- Email: [email protected]
Binding of ions by myosin. II. The effect of neutral salts on the acid-base equilibria of myosin
Binding of Ions by Myosin. II. The Effect of Neutral Salts on the Acid-Base Equilibria of Myosin B. N. Ghosh and E. Mihdlyi’ From the Institute
for Muscle Research, at the Marine Hole, Massachusetts Received
Biological
Laboratory,
Woods
May 26, 1952
Proteins possess to various degrees the capacity to bind different ions. The extent of binding can be determined directly, measuring the activity of the chosen ion in the absence and in the presence of the protein by osmotic pressure (l), membrane potential (2), or specific electrode potential (3, 4), etc. Concentration determinations of ultrafiltrates (5) and ultracentrifugates (6) of protein solutions, or of liquids in dialysis (7) or partition equilibrium (8) with the protein solution may serve the same purpose. The limitations of the direct methods are obvious: With slight affinity for ions, binding will occur only at relatively high concentrations of the free ions, and the analytical methods will not be accurate enough to detect the small differences between the activities or concentrations in the presence and in the absence of the protein. In the latter instances, only indirect methods can be of use. By binding ions the net charge of the protein particle is affected. This change will be reflected in those physicochemical properties of the protein which are related to the magnitude of the charge: electrophoretic mobility (9, lo), acid-base equilibria (11,12), light scattering (13), etc. From the effect of ions upon these properties it is possible to calculate, using adequate theories, the extent of binding. The understanding of interactions between myosin and ions seems to be of great importance for learning the function of this muscle protein. In the present paper, investigations will be described on the effect of neutral salts on the acid-base equilibria of myosin. As was shown by Scatchard and Black (12), the shift of pH of a salt-free protein solution upon addition of neutral salts can give valuable information about the 1 Sponsored by a grant from Armour Heart Association.
& Co. Chicago, 107
Ill., and by the American
108
B. N. GHOSH AND E. MIHALYI
ion-protein interactions. Even if the application of a quantitative presents difficulties, the method can be used to give a qualitative of the binding of ions to proteins.
theory picture
EXPERIMENTAL
Myosin from rabbit muscle was prepared according to Szent-Gyorgyi (14) with slight modifications (15). The extracted protein was precipitated twice by dilution to 0.05 M KC1 concentration, and the second precipitate was dialyzed against a large volume of distilled water under continuous stirring. The dialysis was continued for 3 days, the water being changed daily. After dialysis the pH of the myosin was adjusted to the desired value and the solution was left to equilibrate for a few hours; the pH was then checked again with the glass electrode. Below the isoionic point myosin is soluble in the salt-free state, but above it becomes very viscous and difficult to handle. Thorough mixing is very important with these solutions. The gel particles present were eventually removed by a brief centrifugation. Stock salt solutions, prepared from Merck’s analyzed reagents, were not specially purified of excess acid or alkali, but, if necessary, their pH was brought to 6.0-6.5, the pH of the distilled water used. The salt-free myosin solution was mixed with an equal volume of salt solution of different concentrations; thus at each salt concentration an individual mixture was prepared. They were allowed to equilibrate for 12 hr. in the ice box, then brought to room temperature and their pH values determined. A Beckman glass electrode No. 290, with a saturated KCl-calomel electrode and agar bridge, was used in connection with a Cambridge model R potentiometer at 26 f 1°C. The final myosin concentration in different experiments ranged from 6 to 8 mg./ml. Three series of determinations were conducted with starting pH’s in the salt-free state of 3.95,5.73, and 6.40. From titration experiments (15), the net charge/lo5 g. myosin at the above pH values was approximately +33,0, and -6, respectively. A special experiment was performed with potassium halides below the isoionic point. In these experiments a somewhat higher starting pH of 4.3 was selected to avoid iodine liberation from the KI-containing solutions. The latter were kept in the dark, and no free iodine could be detected in them. The pH determinations after 1 hr. checked very closely with those made after 12 hr., showing that no substitution of iodine occurred during this time. The results are summarized in the accompanying figures (Figs. l-4),
BINDING OF IONS BY MYOSIN. II
109
