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Purification of the acid form of phosphatidylserine from beef brain by countercurrent distribution
ARCHIVES
OF
Purification
BIOCHEMISTRY
AND
of the Acid
BIOPHYSICS
89,
Form
91-96 (1960)
of Phosphatidylserine
by Countercurrent DONALD
THERRIAULT,
from
Beef Brain
Distribution’
THOMAS
NICHOLS
AND
H. JENSEN2
From the Biochemistry Division, United States Army Medical Research Laboratory, Fort Knox, Kentucky, and Department of Biochemistry, University of Louisville School of Medicine, Received
Louisville, December
Kentucky 28, 1959
The acid form of phosphatidylserine has been isolated state by means of countercurrent distribution.
from beef brain
in a pure
finding that phosphatidylserine is one of the phospholipids from brain which, when mixed with lecithin, yields a preparation with procoagulant activity.
INTRODUCTION
Earlier work from this laboratory dealing with the purification and identification of brain phospholipids associated with procoagulant activity3 indicated that a phosphatidylserine fraction can be obtained from beef brain tissue which by itself has little or no procoagulant activity but which on combination with lecithin results in a preparation possessing pronounced procoagulant potency (1). Although the phosphatidylserine fraction examined in the earlier study was not pure, the evidence obtained indicated that phosphatidylserine was the factor which, in combination with lecithin, was responsible for the procoagulant activity. In this paper, a method is described for obtaining the acid form of phosphatidylserine from beef brain in a pure state. Evidence is also presented substantiating the earlier
EXPERIMENTAL
FRACTIONATION BY COUNTERCURRENT DISTRIBUTION Folch’s Fraction III, obtained from fresh beef brain (2), was used as the starting material for further purification by countercurrent distribution as reported previously (1). An all-glass, Craig lOO-cell countercurrent distribution apparatus was used. The solvent system consisted of carbon tetrachloride-methanol-water in the ratio of 62:35:4 by volume. The content of the individual tubes was measured in one of two ways.
1. TOTAL WEIGHT DETERMINATION The entire content of the individual tubes was transferred to small aluminum dishes which had been predried to constant weight in a vacuum desiccator. The solvent was evaporated by means of infrared lamps and the dishes were placed in a vacuum desiccator and dried to constant weight. The difference in weight is reported as total weight in mg.
1 This work is based upon a portion of a thesis submitted by D. Therriault to the Graduate School of the University of Louisville in partial fulfillment for the requirements for the Ph.D. degree. * Deceased, September 30, 1959. 3 The terms “thromboplastin,” “thromboplastin-like,” and “partial thromboplastin” have been used for such active phospholipid preparations. Inasmuch as thromboplastin, as presently used, does not have a specific meaning, the term procoagulant is proposed for a phospholipid preparation which on interaction with certain clotting factors in the presence of calcium ions yields a prothrombin activator.
2. PHOSPHORUS DETERMINATION Two milliliters of the solution (1 ml. from each phase) collected from the various tubes were transferred to Pyrex test tubes. The solvent was evaporated in a hot water bath using a glass bead in each tube to prevent bumping. After addition of 1 ml. of 7Oye perchloric acid and 5 drops of cont. nitric acid, the tubes were heated 91
92
THERRIAULT,
NICHOLS AND JENSEN
for a period of 2 hr. or until the solutions became colorless. Aliquots from these solutions were removed for the phosphorus determination by a modification of the method developed by Chen, Toribara, and Warner (3). Aliquots were made up to 1 ml. with 70% perchloric acid; 7 ml. of distilled water was added, followed by 1 ml. of 2.5% ammonium molybdate. The contents of the tubes were mixed, and 1 ml. of 10% ascorbic acid was added; after mixing, the color was developed by heating at 70°C. for 10 min. The optical density was determined in a Beckman model DU spectrophotometer at 820 rn+ On the basis of these values, distribution curves of the lipid phosphorus were plotted which agreed well with the curves obtained by weight analysis.
