THE CHEMICAL STRUCTURE OF PHOSPHATIDYL SERINE

BY JORDI FOLCH*

(From the Hospital of The Rockefeller Institute for Medical Research, New York, the McLean Hospital Research Laboratories, Waverley, Massachusetts, and the Department of Biological Chemistry, Harvard Medical School, Boston)

(Received for publication, December 20, 1947)

Phosphatidyl serine is the name given to a phosphatide which has been isolated from brain (1, 2). This paper reports work according to which the chemical structure of phosphatidyl serine, prepared by the method described, corresponds to one of the two accompanying formulas in which the position of the fatty acids and phosphoryl radicals is arbitrary, since there is as yet no reliable way of ascertaining whether glycerophosphoric acid in phosphatides belongs to the (Y or the /3 form (3). The name of phosphatidyl serine was chosen because this compound appears to be an ester of serine and a phosphat.idic acid (4).

HZ-0-CO--CiTHa

H&O-PO-0-CHo-CH-COOH I I

OH NHn rr-Phosphatidyl serine

HzC-0-CO--ClrHs I

HC-0---------PO-0-CHS-CH-COOH

I I I

OH NH, H&-O-CO-C~~HI~

fl-Phosphatidyl serine

The postulated formula is based on the following facts: (1) Chemical analyses of phosphatidyl serine (freed of base) for C, H, N, P, carboxyl N, NHz-N, and fatty acids agree very closely with the theoretical values calculated from the postulated formula. (2) Glycerophosphoric acid, n-serine, and fatty acids have been isolated as cleavage products of phos- phatidyl serine in molecular proportions approximately 1: 1: 2. (3) Phos- phatidyl serine reacts with ninhydrin (5) and with HNOB (6) in the same way as an a-amino acid. This shows that both the -COOH and the --NH2 groups of serine are free in the intact molecule of phosphatidyl serine.

* Present address, McLean Hospital, Waverley, Massachusetts.

439

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

440 PHOSPHATIDYL SERINE

On the other hand, phosphatidyl serine does not react with HIO,. Since HI04 is known to react with compounds with a -CH(NH2)-CH(OH)- group, and specifically with serine (7), the fact that phosphatidyl serine does not react with HI04 is conclusive evidence that either its -OH or its -NH2 group is combined. And since its -NH2 group is shown to be free both by the ninhydrin (5) and the HMO2 reaction (B), it is obvious that the -OH group is combined. (4) From the postulated formula, phosphatidyl serine exhibits one free basic group, namely the -NH2 group, and two acidic groups, namely the -COOH group of serine and one group from phosphoric acid. This should give a strongly acidic compound that would bind 1 equivalent of base at the physiological pH. That this is the case is shown by the fact that phosphatidyl serine, isolated from brain by the use of neutral solvents and freed of water-soluble im- purities by dialysis, contains K and Na, and that the ratio (equivalence of base)/(atoms of P) is 1.00. That these bases are combined with phosphatidyl serine in a salt-like combination is shown by the fact that they can be removed by treatment with 0.05 N HCI, base-free phosphatidyl serine being thereby obtained. From the free acid the original phos- phatidyl serine K-Na salt can be prepared by the addition of the amount of base theoretically required.

Serine has been isolated as serine p-hydroxyazobenzene-p-sulfonate (8). It accounts for 80.7 per cent of the. carboxyl N present in the starting material. From it L-serine has been recovered in a yield that accounts for 73.5 per cent of serine present in phosphatidyl serine, according to the postulated formula. The impure barium glycerophosphate obtained accounts for 81 per cent of P in starting material, and the analytically pure barium glycerophosphate for 60 per cent of the original constituent P.

Estimation of fatty acid gives for phosphatidyl serine a (moles of fatty acid)/(atoms of P) ratio of 2.00. The neutral equivalent found for the fatty acids was 283, which is the theoretical value for an equimolar mixture of oleic and stearic acids. By the lead precipitation method, phosphatidyl serine fatty acids were divided equally between saturated and unsaturated acids. From the former, pure stearic acid has been obtained. The amount obtained accounts for 69 per cent of the amount present in phos- phatidyl serine, according to the postulated formula. The unsaturated fatty acid fraction appears to be mostly oleic acid, although attempts to obtain this acid in pure form have failed so far.

The iodine number calculated from the postulated formula is 31.5. The iodine number found varied between different preparations, ranging from 33.0 to 40. This discrepancy can be explained, at least in part, by the fact that our preparations were only between 92 and 97 per cent pure (as evaluated by their (carboxyl N ratio)/(total N)) and that the main

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

J. FOLCH 441

contaminant appeared to be phosphatidyl ethanolamine, which has a higher iodine number than phosphatidyl serine (2).

