J. Biochem., 69, 255-263 (1971) The Elution Behaviors of Acidic Phospholipids on Column Chromatography* Tei SHIMOJO, Hideo KANOH and Kokichi OHNO The Department of Biochemistry, Sapporo Medical College, Sapporo Received for publication, January 6, 1969 1. Elution behavior of acidic phospholipids on silicic acid-, cellulose-, and Sephadex LH-20-column chromatography was investigated with special attention to the cations bound to phospholipids and to their purity, and the following results were obtained. 2. The binding cations are not a primary factor determining their elution behavior but rather cause a secondary effect, affecting micelle formation of phospholipids. 3. The difference in the chromatographic elution behavior between different cationic forms of acidic phospholipids seemed to be due to a difference in the stability and polarity of micelles. 4. Na and K-forms of isolated acidic phospholipids tend to form micelles which are smaller, more polar and, probably, less stable than those of their Ca or Mg-forms in organic solvents. 5. Acidic phospholipids with mixed cations in tissue extract are apt to form less stable or more polar micelles than those with a single species of cation. 6. The mixed cation form is readily converted to the Ca-form by treatment of tissue extracts with CaCl2 There have been reported differences in the behavior of acidic phospholipids on chro matography. On silicic acid column chro matography of animal tissue lipids, for ex ample, Rose (1) and Shimojo and Ohno (2) observed that cardiolipin of heart is eluted with a chloroform-methanol mixture of a volume ratio of 3: 1 to 3: 2 (designated as a "m ore polar" solvent in the present paper), while Gray and Macfarlane (3) reported that * This work w as supported in part by the scientific research grant from the Ministry of Education (091452). cardiolipin of liver is eluted with a chloroform - methanol mixture of 98 : 2 to 95 : 5 (designated as a "less polar" solvent). Similar disagree ment has been observed on elution of phos phatidylserine (4, 5) and phosphatidic acid ( (5,)) from a silicic acid column. It has been also known that some acidic phospho lipids are separated into two or more fractions on DEAE-cellulose column chromatography (9, 10). These irregularity and fluctuation in the chromatographic behavior of acidic phospholipids have made it difficult to isolate the acidic phospholipids quantitatively and to trace the radioactivity in the phospholipids. Vol. 69, ho. 2, 1971 255
256 T. SHIMOJO, H. KANOH and K. OHNO The present study was undertaken to search for the cause for the irregularity and fluctua tion. MATERIALS AND METHODS Acidic Phospholipids-Pig kidney lipids were extracted from 200 g of fresh tissue with a chloroform-methanol mixture (2: 1 by vol ume) and chromatographed on a DEAE-cel lulose column by the method of Rouser et at. ( 11). The mixed acidic phospholipids ob tained by the column chromatoraphy were separated into individual components by re peated chromatography on a silicic acid col umn. Each fraction of cardiolipin, phosphati dylinositol and phosphatidylserine thus ob tained gave a single spot upon thin-layer chromatography with Kieselgel G, and showed a purity more than 95% as determined by Dawson's paper chromatographic method (12). Cationic Forms of Acidic Phospholipids - An acidic phospholipid (less than 100 mg) dis solved in 30 ml of a chloroform-methanol mixture (2: 1) was passed through a column (5 cm x 1 cm) packed with Dowex 50(H+), and the column was washed with 10 ml of the same solvent. To the combined effluent, was added 8 ml of an aqueous salt solution (0.1 M CaCl2, 0.1 Mt MgCl2, 0.2 M NaHCO3, or 0.2,-,1 KHCO3). The mixture was centrifuged and the upper layer was discarded. The lower layer was washed three times with the salt solution. The lower layer was then passed through a small cellulose column (3 cm x 1 cm) in order to remove excess salts and the col umn was washed with 5 ml of a chloroform - methanol mixture (1: 1). The combined effluent was evaporated to dryness and the residue was dissolved in chloroform at a con centration of about 0.1 to 0.2%. Analysis of Cations-An aliquot of acidic phospholipids (less than 20 mg) dissolved in 15 ml of a chloroform-methanol mixture (2: 1) was passed through a Dowex 50(H+) column (5 cm x 0.5 cm). After washing with each 2 mi of methanol and water successively, the cat ion was eluted with 15 ml of 2 N HCl. The effluent was subjected to flame photometric TABLE I. Analytical data of various cationic forms of acidic phospholipids. J. Biochem.