where the shift in pH caused by salt addition, ApH, is plotted against the logarithm of the ionic strength. The properties of a myosin solution are largely affected by pH and ionic strength. Increasing the ionic strength of an acidic myosin solution up to 10-2-10-1 precipitates the myosin. On the contrary, an isoionic, or alkaline salt-free myosin, which forms a thixotropic, gel-like solution, upon salt addition becomes less viscous, and at 10-2-10-1 ionic strength a normal solution is obtained. With the verylargemyosinmolecules, there is probably but little difference in their interaction with small ions, whether they are in a gel-like structure, or free in the solvent. Therefore, in the interpretation of the results, these changes in the structure of the solution were not taken into consideration. The equilibrium dialysis experiments were performed as described by Klotz, Walker, and Pivan (7). Ten milliliters of the isoionic salt-free myosin was poured into a cellophane bag and equilibrated against 20 ml. of NaSCN solution for 4 days at +l”C. At every thiocyanate concentration a blank was also prepared, with distilled water instead of myosin solution in the cellophane bag. All solutions were measured by weight. The SCN concentration was estimated calorimetrically, using Schreiber’s (16) reagent. Optical densities were read in a Beckman model DU spectrophotometer. Since myosin is uncharged at pH 5.73, no Donnan correction was necessary in the calculation of the binding. Figure 5 shows the amount of thiocyanate ions bound by 1 mole of myosin as a function of the free SCN concentration. DISCUSSION
In the relatively simple case of a spherical protein molecule with the charge spread uniformly over its surface, the electrostatic theory can be applied successfully to describe the ionization of acidic and basic groups as a function of the net charge of the molecule and of the ionic strength (17). The pH shift of a salt-free protein solution upon salt addition is given by the relation (18) :
az 2.303dC
~PH __ z------ 2~
ac
22 at0 2.303aC
(1)
where C is the ionic strength, Z the net charge of the molecule, and w is the electrostatic free energy of the molecule divided by Z%T (Ic is the Boltzmann constant, and T is the absolute temperature). This equation holds in the middle portion of the pH scale, with not very di-
110
B.
N.
GHOSH
AND
E.
MIHdLYI
lute protein solutions, where the pH change can be brought about by a negligible change of the degree of dissociation of the protein. The change in net charge in this case is entirely due to the bound ions (first righthand side term of Eq. 1). With isoionic protein, Z is zero; therefore the second right-hand-side term vanishes. The resulting much simpler relation: ApH = - 2g3
AZ
was used by Scatchard and Black (12) to calculate the binding of different ions by isoionic human serum albumin. The results were in satisfactory agreement with the binding of Cl and SCN determined by direct methods. The second right-hand term of Eq. 1 represents the pH change caused by the effect of the ion atmosphere upon the electrostatic free energy of the molecule. Its contribution increases linearily with Z, which makes the analysis of the data obtained outside of the isoionic state more complicated. The successful application of the theory thus depends on the calculation of the electrostatic free energy of the molecule. With spherical molecules the Debye-Huckel approximation gives results in very good agreement with the experimental data. There are also successful attempts to calculate the electrostatic potential of long-chain polymers (19, ZO), but the calculations for asymmetric protein molecules in the presence of neutral salts present yet unsolved difficulties. Myosin is a very asymmetric molecule (21). Its charge distribution and its structure, i.e., whether it is a rigid rod or a flexible filament, are unknown. Theoretical evaluation of the electrostatic free energy of the molecule is therefore impossible. An attempt was made to calculate an empirical value of w by comparing the ApH values based on Eq. 2 with the directly determined binding of SCN (see Fig. 5) and with the binding of Ca deduced from electrophoretic measurements. Limitations of the analytical method do not permit the use of the equilibrium dialysis method above 0.01 M free SCN concentration, the fraction bound becoming smaller and smaller as the concentration of the free ion increases. The ApH determinations were extended up to 1 M salt concentration, but the common territory of the two curves is rather narrow. The electrophoretic data permit the extension of the calculations to higher ionic strengths. Comparing, in the same salt solution and ionic strength, with the net charge determined from the titration curve, one can calculate the
BINDING
OF
IONS
BY
MYOSIN.
111
II
mobility increment Au/AZ (9). This calculation requires that no ions other than H+ should be bound to the protein, or if there are others bound, their amount should be constant over the whole pH range considered. Plotting the mobilities obtained with myosin in 0.15 M KC1 by Erdos and Snelhnan (22) against the net charge deduced from the titration curve in the same solvent (15), a very good linear relation is obtained between pH 3.53 and 8.30, showing that in this pH interval the above conditions are fulfilled. The slope is 1.0 X lO-‘, a much smaller value than was obtained with other proteins (9,23), a result anticipated on the basis of the large dimensions and asymmetry of the myosin molecule. Substituting calcium for potassium, at the same ionic strength and degree TABLE Inftuenee
of ionic
I
strength and pH on the w values of myosin and serum albumin
Ionic strength
ApHe
Azb
wmyosine
0. 0.001 0.005 0.010 0.150
0. 0.08 0.09 0.11 0.29
0. 6.5 16.8 21.8 264.w
0.0168~ 0.0140 0.0068 0.0058 0.0012
0 Interpolated from Fig. 3. b Interpolated from Fig. 5. c Theoretical value. d Calculated from electrophoretic data. 8 For explanation of w values of myosin
and human serum albumin,
WSWUm&lb.’