CHEMICAL
AND CHROMATOGRAPHIC ANALYSES
In order to identify and characterize the phospholipid fractions obtained by countercurrent distribution, the individual fractions were chromatographed at 0°C. according to the method of Marinetti et al. (4) on silicic acid-impregnated paper with a solvent mixture of diisobutyl ketone-acetic acid-water 40:20:3 by volume. The chromatograms were dipped into a O.OOl$& aqueous solution of Rhodamine 6 G (color index 752), examined under ultraviolet light while still wet, then allowed to dry and again examined under ultraviolet light. The lipids appeared as blue or yellow fluorescent spots (5). Lipids which contain a free amino group were detected by spraying the chromatograms with a 0.3% solution of ninhydrin in n-butanol saturated with water and containing 10% lutidine. After being kept in subdued light at room temperature for approximately 30-60 min., a blue color developed in areas containing primary amino groups (ethanolamine and serine). Choline-containing lipids were identified by the phosphomolybdic acid method (6). Acetal phosphatides were determined by Schiff’s reagent or with 2,4-dinitrophenylhydrazine (4). The determination of the nitrogenous phospholipid bases was carried out on acid hydrolyzates according to the method of Marinetti and his associates (7).
PREPARATION OF THE ACID FORM OF PHOSPHATIDYLSERINE Phosphatidylserine was obtained in the acid form by acid precipitation as described by Folch (8). Three grams of the phospholipid preparation were emulsified with 150 ml. water, and 15 ml. of N HCl was added to the emulsion. The preobtained after centrifugation, cipitate, was
washed distilled
with 0.05 N HCl, then water and lyophilized.
dialyzed
against
DETERMINATIONOFPROCOAGULANT ACTIVITY The assay method used to determine procoagulant activity was similar to that described in an earlier paper (9). A reaction mixture was prepared containing the various clotting factors, and the amount of thrombin formed at specified time intervals was determined by adding a measured amount of the reaction mixture to a standard fibrinogen solution. The reagents were prepared in the same manner as described previously (9) with the exception of the antihemophilic factor (Factor VIII) and plasma accelerator-globulin (Factor V) preparations. These factors were substituted by a preparation obtained in the following manner: Eight hundred milliliters of fresh oxalated beef plasma was stirred for 30 min. with BaS04 (Baker’s; 100 mg./ml.). The supernatant solution obtained after centrifugation was diluted 1:20 with deionized water at 4”C., and the pH was adjusted to 5.3 by the addition of 1% acetic acid, allowed to stand overnight at 4”C., and then centrifuged. The precipitate was extracted at -3°C. with 250 ml. of 1 A4 glycine in citrate buffer (pH 6.0, ionic strength 0.3) containing 6.5% ethanol. The extract was dialyzed against 0.85yo NaCl, and the NaCl solution was divided into aliquots and kept in the frozen state. These stock solutions were thawed and diluted 1:5 with 0.85% NaCl solution immediately before use. The antihemophilic factor (Factor VIII) and plasma accelerator-globulin (Factor V)-containing preparation will be referred to as “plasma extract.” RESULTS
FRACTIONATION AND CHARACTERIZATION
Countercurrent distribution of Folch’s Fraction III resulted in the heterogenous distribution curve shown in Fig. 1. Data presented in a previous publication (1) indicated that Folch’s Fraction III contained, in addition to phosphatidylserine, several other phospholipids including phosphatidylethlysophosphatidylethanolamine, anolamine, inositol phosphatide and another lipid which may be polyglycerol phosphatide. Although fractionation by countercurrent distribution did not result in a complete separation of the individual components of the mixture, paper chromatographic analysis (Fig. 2B) indicated that this procedure yielded a fraction
COUNTERCURRENT
DISTRIBUTION
OF
93
PHOSPHATIDYLSERINE
TUBE NUMBER FIG.
1. Countercurrent
distribution
of Folch’s Fraction
III.