CHROMATOGRAPHY OF ACIDIC PHOSPHOLIPIDS 257 analysis for Na, K, Ca and Mg with a Hitachi - Parking Elmer Spectrophotometer. Analytical data of the acidic phospholipids with various cations are shown in Table I. Phosphorous Assay-This was performed by Bartlett's method (13 ). Column Chromatography with Silicic Acid -About 8 to 20 mg of phospholipid was ap plied to a column (10 cm x 1 cm) packed with Mallinckrodt silicic acid (100 mesh), which had been activated at 110 C for 12 hr and washed with methanol to remove colloidal fine parti cles. Elution was carried out in a usual manner at a flow rate of 1 ml per minute. Column Chromatography with Cellulose - About 10 to 12 mg of phospholipid was applied to a column (15 cm x 1 cm) packed with cel lulose powder (300 mesh). Elution was carried out at a flow rate of 0.5 ml per minute by the method reported previously (14 ). Column Chromatography with Sephadex LH-20-A slurry of 90 g of Sephadex LH-20 in 500 ml of chloroform was poured into a tube of 3 cm in diameter and 60 cm in height. The Sephadex bed was pressed with a glass rod to obtain a column height of 45 cm. The lipids (less than 50 mg) dissolved in 10 ml of chloro form were applied to the column and the elution was carried out with 50 ml of chloro form and then with 350 ml of methanol, re spectively. Fractions of 10 ml each were collected at a flow rate of 5 ml per minute. The solvent front appeared at fraction 10 and methanol appeared fraction 31. Thin-Layer Chromatography-Kieselgel G (Merck) was spread on a plate as a thin layer, and a chloroform-methanol-water mixture of a volume ratio of 65: 25: 4 was used as a developing solvent. Spots were detected by spraying 5% phosphomolybdic acid in ethanol, followed by heating at 120 C for 30 min. similar, and most of them was eluted with the "less-polar" solvent (chloroform-methanol of 98: 2 or 95: 5). The Na and K-forms of cardiolipin were, however, eluted with the "m ore-polar" solvent (chloroform-methanal of 7 : 3), although a part of these forms appeared over a wide range of fractions. Elution patterns of phosphatidylinositol are shown in Fig. 2. The Ca and Mg-forms were eluted with the "less-polar" solvent (chloroform-methanol of 95: 5 to 8: 2), al though the peaks of these forms appeared at slightly different solvent ratios of 9 : 1 and 8 : 2, respectively. The Na and K-forms of this phospholipid behaved like those of cardiolipin, and appeared with the "more-polar" solvent. Elution behavior of phosphatidylserine is shown in Fig. 3. The Na and K-forms were mostly eluted with the "more-polar" solvent, although a considerable fraction of these con taining less Na or K was eluted in an inter- RESULTS Chromatographic Behavior of Isolated Acidic Phospholipids Silicic Acid Column Chro matography-elution patterns of cardiolipin with various cations on silicic acid column chromatography are shown in Fig. 1. The patterns of both Ca and Mg-forms were Fig. 1. Silicic acid column chromatography of cardiolipin. Column, 49 of Mallinckrodt silicic acid (10 cm by 1 cm) ; each fraction, 10 ml ; flow rate, 1 ml per minute. Vol. 69, No. 2, 1971
4. Elution 258 T. SHIMOJO, H. KANOH and K. OHNO mediate region between these solvents. The elution behaviors of the Ca and Na forms of phosphatic acid presented previously by Akino (7) in our group are similar to those of the above three acidic phospholipids. Especially their similarity to the Ca-form of cardiolipin and to the Na-form of phosphati dylserine was very close as shown in Fig. behavior of a mixture of acidic phospholipids obtained from pig kidney with ammonium, Na, and Ca cations are shown in Fig. 5. As examined as the Na or Ca-form, the elution peaks of cardiolipin, phosphati dylinositol and phosphatidylserine in the mix ture appeared at positions almost similar to those found for individual isolated phospho lipid in the Na or Ca-form. In the case of ammonium form, however, almost all of the acidic phospholipids in the mixture were eluted with the "more-polar" solvent. Cellulose Column Chromatography-As Fig. 3. Silicic acid column chromatography of phosphatidylserine. Conditions are the same as those described in Fig. 1. Fig. 2. Silicic acid column chromatography of phosphatidylinositol. Conditions are the same as those described in Fig. 1. Fig. 4. Silicic acid column chromatography of phosphatidic acid prepared from egg phosphatidyl choline. Column, 11 cm; each fraction, 100 ml; flow rate, 5 ml/min. (A), Ca-salt of phosphatidic acid ; (B), Na-salt of phosphatidic acid. J. Biochem.