0.1190 0.0914 0.0720 0.0624 0.0308
see text.
of dissociation of myosin, a marked change in electrophoretic mobility occurs (22). This may be attributed to the change in the net charge of the protein caused by the binding of calcium. Myosin is isoionic at pH 5.73 in 0.15 M KCl, and at pH 5.45 in CaClz of the same ionic strength, according to the ApH curves of Fig. 3. The difference of electrophoretic mobilities interpolated from Erdos and Snellman’s data for the above conditions is 2.64 X lO+. This value divided by the mobility increment gives a net charge difference of 264 per mol of myosin. Supposing that the number of bound chloride ions is the same with both these salts, and there is no displacement of K by Ca, the difference in net charge should correspond to the number of equivalents of Ca bound. The values of W, calculated with the data on the binding of SCN and Ca by isoionio myosin, and the corresponding pH values are listed in Table I. In these calculations the molecular weight of myosin was considered to be 840,000 (21).
112
B.
N.
GHOSH
AND
E.
MIHALYI
All the w values fall on the same curve and show the theoretically expected decreasewith an increase of the ionic strength. Both the pH shift and the amount of SCN bound are relatively small, with a considerable experimental error in their determination. Therefore, the calculated w values can be considered but approximate. For comparison, the theoretically calculated values of w for human serum albumin are also included (3). It is apparent that with isoionic myosin the values of w are 6 to 25 times smaller than with serum albumin, a result expected because
1.5 NaSCN 1.0
KCI Na,SO, WA CaCI,
0.5
3.0
2.0 1.0 -log ionic strength
0
ha. 1. Effects of salts on the pH of myosin below its ieoionic point.
of the large dimensions and asymmetry of the myosin molecule. An approximate theoretical value of u, for the myosin molecule in the salt-free state can be calculated without difficulty by using a rigid rod-shaped model. The electrostatic potential at the surface of a cylinder in its central section is given by the relation:
where 2~ is the net charge, I the length, and a the radius of the cylinder (24). NO attempt was made to obtain an average value over the whole surface of the cylinder. The potential was found, and from this the value
BINDING
OF
IONS
BY
MYOSIN.
113
II
of w was calculated, using the dimensions of the myosin molecule [I = 2200 A.; a = 11 A. (21)]. As can be seen in Table I, the calculated value fits very well into the empirically determined ones.
2.5
KF
2.0
1.5
IKi )KBr IKCI
1.0
0.5
,
1 --
3.0
-
-
20
IO
-log ionic FIG. 2.
Effect of potassiumhalides
0
strength
on the pH of myosin below its ieoionic point.
The complications, both with the direct determinations of the binding of ions (Donnan effects) and the interpretation of the ApH values, do not permit, at the present time, the extension of the above considerations to myosin outside of the isoionic state. As was pointed out previously, the ApH curves represent both the specific effect of the bound ions and the general effect of the ionic
114
B.
N.
GHOSH
AND
E.
MIHdLYI
strength. If there is no binding of ions the curves should fall together with different salts when ApH is plotted against the ionic strength. The deviation from this reference curve is due to the bound ions. Evidently only the difference of the bound positive and negative charges will manifest its effect, the pH being shifted in an alkaline direction when the bound anions are in excess, and in an acidic direction when the bound cations are in excess. Examining the curves obtained below the isoionic point (Figs. 1 and 2) it is apparent that different anions, have distinct
ADH
6.5
/O-o* NaSCN.---“o-o-o-o-o-o-o-o-oD 0 ‘KCI G 8”0-8-8 CaCI, -w&8,,
ac
-0.5 t I
I
I
3.0 20 1.0 -log ionic strength
I
0
FIG. 3. Effect of salts on the pH of isoionic myosin.
specific effects. Sulfate ion is the least, and fluoride the most effective. With the halides the sequence of effectiveness clearly follows the Hofmeister series F > I > Br > Cl. It is interesting to note the action of alkaline earth cations. Calcium and magnesium act in the same way, depressing the ApH curve at higher concentrations. Since the anion is common and probably bound to the same extent, the difference between the KCI, CaC12, and MgClz curves should be due to the binding of the bivalent cations. The difference is unlikely to be due to an increase in the weight of higher terms in the electrostatic theory. With serum albumin (12) at pH 3.45 the ApH curves with chlorides of the alkali and alkaline earth metals fall practically together.
BISDING
OF
IONS
BY
ii5
II
MYOSiN.
At pH 5.73, considered as the isoionic point of myosin (15) (Fig. 3), there is binding of thiocyanatc and calcium. With KC1 the pH remains
APH CaCI, MgCL KCI Na,SO, NaSCN
3.0
2.0
1.0
0
-log ionic strength Fro. 4. Meet
of salts
on the pH of myosin above its isoionic point.
