THETHERMODYNAMICS OF ABSORPTION OF XENON BY MYOGLOBIN tion isotherms of acetone on 15.8% TOTP-coated AWFB obtained at various temperatures, the total heat corresponding to the sorption process was calculated (Figure 8). The heat of adsorption of acetone on TOTP modified AWFB (10.3%) was measured and the value of approximately -9.0 kcal/mol was obtained (Figure 8). The total heat observed was reproduced from the heat of solution and the heat of adsorption individually measured using eq 17. The results are shown in Figure 9. The agreement of the observed heats and the calculated heats was extremely good. This also helps sup-
2341
port the additive nature of the sorption mechanism of the solute on a liquid-coated adsorbent. Figure 10 shows the same type of plot for the adsorption of acetone on 2.15% TOTP-coated AWFB. The observed and the calculated heats agreed within the experimental error range for all points.
Acknowledgment. This study was made possible by National Science Foundation Grants No. G P 9456, G P 5400, and GY 4093. Presented at the 157th National Meeting of the American Chemical Society in Minneapolis, Minn., April 1969.
The Thermodynamics of Absorption of Xenon by Myoglobin’ by Gordon J. Ewing and Sigfredo Maestasl Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88001 (Received December 6 , 1969)
The interaction of xenon with solutions of myoglobin, metmyoglobin, and cyanometmyoglobin has been studied at 20, 25, and 30” and at various xenon pressures up to 4.85 atm. Evidence is presented for the formation of HpXe for all three myoglobins and HpXez for myoglobin and metmyoglobin (Hp = hemoprotein). The equilibrium constants for the first step were between 100 and 200 m-l and between 1 and 10 m-1 for the second step, depending upon the temperature and the particular myoglobin utilized. Introduction The physiological activity of xenon and other “inert” gases has led to investigations of the interaction of these gases with biological materials.2 Specifically, the interaction of xenon with hemoglobin and myoglobin has been the subject of several recent investigations. 3-9 The general conclusion to be drawn from the investigations pertaining to hemoglobin-xenon and myoglobin-xenon interactions is that there exists a seeming uncertainty regarding the stoichiometry (or lack of the same) for these “reactions.” Mortimer and Bauer’O first proposed that a definite stoichiometry involving an inert gas and a heme compound may indeed exist but is difficult to establish for very weak interactions. The interaction is indistinguishable from a “solubility” phenomenon when a small percentage of the protein molecules has combined with the inert gas. Thus far the X-ray studies reported indicate that a specific site for xenon (and perhaps other inert gas molecules) exists in myoglobin and hemoHowever, the data for absorption studies are generally reported as Henry’s law solubilities. This report concerns the attempt to study the absorption of xenon by myoglobin over a substantial pressure range (0-4.8 atm). The studies were conducted a t
various temperatures (20, 25, and 30”) so that the heat of the interaction could be determined. It was considered of particular importance to determine if a difference existed between the interaction of xenon with ferromyoglobin and with ferrimyoglobin (hereafter referred to as metmyoglobin). The absorption of xenon by myoglobin and metmyoglobin was studied as well as the absorption of the gas by cyanometmyoglobin, in which the coordination sphere of the Fe(II1) atom contains a CN- ion. The latter experiments were conducted in order to determine whether the (1) This work was supported by grants from NASA (NGR 32-003027) and NSF (GB-7829). (2) A. P. Rinfret and G. F. Doebbler in G. A. Cook, “Argon, Helium, and the Rare Gases,” Vol. 11, Interscience Publishers, New York, N. Yo, 1961, PP 727-764. (3) H. L. Conn, Jr., J. AppZ. Physwl., 16, 1065 (1961). (4) 6 . Y. Yeh and R. E. Peterson, ibid., 20, 1041 (1965). (5) B. P. Schoenborn, H. C. Watson, and J. C. Kendrew, Nature, 207, 28 (1965).
(6) B. P. Schoenborn, ibid., 208, 760 (1965). (7) S. P. Schoenborn and C. L. Nobbs, Mol. Pharmacol., 2, 491 (1966). (8) J. F. Catohpool, Fed. Proc., 27, 884 (1968). (9) M. Keyes and R. Sumry, ibid., 27, 898 (1968). (10) R. G. Mortimer and N. Bauer, J . Phys. Chem., 64, 387 (1960). The Journal of Physical Chemistry, Vol. 74, N o . 11, 19YO
2342
GORDON J. EWINGAND SIGFREDO MAESTAS
formation of a nonlabile compound hindered the absorption of xenon by the hemoprotein.