CHROMATOGRAPHY OF ACIDIC PHOSPHOLIPIDS 259 shown in Figs. 6, 7, and 8, cardiolipin, phos phatidylinositol and phosphatidylserine in the Ca-form were eluted with the "less-polar" solvent (chloroform alone). However, the Na forms of these phospholipids were eluted over Fig. 5. Silicic acid column chromatography of mixed acidic phospholipids isolated by DEAE-cellulose column chromatography. Column, 20 cm by 4 cm ; each fraction, 500 ml ; flow rate, 10 ml/min ; phos pholipid applied, ca. 23 mg lipid-p. (A), NH,-salt; (B), Na-salt; (C), Ca-salt. Abbreviations: CL cardio lipin, PS phosphatidylserine, PI phosphatidylinositol. Fig. 7. Cellulose column chromatography of phos phatidylinositol. Column. 5 g of cellulose powder (Togo Roshi, 300 mesh), 15 cm by 1 cm, each fraction, 10 ml, flow rate 0.5 ml per minute. Fig. 6. Cellulose column chromatography of cardio lipin. Column, 13 g of cellulose powder (Toyo Roshi, 300 mesh), 10 cm by 2 cm; each fraction, 50 ml, flow rate, 2 ml/min. Fig. 8. Cellulose column chromatography of phos phatidylserine. Conditions are the same as those described in Fig. 7. Vol. 69, No, 2, 1971
260 T. SHIMOJO, H. KANOH and K. OHNO a wide range of fractions, while bulk was eluted either with the "less-polar" solvent or with the "more-polar" solvent. Sephadex LH-20 Column Chromatography -Elution patterns of a mixture of phosphati dylinositol and phosphatidylserine obtained on chromatography with Sephadex LH-20 are shown in Fig. 9. The Ca-forms of these lipids were eluted with the "less-polar" solvent (chloroform alone) at the fration 15, and the Na-forms of these with the "more-polar" solvent, at fraction 36. Elution pattern of this phospholipid mix ture with mixed cations of Ca and Na is shown in the lower part of the same figure.,,the two fractions obtained at number 15 and 36 contained both phosphatidylinositol and phosphatidylserine, but the former fraction contained their Ca-form and the latter their Na-form. Chromatographic Behavior of Acidic Phos pholipids in Crude Tissue Extract Silicic Acid Column Chromatography-It was reported in previous papers that most of acidic phospholipids in erythrocyte lipid extract (1 5) as well as those in heart lipid extract (2) were eluted with a "more-polar" solvent, a chloroform-methanol mixture of 8 : 2 to 5 : 5, by silicic acid column chromatography. On the other hand, these acidic phospholipids were eluted with a "less-polar" solvent after treatment of the crude extract with CaCl2 ac cording to the following procedure. To a crude extract of erythrocytes (50 ml) with an ethanol-ether mixture (3: 1) was added 50 Đ moles of CaCl2 containing 45Ca which is equivalent to about two-fold of the acidic Fig. 10. Silicic acid column chromatography of pig erythrocyte phospholipids treated with CaCl2. The solid line indicates amounts of phosphorous in frac tions and the dotted line indicates "Ca-activity. Phospholipid applied: 110 mg. Other details are in the text. Fig. 9. Sephadex LH-20 column chromatography of phosphatidylserine and phosphatidylinositol. Elution of a mixture of phosphatidylserine and phosphatidyl inositol in Ca-form (A), in Na-form (B), and in a mnixed-cationic form (C). Fig. 11. Thin-layer chromatography of the silicic acid column fractions (ref. Fig. 10). Details are in the text. Spots: I phosphatidylserine, 3 phos phatidylinositol, 4 phosphatidylcholine, 5 sphingo myelin, 6, phosphatidylethanolamine, 7 cardiolipin, 8 ceramide. J. Biochem.