.-Ei ---.r F2.0’ c
0
5 E \ -0 = 1.0
/
0
0 / 0 0
2 5 m
L
0co-O’
z
5
4
Q
0 / /o OO 1
3
2
Jog [SCN] free FIG. 5. Binding
of SCS by
isoionic myosin.
practically unchanged, i.e., there is either no binding of potassium or chloride, or they are bound in equivalent quantities. The final series of determinations was conducted with slightly anionic
116
B. N. GHOSH .4ND E. MIdLYI
myosin. As shown in Fig. 4, the curves with KC1 and Na2S04 practically coincide, the effect being manifest below 0.1 ionic strength and absent above it. This behavior suggeststhat it is entirely due to the nonspecific effect of the ionic strength. The curve with thiocyanate behaves differently: It starts with the previously mentioned onesbut later bends down, showing the binding of thiocyanate at concentrations higher than 0.05 M. The curve with CaCL and MgClz is almost parallel to the KC1 curve but is higher by about 0.2 pH unit. The electrophoretic investigations of Erdiis and Snellman (22) support the conclusion that the above shift of the curve is due to the binding of the alkaline earth cations. The binding is apparent at the lowest ionic strength investigated and changes very little further,, i.e., the affinity of myosin for alkaline earth cations should be very pronounced. SUMMARY
The shift of pH of salt-free myosin solutions up011 addition of different neutral salts has been investigated. The results were compared with the directly determined binding of thiocyanate and the binding of calcium deduced from electrophoretic measurements. From t’hese, conclusions were drawn as to the electrostatic free energy of the myosin molecule. The results show a considerable interaction between anions and myosin below the isoionic point, and with bivalent cations and thiocyanate over the whole pH range investigated (3.95-6.40). REFERENCES 1. SCATCHARD, G., BATCHELDER, A. C., AND BROWN, A., J. Am. Chem. Sot. 68, 2326 (1946). 2. CARR, C. W., AND TOPOL, L., J. Phys. & Colloid Chem. 64,176 (1950). 3. SCATCHARD, G., ~HEINBERG, I. H., AND ARMSTRONG, S..H., JR., J. Am. Chem. sot. 72, 535 (1950). 4. SCATCHARD, G., SCHEINBERG, I. H., AND ARMSTRONG, S. H., JR., J. Am. Chem. sot. 74, 540 (1950). 5. GREENBERG, D. M., AND GREENBERG, M., J. Biol. Chem. 94, 373 (1931-2). 6. CHANUTIN, A., LUDEWIQ, S., AND MASKET, A. V., J. Biol. Chem. 148,737 (1942). 7. KLOTZ, I. M., WALKER, F. M., AND PIVAN, R. B., J. Am. Chem. Sot. 66, 1486 (1946) * 8. KARUSH, F., J. Am. Chem. Sot. 73, 1246 (1951). 9. LONOSWORTH, L. G., AND JACOBSEN, C. F., J. Phys. h Colloid Chem. 63, 1’26 (1949). 10. SMITH, R. F., AND BRIGGS, D. R., J. Phys. & Colloid Chem. 64, 33 (1950). II. STEINHARDT, J., AND HARRIS, M., J. Research Nail. Bur. Standards 24, 335
(1Q‘w.
BINDING
OF
IONS
BY
MYOSIN.
II
117
12. SCATCHARD, G., AND BLACK, E. S., J. Phys. & Colloid Chem. 63.88 (1949). 13. EDSALL, J. T., EDELHOCH, H., LONTIE, R., AND MORRISON, P. R., J. Am. Chem. Sot. 72,464l (1950). 14. SZENT-GY~RGYI, A., Chemistry of Muscular Contraction, 2nd Ed.;p. 146. Academic Press, New York, 1951. 15. MIH~LYI, E., Enz~moEogia 14, 224 (1950). 16. SCHREIBER, H., Biochem. 2. 163, 241 (1925). 17. CANNAN, R. K., KIBRICK, A., AND PALMER, A. H., Ann. N. Y. Acad. Sci. 41, 243 (1941). 18. TANFORD, C., Proc. Iowa Acad. Sci. 67,225 (1950). 19. KAWHALSKY, A., AND GILLIS, J., Rec. trau. chim. 68; 879 (1949). 20. OVERBEEK, J. Th. G., Bull. sot. chim. Beiges 67, 252 (1948). 21. PORTZEHL, H., 2. Naturjorsch. Sb, 75 (1950). 22. ERD~S, TH., AND SNELLMAN, O., Biochim. et Biophys. Acta 2, 642 (1948). 23. LONGSWORTH, L. G., Ann. N. Y. Acad. Sci. 41. 267 (1941). 24. PIDDUCK, F. B., A Treatise on Electricity, p. 68. Cambridge University Press, 1925.