0. 0 0. 7 0.6
!LJ 0.5 a >
0.4
2
.O6 5 8
-
4
0. 3
.o
0.2
-0
0 5
-
0 5
3 2
0. 1
-0
2
-0
-
0
-
2
A
n ”
l-7
A
B
c
0 *v D
FIG. 2. Diagrammatic representation of the phospholipid chromatograms. The diagrams represent tracings of the original chromatograms. Chromatography was carried out on silicic acid-impregnated paper. The identification of the lipid spots was as follows: 1) unidentified; 2) inositol phosphatide; 3) sphingomyelin; 4) lecithin; 5) phosphatidylserine; and 6) phosphatidylethanolamine. A: Standard mixture. B: Tubes 65-160, Folch’s Fraction III (Fig. 1). C: Tubes 2&45, free acid preparation; after additional countercurrent distribution (Fig. 3). D: Tubes 75166, free acid preparation; after additional countercurrent distribution (Fig. 3).
94
THERRIAULT,
NICHOLS AND JENSEN
6
4
12
20
28
36
44
52
60
68
76
84
92
100
TUBE NUMBER FIG.
3. Countercurrent
distribution
containing phosphatidylserine contaminated by only two other components, namely inositol phosphatide and an unidentified phospholipid. The components of low partition coefficients were apparently removed from the original Fraction III preparation. Acid hydrolysis of the phospholipid material contained in tubes 65-100 (Fig. 1) and analysis of the water- and chloroform-soluble fractions by paper chromatography (7) showed that the only nitrogen base in this preparation was serine; choline, ethanolamine, and sphingosine were not detected. The material obtained from tubes 65-100 (Fig. 1) gave a positive test for sodium. However, following precipitation with acid from an aqueous emulsion, the flame test for sodium was negative. The acid-treated material was fractionated by countercurrent distribution in the same manner as described above, and the distribution curve is given in Fig. 3. Chromatographic analyses of the material contained in the tubes which represent the two major peaks are shown in Fig. 2 (C and 0). It can be seen that the fractionation resulted in the separation of inositol phosphatide and of the unidentified phospholipid from the phosphatidylserine.
of free
acid preparation.
Results of the chemical analysis4 of phosphatidylserine, isolated from tubes 20-45 (Fig. 3), agree closely with the values calculated for oleylstearylglycerylphosphorylserine. Anal. Calcd for C42H30010NP: C 63.9, H 10.1, N 1.78, P 3.92, N/P 1.00. Found: C 64.5, H 9.6, N 1.82, P 3.89, N/P 1.04. The negative aldehyde test indicated the absence of any acetal type phosphatidylserine in the preparation. All of the nitrogen could be accounted for as serine aminonitrogen. The preparation was found to be completely free of any choline- or ethanolaminecontaining phospholipids. PROCOAGULANT ACTIVITY
The results of the procoagulant activity determinations are shown in Fig. 4. The purified phosphatidylserine fraction, when mixed with pure beef brain lecithin (l), exhibited pronounced procoagulant activity. Whereas the control reaction mixture, in which saline was substituted for the phos4 Carbon and hydrogen analyses were carried out by Clark Microanalytical Laboratory, Urbana, Illinois. The nitrogen determinations were made by micro-Kjeldahl method.
COUNTERCURRENT
DISTRIBUTION
OF
phatidylserine-lecithin, showed less than 5% conversion at the end of 60 min., the reaction mixture containing phosphatidylserine-lecithin caused the complete conversion of prothrombin to thrombin in less than 15 min. In reaction mixtures containing either phosphatidylserine or lecithin alone, only traces of t#hrombin could be detected.