It must be emphasized that this paper deals only with phosphatidyl serine isolated by the method described. It accounts for about 60 per cent of all the lipide carboxyl N from brain. However, the possible existence of phosphatidyl serine having fatty acids other than stearic and oleic acids as constituents must be kept in mind. From another fraction isolated from brain cephalin, to which the name of inositol phos- phatide was given for descriptive purposes (2), other preparations of phosphatidyl serine have been isolated, incidental to the isolation of diphosphoinositide (9). These phosphatidyl serine preparations are found to contain Na instead of K as the most abundant inorganic base.

EXPERIMENTAL

Analytical Methods-Manometric methods were routinely used for the estimation of C (lo), P (II), N (12), and carboxyl N (5). Carboxyl N analysis on phosphatidyl serine gives better results when 1 cc. of water is added to the weighed dry material and the sample allowed to stand for a few minutes with occasional gentle shaking, so as to allow it to form an emulsion. In the case of base-free phosphatidyl serine, 1 or 2 drops of 0.1 N NaOH are added to facilitate emulsification. 13 minutes in the boiling water bath are allowed for reaction with ninhydrin, this length of time having been found to yield good checks between parallel esti- mations. In the later stages of work P has been estimated by Sperry’s method (13), iodine numbers by Yasuda’s method (14), and barium by weighing it as BaS04. NHZ-N was estimated manometrically on acid hydrolysates, as described elsewhere (2), to eliminate interference from unsaturated fatty acids (15). K was estimated as potassium phospho- tungstate (16), and Na as sodium uranyl zinc acetate by a microgravimetric modification of the method of Salit (17). Ca and Mg were estimated by standard methods (18), modified to suit the type of material dealt with.

In cases in which elementary composition has been used for identification of compounds or in which values are recorded as evidence, C and H were estimated by dry combustioql lead chromate being used in compounds that had base, N by the Dumas method, and P gravimetrically as am- monium phosphomolybdate. It has been found that C values by the wet combustion manometric method of Van Slyke and Folch (10) agreed with those obtained by dry combustion.

Glycerol was estimated by the Blix method (19), which has been found to give recoveries of only 95 to 96 per cent when tested with standard glycerol, or glycerophosphate solutions.

1 Analyses run by Dr. E. W. Elek.

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

442 PHOSPHA!MDYL SERINE

Preparation and Properties of Phosphatidyl Serine

Phosphatidyl serine is prepared from brain cephalin by the chloroform- alcohol method of fractionation, freed of water-soluble impurities by dialysis, and lyophilized as described elsewhere (2). Usually this method yields preparations of 85 to 99 per cent purity; i.e., between 85 and 90 per cent of the nitrogen present is carboxyl N. When the percentage of total N as carboxyl N is less than the stated percentage, it is easy to bring it up to this level by the following procedure.

1 gm. of the preparation under study is dissolved in 10 cc. of chloroform, and 16.5 cc. of absolute ethyl alcohol are added to the solution. A tur- bidity develops, and on standing or by centrifugation the system resolves itself into an underlying viscous layer and a clear supernatant solution. The clear supernatant solution is decanted, and to it are added 30 cc. of absolute ethyl alcohol. A precipitate separates which is collected and dried. On analysis it is found to contain carboxyl N at a higher concentra- tion than the mother substance. In a typical case, by this procedure the following fractions have been obtained from 22.G gm. of a preparation containing 1.25 per cent carboxyl N: (1) recovered from the viscous underlying layer, 7.4 gm. of material containing 1.29 per cent carboxyl N; (2) the precipitate collected from the supernatant solution, 11.7 gm. of material containing 1.43 per cent carboxyl N; (3) recovered from the supernatant solution (2), 3.2 gm. of material containing 0.35 per cent carboxyl N.

We have been unable to obtain consistently phosphatidyl serine having more than 92 per cent of its N as carboxyl N. Occasionally, preparations have been obtained showing concentrations of carboxyl N as high as 97 per cent of the total N. The main contaminant appears to be phosphatidyl ethanolamine, as is shown by the fact that all N present is NHz-N. In search of the nature of other contaminants phosphatidyl serine was found to be essentially free of cerebrosides (carbohydrates <O.l per cent), lecithin, or sphingomyelin (choline <O.l per cent) and cholesterol (<O.l per cent).

Phosphatidyl serine is obtained as a loose white powder. On being dissolved in organic solvents and recovered from solution, it acquires a light tan color. After lyophilization, phosphatidyl serine keeps fairly well if stored in vacua in the dark. Under these conditions it retains a certain amount of water. For instance, 401.75 mg. of phosphatidyl serine that has reached constant weight in vacua over CaCl2 at room temperature show a loss of Lveight of 6.G mg. (i.e., 1.65 per cent) on being heated in a vacuum at 80” to constant weight. Restored to former condi- tions (i.e., in a vacuum at room temperature), it regains its former weight within 48 hours.