for Muscle Research, at the Marine Hole, Massachusetts Received
Biological
Laboratory,
Woods
May 26, 1952
Proteins possess to various degrees the capacity to bind different ions. The extent of binding can be determined directly, measuring the activity of the chosen ion in the absence and in the presence of the protein by osmotic pressure (l), membrane potential (2), or specific electrode potential (3, 4), etc. Concentration determinations of ultrafiltrates (5) and ultracentrifugates (6) of protein solutions, or of liquids in dialysis (7) or partition equilibrium (8) with the protein solution may serve the same purpose. The limitations of the direct methods are obvious: With slight affinity for ions, binding will occur only at relatively high concentrations of the free ions, and the analytical methods will not be accurate enough to detect the small differences between the activities or concentrations in the presence and in the absence of the protein. In the latter instances, only indirect methods can be of use. By binding ions the net charge of the protein particle is affected. This change will be reflected in those physicochemical properties of the protein which are related to the magnitude of the charge: electrophoretic mobility (9, lo), acid-base equilibria (11,12), light scattering (13), etc. From the effect of ions upon these properties it is possible to calculate, using adequate theories, the extent of binding. The understanding of interactions between myosin and ions seems to be of great importance for learning the function of this muscle protein. In the present paper, investigations will be described on the effect of neutral salts on the acid-base equilibria of myosin. As was shown by Scatchard and Black (12), the shift of pH of a salt-free protein solution upon addition of neutral salts can give valuable information about the 1 Sponsored by a grant from Armour Heart Association.
& Co. Chicago, 107
Ill., and by the American
108
B. N. GHOSH AND E. MIHALYI
ion-protein interactions. Even if the application of a quantitative presents difficulties, the method can be used to give a qualitative of the binding of ions to proteins.
theory picture
EXPERIMENTAL
Myosin from rabbit muscle was prepared according to Szent-Gyorgyi (14) with slight modifications (15). The extracted protein was precipitated twice by dilution to 0.05 M KC1 concentration, and the second precipitate was dialyzed against a large volume of distilled water under continuous stirring. The dialysis was continued for 3 days, the water being changed daily. After dialysis the pH of the myosin was adjusted to the desired value and the solution was left to equilibrate for a few hours; the pH was then checked again with the glass electrode. Below the isoionic point myosin is soluble in the salt-free state, but above it becomes very viscous and difficult to handle. Thorough mixing is very important with these solutions. The gel particles present were eventually removed by a brief centrifugation. Stock salt solutions, prepared from Merck’s analyzed reagents, were not specially purified of excess acid or alkali, but, if necessary, their pH was brought to 6.0-6.5, the pH of the distilled water used. The salt-free myosin solution was mixed with an equal volume of salt solution of different concentrations; thus at each salt concentration an individual mixture was prepared. They were allowed to equilibrate for 12 hr. in the ice box, then brought to room temperature and their pH values determined. A Beckman glass electrode No. 290, with a saturated KCl-calomel electrode and agar bridge, was used in connection with a Cambridge model R potentiometer at 26 f 1°C. The final myosin concentration in different experiments ranged from 6 to 8 mg./ml. Three series of determinations were conducted with starting pH’s in the salt-free state of 3.95,5.73, and 6.40. From titration experiments (15), the net charge/lo5 g. myosin at the above pH values was approximately +33,0, and -6, respectively. A special experiment was performed with potassium halides below the isoionic point. In these experiments a somewhat higher starting pH of 4.3 was selected to avoid iodine liberation from the KI-containing solutions. The latter were kept in the dark, and no free iodine could be detected in them. The pH determinations after 1 hr. checked very closely with those made after 12 hr., showing that no substitution of iodine occurred during this time. The results are summarized in the accompanying figures (Figs. l-4),
BINDING OF IONS BY MYOSIN. II
109
where the shift in pH caused by salt addition, ApH, is plotted against the logarithm of the ionic strength. The properties of a myosin solution are largely affected by pH and ionic strength. Increasing the ionic strength of an acidic myosin solution up to 10-2-10-1 precipitates the myosin. On the contrary, an isoionic, or alkaline salt-free myosin, which forms a thixotropic, gel-like solution, upon salt addition becomes less viscous, and at 10-2-10-1 ionic strength a normal solution is obtained. With the verylargemyosinmolecules, there is probably but little difference in their interaction with small ions, whether they are in a gel-like structure, or free in the solvent. Therefore, in the interpretation of the results, these changes in the structure of the solution were not taken into consideration. The equilibrium dialysis experiments were performed as described by Klotz, Walker, and Pivan (7). Ten milliliters of the isoionic salt-free myosin was poured into a cellophane bag and equilibrated against 20 ml. of NaSCN solution for 4 days at +l”C. At every thiocyanate concentration a blank was also prepared, with distilled water instead of myosin solution in the cellophane bag. All solutions were measured by weight. The SCN concentration was estimated calorimetrically, using Schreiber’s (16) reagent. Optical densities were read in a Beckman model DU spectrophotometer. Since myosin is uncharged at pH 5.73, no Donnan correction was necessary in the calculation of the binding. Figure 5 shows the amount of thiocyanate ions bound by 1 mole of myosin as a function of the free SCN concentration. DISCUSSION
In the relatively simple case of a spherical protein molecule with the charge spread uniformly over its surface, the electrostatic theory can be applied successfully to describe the ionization of acidic and basic groups as a function of the net charge of the molecule and of the ionic strength (17). The pH shift of a salt-free protein solution upon salt addition is given by the relation (18) :
az 2.303dC
~PH __ z------ 2~
ac
22 at0 2.303aC
(1)
where C is the ionic strength, Z the net charge of the molecule, and w is the electrostatic free energy of the molecule divided by Z%T (Ic is the Boltzmann constant, and T is the absolute temperature). This equation holds in the middle portion of the pH scale, with not very di-
110
B.