Experimental Section
0.8
The absorption of xenon by myoglobin, metmyoglobin, and cyanometmyoglobin was studied by measuring amounts of the gas absorbed by 10% aqueous solutions of the hemoproteins by a manometric method. The method involved placing a known quantity of the gas above a solution and then determining the quantity dissolved by measuring the pressure change. This was done in a stainless steel manifold. The pressures of the gas were measured by either of two gauges; one gauge was a 0-3 atm pressure gauge, the other covered the range from 0 to 5 atm. Both were built by the Heise Bourdon Tube Co., Inc. of Newton, Conn. The gauges were sensitive to the nearest 0.5 mm and exhibited maximum hysteresis of 1.5 mm and 3.5 mm, respectively. Both gauges were temperature compensated in the temperature range -31.5 to 51.5". Since bourdon gauges respond to the difference in pressure between the system and its surroundings, absolute pressures were obtained by adding the barometric pressure to the bourdon gauge reading. This pressure was then corrected for the partial pressure of water. The protein solutions were kept to within 0.1" of the desired temperature while the gauge (not in the constant temperature bath) was regulated to within 1.0" of the desired temperature. The solutions were stirred during the absorption runs by a magnetic bar driven by an immersed stirrer. The length of an absorption run was approximately 6-8 hr. The amount of xenon absorbed by the solution was determined by measuring the initial pressure and the final pressure to obtain the pressure change, AP, and applying the ideal gas law
APV RT
An = -
To determine the amount of xenon absorbed by the hemoprotein, the amount of xenon calculated to dissolve in water a t the temperature and equilibrium pressure of the experiment was subtracted from An. This quantity divided by the number of moles of myoglobin in the solution (usually mol) yielded the amount of xenon dissolved per mole of myoglobin. The amounts of xenon attributed to dissolution in water for each run were calculated from the Henry's law constants, 0.85 X lo7 mm/mole fraction (20"), 0.96 x lo7 mm/mole fraction (25"), and 1.13 X lo7 mm/mole fraction (30"), determined in this laboratory. These values are in fair agreement with the data of Wood and Caputi.'l The solutions were prepared to contain approximately 10% horse heart myoglobin obtained from Calbiochem. Some of the metmyoglobin solutions included phosphate buffer (pH S.l), 0.63 mmo1/25 ml of solution. The The Journal of Physical Chemistry, Vol. 74, No. 11, 10'70
C .a
-0
0" 0.6
e
+
e,
E -
0.4
\
e,
-
X
s 0.2 1
2 PXCI
3
4
atm
Figure 1. Metmyoglobin adsorption of xenon: 25'; 0, 30'.
0, 20'; A,
unbuffered solutions had a pH of 6.96. No appreciable difference in the absorption of xenon by the buffered and unbuffered solutions was observed. The myoglobin solutions were prepared by reduction of metmyoglobin with sodium dithionite (approximately 4 mol of dithionite/mol of myoglobin). The cyanometmyoglobin solutions were prepared by adding to 25 ml of metmyoglobin solution, 0.50 ml of KCN solution (9.6 g of KCN/5O ml), 0.50 ml of K3Fe(CN)s solution (3.0 g/50 ml), and 0.5 ml of KH2P04Na2HP04 buffer solution (8.5 g of Ei"P04/50 ml and 8.9 g of Na2HP04/50 ml). All of the solutions were analyzed by the o-phenanthroline method for iron or by analyzing cyanometmyoglobin spectrophotometrica1ly.l2
Results and Discussion The xenon absorption data for all three myoglobins show approximately the same characteristics. I n the pressure ranges studied, large deviations from Henry's law were observed a t the different temperatures. When corrections mere made for the solubility of xenon in water, the net interaction with myoglobin produced a curve typical of stoichiometric interactions. Generally, metmyoglobin and cyanometmyoglobin showed slightly larger absorption of xenon than did myoglobin for similar pressures. These differences were small, however. Figure 1 shows the absorption of xenon by metmyoglobin at 20, 25, and 30". Figures 2 and 3 show the absorption of xenon by myoglobin and cyanometmyoglobin, respectively, at similar temperatures. (11) D. Wood and R. Caputi, U. S. Naval Radiological Defense Laboratory Report TR-988,San Francisco, Calif., Feb 1966. (12) D. Drabkin, Amer. J. Med. Sci., 209, 268 (1946).
2343
THETHERMODYNAMICS OF ABSORPTION OF XENON BY MYOGLOBIN
t
A plot of mHpxe/mHp vs. Pxe should yield a straight line with a slope of K , and an intercept of zero. K , values can be readily converted to K values, where
K = - mHpxe
mHpmxe
by multiplying K p by the Henry's law constant for that particular temperature. Plots of the type described gave the expected straight line with an intercept very near zero. The least-squares method was utilized in determining the slopes. Values of K for the xenoncyanometmyoglobin interaction are given in Table I. 1
2
3
4
P x r ,atm Figure 2. Myoglobin adsorption of xenon:
0, 20"; A, 25';
Table I : Equilibrium Constants for Cyanometmyoglobin Absorption of Xenon Temp, OC
KW, m -1
20 25 30
145 115
186
The data for the xenon interaction with metmyoglobin and with myoglobin gave some indication of interactions beyond the one to one interaction with cyanometmyoglobin. We were able to obtain a better explanation of our observations by assuming a two-step equilibrium Hp
K
+ Xe
HpXe
and Y
I
I
I
I
1
2
3
4
Pa,ATM.