PHOSPHATIDYLSERINE
95
100 -I
DISCUSSION
The procedure of countercurrent distribution makes use of the slight differences in solubility of various components of a mixture. Since the sodium and potassium salts of phosphatidylserine exhibit solubility characteristics different from those of the acid form, countercurrent distribution seems to offer a convenient method of separating phosphatidylserine from contaminating substances. Since the partition coefficient of the salt form of phosphatidylserine is high, all contaminant’s having lower partition coefficients can be removed. On the other hand, the partition coefficient of the acid form is low and, therefore, the remaining contaminants of high partition coefficients can be separated. The high partition coefficient of the salt form, as compared to that of the acid form, indicates that the salt form is more soluble in the phase of higher solvent polarity whereas the acid form is more soluble in the relatively nonpolar phase. This is in good agreement with the results of Marinetti et al. (10) who found that the elution of the sodium salt of phosphatidylserine from a silicic acid column required a more polar solvent. The present results indicate that the phosphatidylserine in Folch’s Fraction III is present mainly as the salt. Marinetti et aZ.‘s results, on the other hand, seem to indicate that their phosphatidylserine preparation was a mixture of the sodium salt and acid form. The present findings seem to be more compatible with those of Folch (8) who reported a ratio (equivalence of base/ atoms of phosphorus) of 1.00 for the phosphatidylserine isolated from brain. Comparison of the data of the elemental analysis of the purified phosphatidylserine oleylstearylglycerylphosphorylserine, with the structure postulated by Folch (8), shows very good agreement. However, the compo-
g
20
10 i/
/
4
6
8 TIME
10
12
I4
16
MINUTES
FIG. 4. Rate of conversion of prothrombin to thrombin. The reaction mixture consisted of the following reactants: 0.1 ml. of a purified preparation obtained from a BaS04 eluate of rabbit serum [containing proconvertin (Factor VII), plasma thromboplastin component (Factor IX), as well as Stuart factor], 0.1 ml. of phosphatidylserine-lecithin (0.025 mg./ml.), 0.1 ml. of plasma extract [containing antihemophilic factor (Factor VIII) and plasma accelerator-globulin (Factor V)l, and 0.1 ml. CaClz (0.025 M). After 10 min. incubation at 37”C., 0.2 ml. prothrombin (1000 units/ml.) and 0.1 ml. of CaClz (0.025 M) were added. At time intervals indicated, 0.1 ml. of the reaction mixture was added to 0.4 ml. of the fibrinogen solution at 37°C.
nent fatty acids of this phospholipid have not been isolated and characterized as yet. Although the method of countercurrent distribution may not be adaptable to the separation of complex phospholipid mixtures into their component parts, it offers a valuable tool for the purification of individual phospholipids. From the present observations, it appears that phospholipids which are first fractionated by other methods, such as column chromatography (ll), may lend themselves very well to additional purification by means of countercurrent distribution. Examination of the procoagulant activity of the pure acid of phosphatidylserine substantiated earlier findings obtained with a
96
THERRIAULT,
NICHOLS
partially purified preparation (1). Although phosphatidylserine alone was comparatively inactive, when mixed with lecithin it was found to exert a pronounced procoagulant activity. REFERENCES 1. THERRIAULT, D., NICHOLS, T., AND JENSEN, H., J. Biol. Chem. 233, 1061 (1958). 2. FOLCH, J., J. Biol. Chem. 146, 35 (1942). 3. &EN, P. S., TORIBARA, T. Y., AND WARNER, H., Anal. Chem. 28, 1756 (1956). 4. MARINETTI, G. V., ERBLAND, J., AND KOCHEN, J., Federation PTOC. 16, 837 (1957).
AND
JENSEN
5. ROUSER, G., MARINETTI, G. V., WITTER, R. F., BERRY, J. F., AND STOTZ, E., J. Biol. Chem. 223, 485 (1956). 6. CHARGAFF, E., LEVINE, C., AND GREEN, C., J. Biol. Chem. 176, 67 (1948). 7. MARINETTI, G. V., S~ARAMUZZINO, D. J., AND STOTZ, E., J. Biol. Chem. 224, 819 (1957). 8. FOLCH, J., J. Biol. Chem. 174, 439 (1948). 9. THERRIAULT, D. G., GRAY, J. L., AND JENSEN, H., Proc. Sot. Ezptl. Biol. Med. 96,207 (1957). 10. MARINETTI, G. V., ERBLAND, J., AND STOTZ, E., Biochim. et Biophys. Acta 90, 40 (1958). 11. RHODES, D. N., Chem. & Ind. (London) 13, 1010 (1956).
