Oct., 1963
DIFFERENTIAL SOLUBILIZATION OF K
ASD
Na DYESALTSBY LECITHIN
2085
THE DIFFERENTIAL SOLUBILIZATION OF POTASSIUM AND SODIUM DYE SALTS BY LECITHIN MICELLES I N BENZENE BY IRABLEI AND R. E. LEE, JR. Melpur, Incorporated, Falls Church, Virgimu Received March 8, 1963
A hypothesis of biological differentiation of sodium and potassium through lipid phase transitions in the bimolecular Leaflets of biological membranes has been probed using lecithin micelles in benzene as a model system. It wa3 found that in micelles which may be considered ordered, the temperature coefficient of the sohbilizing power of lecithin for a sodium dye salt is negative, and that for the potassium salt of the same dye is positive.
The events which occur in nerve membranes may be classified into two groups of processes. One category is associated with certain properties of the membrane which enable it to create a concentration gradient with respect to sodium and potassium ions and to maintain that difference. The other category is concerned with the physico-chemical changes occurring in the membrane which give rise to the sudden increase in ion flux.1.2 This paper is concerned with the latter category of events, whose nature is obscure a t present. A membrane may be considered to consist of alternating apolar and polar sheets containing a high population of oxygen a t o m (from the phosphate groups of lecithin or cephalin). If attention is focused on the structure of crystalline solids containing sodium or potassium surrounded by oxygen atoms, considerable differences can be found in the nature of the complexing or coordinating ability of cations for oxygen. Sodium can only form structures with coordination numbers of 6 and 8 with respect to oxygen. On the other hand, the coordination number of potassium for oxygen has been One could say that found to be 6, 7, 8, 9, 10, and there is a relatively wide array of structures which can accommodate potassium and in comparison, sodium requires more rigorous spatial arrangements of oxygen atoms. It therefore appears possible that this discrimination of sodium from potassium in crystalline solids containing oxygen atoms is at the root of the biological clifferentiation of these two ions; and that sodium is differentiated from potassium and prevented from entering the resting cell through order-disorder transitions in the ionic or polar portions of the nerve membrane. The structures of polar lipid micelles in nonaqueous media bear strong resemblances to nerve membranes. The principal characteristic of both is the bimolecular leaflet. Lecithin, a principal component of nerve membranes, forms micelles in benzene. The solubilising effects of these lipid structures on the sodium and potassium salts of a water-soluble dye in benzene was chosen as a model system to probe experimentally the hypothesis presented above. Experimental In order to use a natural product for this study, a purification scheme had to be developcd. The details of this procedure will be presented elsewhere, however; the principal aspects are as follows. The three types of impurities present in lecithin in high concentrations are ninhydrin staining materials, lysolecithin, and alkali and alkaline earth metal cations. Elworthy4 and (1) A. L. Hodgkin a n d R. D. Keynes, J. Physiol., 128, 28 (1955). (2) A. L. Hodgkin, Proc. 120y. Soc. (London), B148, 1 (1957). (3) L. Pauling, “The Nature of the Chemical Bond,” 2nd Ed., Cornel1 Univ. Press, Ithaca, N. Y.,1948. (4) P. H. Elwortby and L Saunders, J . Chem. floe., 330 (1957).
others6 have developed schemes which permit the removal of the first two types, but have not paid adequate attention to the cations. It was found early in this study that cations in the lecithin seriously altered its ability to solubilize the dye salts which were used. The removal of mono- and divalent cations from the natural lecithin used in this work was effected by a nonaqueous ion exchange technique. The lecithin was dissolved in chloroform, to which an excess of strong sulfonic acid resin was added. After a minimum of three days, the metal cation content of the impure lecithin was routinely reduced from over 2070 by weight to less than 0.017,. The storage of the purified lecithin proved to be another serious problem. It was found that after two days of storage in the anhydrous state, significant amounts of lysolecithin could be detected. This could be prevented by storing the purified lecithin in dry benzene solution, or, as a precipitate in dry acetone. The solubilization method employed was similar t o that of Kolthoff and Stricks.6 The solvent used to determine specific absorbance was a mixture of chloroform, methanol, and benzene in the ratio 66:30:4. It was found that absorbancy in this solvent was related to absorbancy in the solubilizing system by a constant. After an initial determination of the specific absorbance of the pure dye in the solvent, the specific absorbance in the solubilizing mixtures was determined. After that, dye concentration was directly measured in the solubilizing solutions. This approach reduced errors in estimating dye concentrations since the equilibrium reaction mixtures required no dilution with solvent capable of dissolving any dye particles accidently picked up in the presumably clear supernatant. Prior to removing metallic cations from lecithin, these systems required up to 400 hr. to reach solubilization equilibrium. Using lecithin purified according to the above procedure, this time was reduced to a maximum of 7 2 hr. The dye used in the experiments reported here was m-(pani1ino)-phenylazobenzenesulfonic acid. It was purchased as the sodium salt, and purified by repeated recrystallizations from water. The potassium salt was prepared by precipitating the sulfonic acid of the dye with HC1. After repeated washing with hot water, an equivalent amount of KOH was added. This salt was recrystallized from water several times. The calcium salt was prepared by neutralizing the sulfonic acid form of the dye with NH40H. This resulted in the soluble ammonium salt which then could react with a soluble calcium salt, CaC12, to form the insoluble calcium salt of the dye. This dye salt was recrystallized from water several times. The procedure in a typical solubilization experiment was as follows. A benzene solution of lecithin was charged with coarse crystals of dye. Two types of closure were used: ( I ) screw-cap test tubes sealed with caulking and sealing wax and (2) test tubes sealed by fusing the glass. The second method proved more reliable and versatile. The tubes were secured t o a tumbling drum which was immersed in a constant temperature water bath. At the end of an experiment the tubes were set upright in the bath overnight and spun down in a centrifuge the next morning. The absorbancy of these reaction mixtures was measured in a Cary 15 spectrophotometer. The cuvettes were equipped with spacers which allowed absorbancy measurements of concentrated solutions.