N.
GHOSH
AND
E.
MIHdLYI
lute protein solutions, where the pH change can be brought about by a negligible change of the degree of dissociation of the protein. The change in net charge in this case is entirely due to the bound ions (first righthand side term of Eq. 1). With isoionic protein, Z is zero; therefore the second right-hand-side term vanishes. The resulting much simpler relation: ApH = - 2g3
AZ
was used by Scatchard and Black (12) to calculate the binding of different ions by isoionic human serum albumin. The results were in satisfactory agreement with the binding of Cl and SCN determined by direct methods. The second right-hand term of Eq. 1 represents the pH change caused by the effect of the ion atmosphere upon the electrostatic free energy of the molecule. Its contribution increases linearily with Z, which makes the analysis of the data obtained outside of the isoionic state more complicated. The successful application of the theory thus depends on the calculation of the electrostatic free energy of the molecule. With spherical molecules the Debye-Huckel approximation gives results in very good agreement with the experimental data. There are also successful attempts to calculate the electrostatic potential of long-chain polymers (19, ZO), but the calculations for asymmetric protein molecules in the presence of neutral salts present yet unsolved difficulties. Myosin is a very asymmetric molecule (21). Its charge distribution and its structure, i.e., whether it is a rigid rod or a flexible filament, are unknown. Theoretical evaluation of the electrostatic free energy of the molecule is therefore impossible. An attempt was made to calculate an empirical value of w by comparing the ApH values based on Eq. 2 with the directly determined binding of SCN (see Fig. 5) and with the binding of Ca deduced from electrophoretic measurements. Limitations of the analytical method do not permit the use of the equilibrium dialysis method above 0.01 M free SCN concentration, the fraction bound becoming smaller and smaller as the concentration of the free ion increases. The ApH determinations were extended up to 1 M salt concentration, but the common territory of the two curves is rather narrow. The electrophoretic data permit the extension of the calculations to higher ionic strengths. Comparing, in the same salt solution and ionic strength, with the net charge determined from the titration curve, one can calculate the
BINDING
OF
IONS
BY
MYOSIN.
111
II
mobility increment Au/AZ (9). This calculation requires that no ions other than H+ should be bound to the protein, or if there are others bound, their amount should be constant over the whole pH range considered. Plotting the mobilities obtained with myosin in 0.15 M KC1 by Erdos and Snelhnan (22) against the net charge deduced from the titration curve in the same solvent (15), a very good linear relation is obtained between pH 3.53 and 8.30, showing that in this pH interval the above conditions are fulfilled. The slope is 1.0 X lO-‘, a much smaller value than was obtained with other proteins (9,23), a result anticipated on the basis of the large dimensions and asymmetry of the myosin molecule. Substituting calcium for potassium, at the same ionic strength and degree TABLE Inftuenee
of ionic
I
strength and pH on the w values of myosin and serum albumin
Ionic strength
ApHe
Azb
wmyosine
0. 0.001 0.005 0.010 0.150
0. 0.08 0.09 0.11 0.29
0. 6.5 16.8 21.8 264.w
0.0168~ 0.0140 0.0068 0.0058 0.0012
0 Interpolated from Fig. 3. b Interpolated from Fig. 5. c Theoretical value. d Calculated from electrophoretic data. 8 For explanation of w values of myosin
and human serum albumin,
WSWUm&lb.’