Figure 3. Cyanometmyoglobin adsorption of xenon: A, 25'; D, 30".
HpXe
+ Xe
K'
HpXez
where the equilibrium expressions are
K = - mHpXe mHpmXe
0, 20";
and The data for the cyanometmyoglobin-xenon interaction were consistent with a very simple interpretation, so they will be treated first. The data in Figure 3 appear consistent with a single-step equilibrium process Hp
+ Xe =+=
HpXe
with the corresponding equilibrium expression mHpxe Kp = mHJ'xe where K , is an equilibrium constant, m's are molalities of the species indicated, Pxe is the partial pressure of xenon, and Hp is the hemoprotein. This may be written
K'
=
WHpXee
m~ pxemxe The m's correspond to the molal concentrations of the subscripted species in solution. To evaluate the equilibrium constants, two mass balance equations were utilized. fHp
fxe
+ mHpxe, mxe f mHpxe + 2 m
=
mHp 4- mnpxe
~
~
The f's correspond to the total (formal) concentration of all forms of the substance indicated. These equations, along with the two equilibrium relationships, can The Journal of Phgsical Chemistry, Vol. 74, No. 11, 1970
~
~
2344
GORDON J. EWING AND SIGFREDO MAESTAS
be utilized to eliminate the concentrations of the various hemoprotein forms ( m ~ mHpxe, ~ , and mHpXe2), Table IV : Thermodynamic Functions for Xenon Absorption by Myoglobins The appropriate algebraic manipulationsl2 yield the AUOas", following relationship
AHo,
Heme protein
kortl/mol
kcrtl/mol
Metmyoglobin Myoglobin Cyanometmyoglobin
-2.9" -2.7" -2.9
-7.2a -5.1" -9.0
a
ASoza0,
eu
- 14a -8.0"
- 20
First equilibrium step.
analogous to y =
KX + KK'
the equation for a straight line. Plots of x vs. y were obtained for each isotherm in Figures 2 and 3, and K was determined from the slope, and K' from the slope and the intercept. These values are listed in Tables I1 and 111. The values of K' are only approximate and merely indicate the possibility for a second site for interaction. Schoenborn and Nobbs' also present some evidence for interactions other than the primary one. Table I1 : Equilibrium Constants for Metmyoglobin Absorption of Xenon
20 25 30
200 146 130
4 . 2 -7.2 -2.3
Table I11: Equilibrium Constants for Myoglobin Absorption of Xenon K'eq,
m -1
20 25 30
109 94 85
2.3 2.6
0.5
The interesting characteristic of the absorption data comparatively is that the equilibrium constants indicate a slightly greater affinity of the Fe(II1)-containing myoglobins for xenon whereas the oxygen interaction is with Fe(I1)-containing M b only. The free energies of reaction, enthalpies, and entropies for each of the absorption processes were evaluated from the equilibrium constants and their rate of change with temperature (Table IV). The most apparent property of the interactions of xenon with the myoglobins is that these are in the (I weak-bond" category. The evidence indicates that
The Journal of Physical Chemistry, Vol. 74, No. 11, 1970
the binding of xenon to myoglobin is not strong iron-toligand bonding as witnessed by (1) the absence of spectral differences in the spectra of xenonated and unxenonated myoglobins, (2) the apparent slow absorption process, and (3) the low heats of reaction. This is an indication that the role of the iron atom in heme proteins is not as prominent in binding "inert" gases as it is in its capacity in binding strong ligands like 02, CO, CN- ion, etc. This quantitative difference between strong ligand xenon interactions with myoglobin is consistent with the X-ray studies of Schoenborn, et ~ l . which , ~ places the xenon on the opposite side of the porphyrin ring from the 0 2 absorption site. In spite of the low enthalpies of absorption, these appear to be the driving forces of the reactions. The large entropies of xenonation of myoglobins are indicative of large conformational changes in the hemoprotein molecules during the absorption process. These have been observed previously in the absorption of other inert gases.1° Chemisorption may indeed be the phenomenon involved here if one defines the process to include the property of saturation of interaction sites. Clathrate formation is also suggested. Although the affinities of the myoglobins for xenon are small, these are large in comparison to their affinities for H2, N2, and Ar.'O I n conclusion, it is stressed that this simple experimental method allows substantial information to be derived for these processes. Correct values for X e uptake by myoglobin depend on several factors, namely, (1) the sensitivity of the measuring instrument (gauge), (2) the amount of gas absorbed by the protein, (3) the correctness of the data for the solubility of the gas in the solvent, and (4)temperature control. The latter seems to be the major source of experimental error in this study. A temperature difference of 1" between the reaction vessel and the gauge would result in an error in the calculated quantity of xenon interacting with myoglobin of about 5% when near saturation. This corresponds roughly with the scatter of our measurements. (13) J. C. Speabman, J. Chem. SOC., 855 (1940).