CHROMATOGRAPHY OF ACIDIC PHOSPHOLIPIDS 261 Fig. 12. Sephadex LH-20 column chromatography of rat liver phospholipids. Phospholipids applied: ca. 30 mg, containing 98>' 101 cpm of '~ P. (A) Puri fied by Folch's procedure with water; (B) with 0.1 M CaCl phospholipid content, and the solvent was re moved. The residue was dissolved in chloro form and chromatographed on a silicic acid column. Most of the acidic phospholipids in the erythrocyte lipid extract as well as those in heart (2) were eluted with a "less-polar" solvent of chloroform-methanol 98: 2, 95: 5 or 9: 1 as shown in Figs. 10 and 11. A phos phatidylserine preparation was isolated from the fraction of 95: 5 by cellulose column chro matography. The binding-cations were mainly Ca (0.91 eq.) and, partly Mg (0.02 eq.). Sephadex LH-20 Column Chromatography -The liver lipid extract purified by Folch's washing procedure (16) with water was dis solved in dry chloroform and applied to a Sephadex column. All of the phospholipids were eluted with a "more-polar" solvent as a peak near fraction 36, as shown in Fig. 12(A). On the other hand, the lipids obtained by the similar procedure, excepting the use of 0.1 M CaCl2 instead of water in the Folch's washing procedure, showed different behavior. In this case most of the acidic phospholipids were eluted with a "less-polar" solvent and other non-acidic phospholipids, phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, were eluted with a "more-polar" solvent as shown in Fig. 12(B). Thin-layer chromatog- Fig. 13. Thin-layer chromatography of Sephadex column fractions (ref. Fig. 12-B). Detection, auto radiography ; Spots, CL cardiolipin, PI phosphatidyl inositol, PS phosphatidylserine, PA phosphatidic acid, PE phosphatidylethanolamine, PC phosphatidyl choline, SM sphingomyeline. Other details are in the text. raphy of these fractions are shown in Fig. 13. DISCUSSIONS Rose (1) has reported that the Na-content of cardiolipin eluted by silicic acid column chromatography with a chloroform-methanol mixture of 6: 4 was higher than that eluted with a mixture of 9: 1 and we have reported previously that there was a distinct difference in the elution behavior between the Na and Ca-forms of cardiolipin (2). A similar differ ence was reported by Akino (7) in our laboratory and recently by Renkonen (8) on the elution of phosphatidic acid. These ob servations indicated different elution patterns on column chromatography with silicic acid Vol. 69, No. 2, 1971
262 T. SHIMOJO, H. KANOH and K. OHNO of acidic phospholipids with different cations, and suggested that the differences in the be havior are caused by their binding cations. The results obtained with isolated phospho lipids in the present study demonstrated this effect of binding cations. It has been shown in privious work (17 ) that the phosphatidylserine preparations ob tained by silicic acid column chromatography of erythrocyte lipids with different solvents of 8: 2, 6: 4 and 5: 5 chloroform-methanol mix tures contained Ca and Mg. It was also shown by the same authors that the cardio lipin preparation (2) isolated from the "more - polar" fraction of silicic acid column chro matography of pig heart lipids contained also Mg, Na, and Ca although the Ca or Mg-forms of isolated cardiolipin appeared in the "less - polar" fraction (2), These results suggested that the binding cations of acidic phospho lipids are probably not a primary factor deter mining their elution behavior but rather cause a secondary effect on the behavior. It has been well known that many am phiphatic molecules form micelles in organic solvent such as benzene (18, 19). Lecithin micelles in organic solvents have been studied by Elworthy and McIntosh (20) who