0.1190 0.0914 0.0720 0.0624 0.0308
see text.
of dissociation of myosin, a marked change in electrophoretic mobility occurs (22). This may be attributed to the change in the net charge of the protein caused by the binding of calcium. Myosin is isoionic at pH 5.73 in 0.15 M KCl, and at pH 5.45 in CaClz of the same ionic strength, according to the ApH curves of Fig. 3. The difference of electrophoretic mobilities interpolated from Erdos and Snellman’s data for the above conditions is 2.64 X lO+. This value divided by the mobility increment gives a net charge difference of 264 per mol of myosin. Supposing that the number of bound chloride ions is the same with both these salts, and there is no displacement of K by Ca, the difference in net charge should correspond to the number of equivalents of Ca bound. The values of W, calculated with the data on the binding of SCN and Ca by isoionio myosin, and the corresponding pH values are listed in Table I. In these calculations the molecular weight of myosin was considered to be 840,000 (21).
112
B.
N.
GHOSH
AND
E.
MIHALYI
All the w values fall on the same curve and show the theoretically expected decreasewith an increase of the ionic strength. Both the pH shift and the amount of SCN bound are relatively small, with a considerable experimental error in their determination. Therefore, the calculated w values can be considered but approximate. For comparison, the theoretically calculated values of w for human serum albumin are also included (3). It is apparent that with isoionic myosin the values of w are 6 to 25 times smaller than with serum albumin, a result expected because
1.5 NaSCN 1.0
KCI Na,SO, WA CaCI,
0.5
3.0
2.0 1.0 -log ionic strength
0
ha. 1. Effects of salts on the pH of myosin below its ieoionic point.
of the large dimensions and asymmetry of the myosin molecule. An approximate theoretical value of u, for the myosin molecule in the salt-free state can be calculated without difficulty by using a rigid rod-shaped model. The electrostatic potential at the surface of a cylinder in its central section is given by the relation:
where 2~ is the net charge, I the length, and a the radius of the cylinder (24). NO attempt was made to obtain an average value over the whole surface of the cylinder. The potential was found, and from this the value
BINDING
OF
IONS
BY
MYOSIN.
113
II
of w was calculated, using the dimensions of the myosin molecule [I = 2200 A.; a = 11 A. (21)]. As can be seen in Table I, the calculated value fits very well into the empirically determined ones.
2.5
KF
2.0
1.5
IKi )KBr IKCI
1.0
0.5
,
1 --
3.0
-
-
20
IO
-log ionic FIG. 2.
Effect of potassiumhalides
0
strength
on the pH of myosin below its ieoionic point.
The complications, both with the direct determinations of the binding of ions (Donnan effects) and the interpretation of the ApH values, do not permit, at the present time, the extension of the above considerations to myosin outside of the isoionic state. As was pointed out previously, the ApH curves represent both the specific effect of the bound ions and the general effect of the ionic
114
B.
N.
GHOSH
AND
E.
MIHdLYI
strength. If there is no binding of ions the curves should fall together with different salts when ApH is plotted against the ionic strength. The deviation from this reference curve is due to the bound ions. Evidently only the difference of the bound positive and negative charges will manifest its effect, the pH being shifted in an alkaline direction when the bound anions are in excess, and in an acidic direction when the bound cations are in excess. Examining the curves obtained below the isoionic point (Figs. 1 and 2) it is apparent that different anions, have distinct
ADH
6.5
/O-o* NaSCN.---“o-o-o-o-o-o-o-o-oD 0 ‘KCI G 8”0-8-8 CaCI, -w&8,,
ac
-0.5 t I
I
I
3.0 20 1.0 -log ionic strength
I
0
FIG. 3. Effect of salts on the pH of isoionic myosin.
specific effects. Sulfate ion is the least, and fluoride the most effective. With the halides the sequence of effectiveness clearly follows the Hofmeister series F > I > Br > Cl. It is interesting to note the action of alkaline earth cations. Calcium and magnesium act in the same way, depressing the ApH curve at higher concentrations. Since the anion is common and probably bound to the same extent, the difference between the KCI, CaC12, and MgClz curves should be due to the binding of the bivalent cations. The difference is unlikely to be due to an increase in the weight of higher terms in the electrostatic theory. With serum albumin (12) at pH 3.45 the ApH curves with chlorides of the alkali and alkaline earth metals fall practically together.
BISDING
OF
IONS
BY
ii5
II
MYOSiN.
At pH 5.73, considered as the isoionic point of myosin (15) (Fig. 3), there is binding of thiocyanatc and calcium. With KC1 the pH remains
APH CaCI, MgCL KCI Na,SO, NaSCN
3.0
2.0
1.0
0
-log ionic strength Fro. 4. Meet
of salts
on the pH of myosin above its isoionic point.