OF
Purification
BIOCHEMISTRY
AND
of the Acid
BIOPHYSICS
89,
Form
91-96 (1960)
of Phosphatidylserine
by Countercurrent DONALD
THERRIAULT,
from
Beef Brain
Distribution’
THOMAS
NICHOLS
AND
H. JENSEN2
From the Biochemistry Division, United States Army Medical Research Laboratory, Fort Knox, Kentucky, and Department of Biochemistry, University of Louisville School of Medicine, Received
Louisville, December
Kentucky 28, 1959
The acid form of phosphatidylserine has been isolated state by means of countercurrent distribution.
from beef brain
in a pure
finding that phosphatidylserine is one of the phospholipids from brain which, when mixed with lecithin, yields a preparation with procoagulant activity.
INTRODUCTION
Earlier work from this laboratory dealing with the purification and identification of brain phospholipids associated with procoagulant activity3 indicated that a phosphatidylserine fraction can be obtained from beef brain tissue which by itself has little or no procoagulant activity but which on combination with lecithin results in a preparation possessing pronounced procoagulant potency (1). Although the phosphatidylserine fraction examined in the earlier study was not pure, the evidence obtained indicated that phosphatidylserine was the factor which, in combination with lecithin, was responsible for the procoagulant activity. In this paper, a method is described for obtaining the acid form of phosphatidylserine from beef brain in a pure state. Evidence is also presented substantiating the earlier
EXPERIMENTAL
FRACTIONATION BY COUNTERCURRENT DISTRIBUTION Folch’s Fraction III, obtained from fresh beef brain (2), was used as the starting material for further purification by countercurrent distribution as reported previously (1). An all-glass, Craig lOO-cell countercurrent distribution apparatus was used. The solvent system consisted of carbon tetrachloride-methanol-water in the ratio of 62:35:4 by volume. The content of the individual tubes was measured in one of two ways.
1. TOTAL WEIGHT DETERMINATION The entire content of the individual tubes was transferred to small aluminum dishes which had been predried to constant weight in a vacuum desiccator. The solvent was evaporated by means of infrared lamps and the dishes were placed in a vacuum desiccator and dried to constant weight. The difference in weight is reported as total weight in mg.
1 This work is based upon a portion of a thesis submitted by D. Therriault to the Graduate School of the University of Louisville in partial fulfillment for the requirements for the Ph.D. degree. * Deceased, September 30, 1959. 3 The terms “thromboplastin,” “thromboplastin-like,” and “partial thromboplastin” have been used for such active phospholipid preparations. Inasmuch as thromboplastin, as presently used, does not have a specific meaning, the term procoagulant is proposed for a phospholipid preparation which on interaction with certain clotting factors in the presence of calcium ions yields a prothrombin activator.
2. PHOSPHORUS DETERMINATION Two milliliters of the solution (1 ml. from each phase) collected from the various tubes were transferred to Pyrex test tubes. The solvent was evaporated in a hot water bath using a glass bead in each tube to prevent bumping. After addition of 1 ml. of 7Oye perchloric acid and 5 drops of cont. nitric acid, the tubes were heated 91
92
THERRIAULT,
NICHOLS AND JENSEN
for a period of 2 hr. or until the solutions became colorless. Aliquots from these solutions were removed for the phosphorus determination by a modification of the method developed by Chen, Toribara, and Warner (3). Aliquots were made up to 1 ml. with 70% perchloric acid; 7 ml. of distilled water was added, followed by 1 ml. of 2.5% ammonium molybdate. The contents of the tubes were mixed, and 1 ml. of 10% ascorbic acid was added; after mixing, the color was developed by heating at 70°C. for 10 min. The optical density was determined in a Beckman model DU spectrophotometer at 820 rn+ On the basis of these values, distribution curves of the lipid phosphorus were plotted which agreed well with the curves obtained by weight analysis.