Results Because the spectrum of a dye reflects its environment, it is often possible to determine the nature of (5) C . H. Lea and D. N. Rhodes, Biochem. J., 59 (1955). (6) I. M. Kolthoff and W. Strioka, J. Phys. Colloed Chsm., 52, 015 (1948).
2086
IRABLEIAND R. E. LEE,JR.
Vol. 67
0.6
z 0
d 0.4
k
gq: 0.3
40 (LECITHIN CONC. X ld)%,
Fig. 1.-The
solubilization of K a and K dye salts by lecithin in benzene as a function of concentration and temperature. Determination of critical micelle concentrations.
t
-NQ
.
Fig. 2.-The
IO
DYE SALT: 4OoC
4000
(LECITHIN CONC x io4) % solubilization of Na and K dye salts by lecithin in benzene over a wide range of lecithin concentration a t 25 and 40".
an unknown environment in which a dye is contained by a simulation process. One examines the spectrum of the dye in a series of solvents, chosen so as to simulate as closely as possible the probable nature
of the unknown enviroiimelit. I n this manner, the spectral characteristics of the dye in various solvents, e.g., decanol, hexanol, butanol, and water, were determined. All these were found to be virtually
Oct., 1963
DIFFERENTIAL SOLUBILIZATION OF K AND ?Sa DYESALTS BY LECITHIN
identical with the spectrum of the dye in lecithin-benzene solutions. I n the light of these data, the precise disposition of the dye within the micellar structure could not be made. However, it is clear that the environment of the dye in lecithin-benzene solutions is similar to that in polar solvents such as water and ethanol, and such an environment is characteristic of the interior of a polar lipid micelle in nonaqueous media.’ Preliminary studies of the ultraceiitrifugal sedimentation behavior of the dye-lecithin-benzene solutions showed that the dye moved with the schlieren peak. This indicates that all the dye in the solutions is associated with the macromolecular species (micelles) present. The ability of lecithin to solubilize the sodium and potassium salts of the dye in benzene as a function of lecithin concentration, and solutioii temperature is illustrated a t low concentrations in Fig. l. It can be seen that there is a clear indication of a critical concentration for the formation of solubilizing aggregates lecithin. (c.m.c.) at 33 X Variation of the cationic species appears to have had no effect on the c.m.c. a t 25’. However, at 40°, the c.m.c. value obtatined with the potassium dye salt was 98 X lo-+% and that of the sodium dye salt was 75 X l O - b % lecithin. I n Fig. 2, the solubilization data over a wide range of lecithin concentrations, at 25 and 40’ are presented. The solubilizing power of the lecithin-benzene system assumes three different values through three successive ranges of lecithin concentration. The first range occurs close to the c.m.c., in which region there is a true linear relationship between the amount of dye solubilized and the lecithin concentration. There is a middle region from ca. 0.01 t o 0.15% lecithin in which the values of solubilizing power change in an approach to a third range of values above 0.15% lecithin. I n this third region, there is again a good linear relationship between dye solubilized and lecithin concentration. It should be noted that the amounts of potassiurn dye salt solubilized are greater than the amounts of sodium dye salt. The temperature coefficient for the solubilization of the potassium dye is positive in the low concentration range, whereas that for the sodium dye salt appears to be negative in the same region. The values of the solubilizing power of lecithin in the low and high Concentration regions a t 25 and 40’ may be found in Table I. In the low concentration region, the ability of lecithin to solubilize the sodium dye salt decreases as the temperature increases. On the other hand, the solubility of the potassium dye salt increases. I n the high concentration range, the solubility of the sodium salt reinains about the same as the temperature increases, and that of the potassium dye salt increases by about the same amount as in the low concentration region. The value of the temperature coefficients of the solubilizing power may be estimated from the ratios of the solubilizing power of lecithin toward the sodium dye salts a t 25 and 40”, and also for the potassium dye salts in a similar manner. These may be found in Table 11. (7) C R.Singletrrry, .I Am. Od Chemzsts’ Soc , 32, 446 (1955).