.-Ei ---.r F2.0’ c
0
5 E \ -0 = 1.0
/
0
0 / 0 0
2 5 m
L
0co-O’
z
5
4
Q
0 / /o OO 1
3
2
Jog [SCN] free FIG. 5. Binding
of SCS by
isoionic myosin.
practically unchanged, i.e., there is either no binding of potassium or chloride, or they are bound in equivalent quantities. The final series of determinations was conducted with slightly anionic
116
B. N. GHOSH .4ND E. MIdLYI
myosin. As shown in Fig. 4, the curves with KC1 and Na2S04 practically coincide, the effect being manifest below 0.1 ionic strength and absent above it. This behavior suggeststhat it is entirely due to the nonspecific effect of the ionic strength. The curve with thiocyanate behaves differently: It starts with the previously mentioned onesbut later bends down, showing the binding of thiocyanate at concentrations higher than 0.05 M. The curve with CaCL and MgClz is almost parallel to the KC1 curve but is higher by about 0.2 pH unit. The electrophoretic investigations of Erdiis and Snellman (22) support the conclusion that the above shift of the curve is due to the binding of the alkaline earth cations. The binding is apparent at the lowest ionic strength investigated and changes very little further,, i.e., the affinity of myosin for alkaline earth cations should be very pronounced. SUMMARY
The shift of pH of salt-free myosin solutions up011 addition of different neutral salts has been investigated. The results were compared with the directly determined binding of thiocyanate and the binding of calcium deduced from electrophoretic measurements. From t’hese, conclusions were drawn as to the electrostatic free energy of the myosin molecule. The results show a considerable interaction between anions and myosin below the isoionic point, and with bivalent cations and thiocyanate over the whole pH range investigated (3.95-6.40). REFERENCES 1. SCATCHARD, G., BATCHELDER, A. C., AND BROWN, A., J. Am. Chem. Sot. 68, 2326 (1946). 2. CARR, C. W., AND TOPOL, L., J. Phys. & Colloid Chem. 64,176 (1950). 3. SCATCHARD, G., ~HEINBERG, I. H., AND ARMSTRONG, S..H., JR., J. Am. Chem. sot. 72, 535 (1950). 4. SCATCHARD, G., SCHEINBERG, I. H., AND ARMSTRONG, S. H., JR., J. Am. Chem. sot. 74, 540 (1950). 5. GREENBERG, D. M., AND GREENBERG, M., J. Biol. Chem. 94, 373 (1931-2). 6. CHANUTIN, A., LUDEWIQ, S., AND MASKET, A. V., J. Biol. Chem. 148,737 (1942). 7. KLOTZ, I. M., WALKER, F. M., AND PIVAN, R. B., J. Am. Chem. Sot. 66, 1486 (1946) * 8. KARUSH, F., J. Am. Chem. Sot. 73, 1246 (1951). 9. LONOSWORTH, L. G., AND JACOBSEN, C. F., J. Phys. h Colloid Chem. 63, 1’26 (1949). 10. SMITH, R. F., AND BRIGGS, D. R., J. Phys. & Colloid Chem. 64, 33 (1950). II. STEINHARDT, J., AND HARRIS, M., J. Research Nail. Bur. Standards 24, 335
(1Q‘w.
BINDING
OF
IONS
BY
MYOSIN.
II
117
12. SCATCHARD, G., AND BLACK, E. S., J. Phys. & Colloid Chem. 63.88 (1949). 13. EDSALL, J. T., EDELHOCH, H., LONTIE, R., AND MORRISON, P. R., J. Am. Chem. Sot. 72,464l (1950). 14. SZENT-GY~RGYI, A., Chemistry of Muscular Contraction, 2nd Ed.;p. 146. Academic Press, New York, 1951. 15. MIH~LYI, E., Enz~moEogia 14, 224 (1950). 16. SCHREIBER, H., Biochem. 2. 163, 241 (1925). 17. CANNAN, R. K., KIBRICK, A., AND PALMER, A. H., Ann. N. Y. Acad. Sci. 41, 243 (1941). 18. TANFORD, C., Proc. Iowa Acad. Sci. 67,225 (1950). 19. KAWHALSKY, A., AND GILLIS, J., Rec. trau. chim. 68; 879 (1949). 20. OVERBEEK, J. Th. G., Bull. sot. chim. Beiges 67, 252 (1948). 21. PORTZEHL, H., 2. Naturjorsch. Sb, 75 (1950). 22. ERD~S, TH., AND SNELLMAN, O., Biochim. et Biophys. Acta 2, 642 (1948). 23. LONGSWORTH, L. G., Ann. N. Y. Acad. Sci. 41. 267 (1941). 24. PIDDUCK, F. B., A Treatise on Electricity, p. 68. Cambridge University Press, 1925.