1413 36. Coburn, R. F. & Mayers, L. B. (1971) Am. J. Physiol. 220, 66-74

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

B A Wittenberg, J B Wittenberg and P R CaldwellRole of myoglobin in the oxygen supply to red skeletal muscle.

1975, 250:9038-9043.J. Biol. Chem. 

  http://www.jbc.org/content/250/23/9038Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/250/23/9038.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from