2087
TABLE I THESOLUBILIZING POWER OF LECITHIN IN BENZENE, AS MOLES OF DYESOLUBILIZED/MOLE OF LECITHIN Molecular weight of lecithin, 853
1500 X
lo-‘%
Na 0.250 K .340
4O0{P
TEMPERATURE
*1500 X 10-4 %
0.97
(0) 0.967
[z]Bj:;
(0)
It is evident that in the low concentration range the temperature coefficient for sodium dye salt solubilization is negative and in the high concentration region it is close to zero. In the case of the solubilization of the potassium dye salt, the temperature coefficient for the low concentration region is positive and close to zero in the high concentration range. In Table 111, ratios of lecithin solubilizing power toward potassium dye salt and sodium dye salt are listed as a function of concentration and temperature. Along with these may be found the ratio of the atomic volumes of anhydrous potassium and sodium ions. TABLE I11 RATIOSO F
SOLUBILIZING POWER O F
VOLUMESOF Na 100
x
10-4 %
LECITHINAND K
O F ATOMIC
AND
Lecithin
25OK/Na 1.36 40’ K/Na 2.30 Atomic volume K/Na: I . 40
1500 X 10-4 %
2.16 2.19
Discussion of Results The formation of lecithin micelles in benzene has been studied by Elworthy using colligatives and hydrodynamicg techniques. He has shown that micelles of lecithin undergo a transition a t about 0.08% lecithin from “small” micelles, mol. tvt. ca. 3500, to “large” micelles, mol. wt. ca. 50,000. The work presented here indicates that there is a true c.m.c. for the formation of aggregates capable of solubilizing water-soluble dyes. This occurs a t far lower concentrations than Elworthy’s transition concentration. Further, the transition from “small” to “large” micelle is readily discerned by the change in solubilizing efficiency of these micellar solutions. This would indicate that there is a structural transition within the micelle in addition to its increase in size. When the large aggregates form, the potassium salt becomes more strongly solubilized, and a t the same time, the sodium salt becomes less soluble. If it is assumed that the larger the lecithin micelle becomes, the more probable is the approach to the liquid-like state, these results can be explained on the basis of an order-disorder transition. That is, that the number of condensed states in which potassium can be accommodated is greater than the number of states in which (8) P. H. Elworthy, J . Chem. Soc., 813 (1959). (9) P. H.Elworthy, ibid., 1951 (1959).
2088
IRABLEIAND R. E. LEE,JR.
sodium can be accommodated. Therefore, more potassium dye can be solubilized in a “disordered” micellar structure than can sodium dye. Using the assumption that the small micelles are more “ordered” than the large micelles, the data in Tables I and I1 are explicable, The temperature coefficients for both potassium and sodium dye salts in the large micelles are both close to zero. It is clear that if the large micelles are considered to be “disordered,” then raising the temperature will not introduce much additional disorder. Therefore, the solubilizing power of the micelles toward sodium and potassium salts should not change much. In the case of the small micelles, the temperature coefficients are negative for sodium dye and positive for potassium dye. Here again, if it is assumed that the small micelles are more ordered than the large micelles, then by increasing the temperature, it should be possible to induce an order to disorder transition. In that case, the ability of the micelles to solubilize the sodium dye salt should decrease, and that for the potassium dye salt should increase, which has been shown to occur. In the ordered state, the only difference between the
Vol. 67
coordination of potassium and sodium in a condensed environment of oxygen atoms should be related to their atomic volumes. The data in Table 111 shorn that in the “ordered” system (small micelle), the ratio of solubilizing power of lecithin towards the potassium dye salt vs. the sodium dye salt is almost identical to the ratio of their respective atomic volumes. In all the other systems, where there is a marked degree of disorder, the solubilizing power ratio is much greater than that of the atomic volumes. Conclusion The intent of this work was to probe the hypothesis that order-disorder transitions within biological membranes may provide the physical basis for the differentiation of sodium from potassium in living systems. Insofar as lecithin micelles in a nonaqueous medium are a reasonable model of the bimolecular leaflcts characteristic of biological membranes, the data presented here is consistent with that concept. Acknowledgments.-The work presented here mas supported by the Air Force Office of Scientific Research on Contract No. A F 49 (638)-1176.