CHEMICAL
AND CHROMATOGRAPHIC ANALYSES
In order to identify and characterize the phospholipid fractions obtained by countercurrent distribution, the individual fractions were chromatographed at 0°C. according to the method of Marinetti et al. (4) on silicic acid-impregnated paper with a solvent mixture of diisobutyl ketone-acetic acid-water 40:20:3 by volume. The chromatograms were dipped into a O.OOl$& aqueous solution of Rhodamine 6 G (color index 752), examined under ultraviolet light while still wet, then allowed to dry and again examined under ultraviolet light. The lipids appeared as blue or yellow fluorescent spots (5). Lipids which contain a free amino group were detected by spraying the chromatograms with a 0.3% solution of ninhydrin in n-butanol saturated with water and containing 10% lutidine. After being kept in subdued light at room temperature for approximately 30-60 min., a blue color developed in areas containing primary amino groups (ethanolamine and serine). Choline-containing lipids were identified by the phosphomolybdic acid method (6). Acetal phosphatides were determined by Schiff’s reagent or with 2,4-dinitrophenylhydrazine (4). The determination of the nitrogenous phospholipid bases was carried out on acid hydrolyzates according to the method of Marinetti and his associates (7).
PREPARATION OF THE ACID FORM OF PHOSPHATIDYLSERINE Phosphatidylserine was obtained in the acid form by acid precipitation as described by Folch (8). Three grams of the phospholipid preparation were emulsified with 150 ml. water, and 15 ml. of N HCl was added to the emulsion. The preobtained after centrifugation, cipitate, was
washed distilled
with 0.05 N HCl, then water and lyophilized.
dialyzed
against
DETERMINATIONOFPROCOAGULANT ACTIVITY The assay method used to determine procoagulant activity was similar to that described in an earlier paper (9). A reaction mixture was prepared containing the various clotting factors, and the amount of thrombin formed at specified time intervals was determined by adding a measured amount of the reaction mixture to a standard fibrinogen solution. The reagents were prepared in the same manner as described previously (9) with the exception of the antihemophilic factor (Factor VIII) and plasma accelerator-globulin (Factor V) preparations. These factors were substituted by a preparation obtained in the following manner: Eight hundred milliliters of fresh oxalated beef plasma was stirred for 30 min. with BaS04 (Baker’s; 100 mg./ml.). The supernatant solution obtained after centrifugation was diluted 1:20 with deionized water at 4”C., and the pH was adjusted to 5.3 by the addition of 1% acetic acid, allowed to stand overnight at 4”C., and then centrifuged. The precipitate was extracted at -3°C. with 250 ml. of 1 A4 glycine in citrate buffer (pH 6.0, ionic strength 0.3) containing 6.5% ethanol. The extract was dialyzed against 0.85yo NaCl, and the NaCl solution was divided into aliquots and kept in the frozen state. These stock solutions were thawed and diluted 1:5 with 0.85% NaCl solution immediately before use. The antihemophilic factor (Factor VIII) and plasma accelerator-globulin (Factor V)-containing preparation will be referred to as “plasma extract.” RESULTS
FRACTIONATION AND CHARACTERIZATION
Countercurrent distribution of Folch’s Fraction III resulted in the heterogenous distribution curve shown in Fig. 1. Data presented in a previous publication (1) indicated that Folch’s Fraction III contained, in addition to phosphatidylserine, several other phospholipids including phosphatidylethlysophosphatidylethanolamine, anolamine, inositol phosphatide and another lipid which may be polyglycerol phosphatide. Although fractionation by countercurrent distribution did not result in a complete separation of the individual components of the mixture, paper chromatographic analysis (Fig. 2B) indicated that this procedure yielded a fraction
COUNTERCURRENT
DISTRIBUTION
OF
93
PHOSPHATIDYLSERINE
TUBE NUMBER FIG.
1. Countercurrent
distribution
of Folch’s Fraction
III.