showed that the molecular weight of the micelle de pends on the polarity of the solvents; 57,000 in benzene, 7,100 in ethanol and 2,300 in methanol. We have suggested that the elution behavior of phospholipids on cellulose column chromatography depends on their micellar state (14 ). It was shown from the Sephadex column chromatography in the present study that the Ca-form of phosphatidylinositol and phospha tidylserine may form larger and more stable micelles than those of the Na-form. Acidic phospholipids binding Ca and Mg ions in a tissue extract were eluted with the "more - polar" solvent on silicic acid column chro matography. This suggests that the micelles of acidic phospholipids with mixed cations are smaller, more polar and less stable than those with a single species of cation. Appearance of acidic phospholipids in a wide range of fractions on column chromatography of tissue lipids may be due to the instability of the large micelles which might be degraded to smaller micelles during chromatographic pro cedures. After treatment of a crude tissue extract with CaCl2, acidic phospholipids were eluted with the "less-polar" solvent on silicic acid and. Sephadex LH-20 column chromatography. The binding cations of the lipids in this frac tion were mainly Ca. This indicates that the treatment with CaCl2 turned the acidic phos pholipids into their Ca-forms. Abramson et al. showed that aqueous dispersion of phos phatidylserine (21) and phosphatidic acid (22) bind bivalent cations such as Ca or Mg preferably to univalent cations and Shimojo and Ohno (2) showed that cardiolipin in or ganic solvents has a similar affinity for bivalent cations. It may therefore be con sidered that the binding cations in acidic phos pholipids can be replaced by excess Ca re gardless of their micellar state. The acidic phospholipids with the single species of Ca ion thus formed may have a property to form more stable and less polar micelles than with mixed bivalent or monovalent cations. Such stable micelles might be eluted with a "less - polar" solvent on column chromatography with silicic acid, Sephadex or cellulose. REFERENCES 1. H.G. Rose, Biochim. Biophys. Acta, 84,109 (1964). 2. T. Shimojo and K. Ohno, J. Biochem., 60, 462 (1966). 3. G.M. Gray and M.G. Macfarlane, Biochem. J., 70, 409 (1958). 4. D.J. Hanahan, J.C. Dittmer and E. Warashina, J. Biol. Chem., 228, 685 (1957). 5. P. Ways and D.J. Hanahan, J. Lipid Res., 5, 318 (1964). 6. G. Hubscher and B. Clark, Biochim. Biophys. Acta, 41, 45 (1960). 7. T. Akino, The Sapporo Medical J., 28, 162 (1965). 8. 0. Renkonen, Biochim. Biophys. Acta, 152, 114 (1968). 9. T.E. Morgan, D.O. Tinker and D.J. Hanahan, Arch. Biochem. Biophys., 103, 54 (1963). 10. H.S. Hendrickson and C.E. Ballou, J. Biol. Chem., 239, 1369 (1964). 11. G. Rouser, G. Kritckevsky, D. Heller and E. Lieber, J. Am. Oil Chemist's Soc., 40, 425 (1963). 12. R.M.C. Dawson, Biochem. J., 75, 45 (1960). J. Biochem,
CHROMATOGRAPHY OF ACIDIC PHOSPHOLIPIDS 263 13. G.R. Bartlett, J. Biol. Chem., 234, 466 (1959). 14. T. Shimojo, T. Orii, H. Yamaguchi and K. Ohno, J. Biochem., 59, 1 (1966). 15. T. Shimojo, A. Kataura, H. Yamaguchi and K. Ohno, The Sapporo Medical J., 28, 85 (1965). 16. J. Folch, M. Lees and G.H.S_ Stanley, J. Biol. Chem., 226, 497 (1957). 17. T. Shimojo and K. Ohno, J. Biochem., 60, 228 (1966). 18. P.H. Elworthy, J. Chem. Soc., 1951 (1959). 19, P.H. Elworthy, J. Am. Oil Chemist's Soc., 42, 451 (1965). 20. P.H. Elworthy and D.S. McIntosh, J. Pharm. Pharmacol., 13, 663 (1961). 21. M.B. Abramson, R. Katzman, C.E. Wilson and H.P. Gregor, J. Biol. Chem., 239, 4066 (1964). 22. M.B. Abramson, R. Katzman and H.P. Gregor, J. Biol. Chem., 239, 70 (1964). Vol. 69, No. 2, 1971