0. 0 0. 7 0.6
!LJ 0.5 a >
0.4
2
.O6 5 8
-
4
0. 3
.o
0.2
-0
0 5
-
0 5
3 2
0. 1
-0
2
-0
-
0
-
2
A
n ”
l-7
A
B
c
0 *v D
FIG. 2. Diagrammatic representation of the phospholipid chromatograms. The diagrams represent tracings of the original chromatograms. Chromatography was carried out on silicic acid-impregnated paper. The identification of the lipid spots was as follows: 1) unidentified; 2) inositol phosphatide; 3) sphingomyelin; 4) lecithin; 5) phosphatidylserine; and 6) phosphatidylethanolamine. A: Standard mixture. B: Tubes 65-160, Folch’s Fraction III (Fig. 1). C: Tubes 2&45, free acid preparation; after additional countercurrent distribution (Fig. 3). D: Tubes 75166, free acid preparation; after additional countercurrent distribution (Fig. 3).
94
THERRIAULT,
NICHOLS AND JENSEN
6
4
12
20
28
36
44
52
60
68
76
84
92
100
TUBE NUMBER FIG.
3. Countercurrent
distribution
containing phosphatidylserine contaminated by only two other components, namely inositol phosphatide and an unidentified phospholipid. The components of low partition coefficients were apparently removed from the original Fraction III preparation. Acid hydrolysis of the phospholipid material contained in tubes 65-100 (Fig. 1) and analysis of the water- and chloroform-soluble fractions by paper chromatography (7) showed that the only nitrogen base in this preparation was serine; choline, ethanolamine, and sphingosine were not detected. The material obtained from tubes 65-100 (Fig. 1) gave a positive test for sodium. However, following precipitation with acid from an aqueous emulsion, the flame test for sodium was negative. The acid-treated material was fractionated by countercurrent distribution in the same manner as described above, and the distribution curve is given in Fig. 3. Chromatographic analyses of the material contained in the tubes which represent the two major peaks are shown in Fig. 2 (C and 0). It can be seen that the fractionation resulted in the separation of inositol phosphatide and of the unidentified phospholipid from the phosphatidylserine.
of free
acid preparation.
Results of the chemical analysis4 of phosphatidylserine, isolated from tubes 20-45 (Fig. 3), agree closely with the values calculated for oleylstearylglycerylphosphorylserine. Anal. Calcd for C42H30010NP: C 63.9, H 10.1, N 1.78, P 3.92, N/P 1.00. Found: C 64.5, H 9.6, N 1.82, P 3.89, N/P 1.04. The negative aldehyde test indicated the absence of any acetal type phosphatidylserine in the preparation. All of the nitrogen could be accounted for as serine aminonitrogen. The preparation was found to be completely free of any choline- or ethanolaminecontaining phospholipids. PROCOAGULANT ACTIVITY
The results of the procoagulant activity determinations are shown in Fig. 4. The purified phosphatidylserine fraction, when mixed with pure beef brain lecithin (l), exhibited pronounced procoagulant activity. Whereas the control reaction mixture, in which saline was substituted for the phos4 Carbon and hydrogen analyses were carried out by Clark Microanalytical Laboratory, Urbana, Illinois. The nitrogen determinations were made by micro-Kjeldahl method.
COUNTERCURRENT
DISTRIBUTION
OF
phatidylserine-lecithin, showed less than 5% conversion at the end of 60 min., the reaction mixture containing phosphatidylserine-lecithin caused the complete conversion of prothrombin to thrombin in less than 15 min. In reaction mixtures containing either phosphatidylserine or lecithin alone, only traces of t#hrombin could be detected.
PHOSPHATIDYLSERINE
95
100 -I
DISCUSSION
The procedure of countercurrent distribution makes use of the slight differences in solubility of various components of a mixture. Since the sodium and potassium salts of phosphatidylserine exhibit solubility characteristics different from those of the acid form, countercurrent distribution seems to offer a convenient method of separating phosphatidylserine from contaminating substances. Since the partition coefficient of the salt form of phosphatidylserine is high, all contaminant’s having lower partition coefficients can be removed. On the other hand, the partition coefficient of the acid form is low and, therefore, the remaining contaminants of high partition coefficients can be separated. The high partition coefficient of the salt form, as compared to that of the acid form, indicates that the salt form is more soluble in the phase of higher solvent polarity whereas the acid form is more soluble in the relatively nonpolar phase. This is in good agreement with the results of Marinetti et al. (10) who found that the elution of the sodium salt of phosphatidylserine from a silicic acid column required a more polar solvent. The present results indicate that the phosphatidylserine in Folch’s Fraction III is present mainly as the salt. Marinetti et aZ.‘s results, on the other hand, seem to indicate that their phosphatidylserine preparation was a mixture of the sodium salt and acid form. The present findings seem to be more compatible with those of Folch (8) who reported a ratio (equivalence of base/ atoms of phosphorus) of 1.00 for the phosphatidylserine isolated from brain. Comparison of the data of the elemental analysis of the purified phosphatidylserine oleylstearylglycerylphosphorylserine, with the structure postulated by Folch (8), shows very good agreement. However, the compo-
g
20
10 i/
/
4
6
8 TIME
10
12
I4
16
MINUTES
FIG. 4. Rate of conversion of prothrombin to thrombin. The reaction mixture consisted of the following reactants: 0.1 ml. of a purified preparation obtained from a BaS04 eluate of rabbit serum [containing proconvertin (Factor VII), plasma thromboplastin component (Factor IX), as well as Stuart factor], 0.1 ml. of phosphatidylserine-lecithin (0.025 mg./ml.), 0.1 ml. of plasma extract [containing antihemophilic factor (Factor VIII) and plasma accelerator-globulin (Factor V)l, and 0.1 ml. CaClz (0.025 M). After 10 min. incubation at 37”C., 0.2 ml. prothrombin (1000 units/ml.) and 0.1 ml. of CaClz (0.025 M) were added. At time intervals indicated, 0.1 ml. of the reaction mixture was added to 0.4 ml. of the fibrinogen solution at 37°C.
nent fatty acids of this phospholipid have not been isolated and characterized as yet. Although the method of countercurrent distribution may not be adaptable to the separation of complex phospholipid mixtures into their component parts, it offers a valuable tool for the purification of individual phospholipids. From the present observations, it appears that phospholipids which are first fractionated by other methods, such as column chromatography (ll), may lend themselves very well to additional purification by means of countercurrent distribution. Examination of the procoagulant activity of the pure acid of phosphatidylserine substantiated earlier findings obtained with a
96
THERRIAULT,
NICHOLS
partially purified preparation (1). Although phosphatidylserine alone was comparatively inactive, when mixed with lecithin it was found to exert a pronounced procoagulant activity. REFERENCES 1. THERRIAULT, D., NICHOLS, T., AND JENSEN, H., J. Biol. Chem. 233, 1061 (1958). 2. FOLCH, J., J. Biol. Chem. 146, 35 (1942). 3. &EN, P. S., TORIBARA, T. Y., AND WARNER, H., Anal. Chem. 28, 1756 (1956). 4. MARINETTI, G. V., ERBLAND, J., AND KOCHEN, J., Federation PTOC. 16, 837 (1957).
AND
JENSEN
5. ROUSER, G., MARINETTI, G. V., WITTER, R. F., BERRY, J. F., AND STOTZ, E., J. Biol. Chem. 223, 485 (1956). 6. CHARGAFF, E., LEVINE, C., AND GREEN, C., J. Biol. Chem. 176, 67 (1948). 7. MARINETTI, G. V., S~ARAMUZZINO, D. J., AND STOTZ, E., J. Biol. Chem. 224, 819 (1957). 8. FOLCH, J., J. Biol. Chem. 174, 439 (1948). 9. THERRIAULT, D. G., GRAY, J. L., AND JENSEN, H., Proc. Sot. Ezptl. Biol. Med. 96,207 (1957). 10. MARINETTI, G. V., ERBLAND, J., AND STOTZ, E., Biochim. et Biophys. Acta 90, 40 (1958). 11. RHODES, D. N., Chem. & Ind. (London) 13, 1010 (1956).