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

J. FOLCH 443

Phosphatidyl serine is freely soluble in chloroform, ethyl ether, and petroleum ether, and insoluble in ethyl alcohol, methyl alcohol, or acetone. On standing in contact with water, it forms an emulsion. These emulsions are very stable and an 8 per cent emulsion of phosphatidyl serine will stand centrifugation at 4000 R.P.M. for an indefinite period of time.

Bases Combined with Phosphaiidyl Se&e--Phosphatidyl serine prepara- tions obtained by the method described are found to contain 1 equivalent of inorganic base for each atom of P. K is by far the most abundant base with a smaller amount of Na, but neither Ca nor Mg is present. Detailed results on this point are given in Table I for the two preparations. Studies carried out on cephalin preparations (20) (presumably containing about

TABLE I Analysis of Two Preparations of Brain Phosphatidyl Serins

per tend

61.50 9.60

1.71 3.78 1.71

per ten; per cent per cent

C 60.8 61.1 61.71 H 9.32 9.59 9.37 N 1.65 1.70 1.65 P 3.75 3.76 3.76 COOH-N 1.52 1.70 1.57 Iodine No. 40 38.0

+ B stands for the cations present. Preparation Oct. III contained 3.76 per cent K and 0.51 per cent Na; (equivalence of base)/(atoms of P) = 0.98. The values given in this column have been calculated for oleylstearylglycerylphosphoryl serine corrected for the cations present.

t B stands for the cations present. Preparation Sept. IIIbII contained 2.93 per cent of K and 0.99 per cent of Na; (equivalence of base)/(atoms of P) = 0.98. The values given in this column have been calculated for oleylstearylglycerylphosphoryl serine corrected for the cations present.

Calculated for BCuHn01oh’P’

Preparation Sept. IIIbII c.mponents

-

50 per cent phosphatidyl serine (21)) have shown that these bases are combined in un-ionized form.

Analyses of Phosphalidyl Serine-The results of chemical analysis of phosphatidyl serine are given in Table I. It is seen that the results ob- tained closely agree with values calculated for the postulated formula corrected in each case for the amount of inorganic bases present in the preparation.

Preparation of Base-Free Phosphaiidyl Scrine

The ability of phosphatidyl serine to form fairly ccncentratcd emulsion8 in water depends upon the presence of K or Na combined in its molecule.

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

444 PHOSPHATIDYL SERINE

Acidification of the emulsion results in progressive precipitation of phos- phatidyl serine as free acid. This precipitation is complete at about pH 1.5 (0.05 N HCl).

The insolubility of the free acid in water is made use of to prepare base- free phosphatidyl serine. The method is as follows.

3 gm. of phosphatidyl serine are emulsified with 150 cc. of water and to the emulsion are added 15 cc. of N HCl. The precipitate that forms is centrifuged off and washed once with 0.1 N HCI. The washed precipitate is transferred to a cellophane sausage casing and dialyzed against distilled water in the ice box for 3 days, the outside liquid being renewed six times. In the course of dialysis some of the precipitate goes back into emulsion. From the contents of the dialysis sack base-free phosphatidyl serine is obtained either by lyophilization or by precipitation with a large excess of alcohol (four to six times as much alcohol as water). In either case the dry substance is taken up in 10 cc. of CHCl, and 70 cc. of acetone are added

TABLE II

Analysis of Base-Free Phosphatidyl Serine

Component

C H P N Base COOH-N

Preparation Sept. IIIbII

- f Xc&ted for GzHso01oNP

per cert #W cent ge, cent per cent ger rend

63.34 63.89 63.53 63.5 63.9 9.92 9.72 9.87 9.82 10.1 3.89 3.83 3.96 3.86 3.92 1.74 1.80 1.81 1.69 1.78 1.0 0.5 0.1 0.4 0.0 1.59 1.65 1.68 1.59 1.78

-

to it. The precipitate that forms is collected and dried, 2.3 gm. of a light tan loose powder being obtained. After being dried to constant weight over CaClz in a vacuum, it still contains about 1.80 per cent water. For instance, 255.54 mg. of base-free phosphatidyl serine that had reached constant weight in vacua over CaCls showed a loss of weight of 4.64 mg. (i.e., 1.80 per cent) on being heated in a vacuum at 80” to constant weight. The dried product, when placed in a vacuum desiccator over CaCL, re- gained its former weight in 24 hours.

The results of chemical analyses of several preparations are given in Table II. They agree closely with values calculated for oleylstearyl- glycerylphosphoryl serine (CQHSOO~ONP) .

After drying, base-free phosphatidyl serine forms emulsions in water with great difficulty. It is only on addition of the amount of bases (KOH, NaOH) required to reform the K or Na salt (1 equivalent of base per atom of P) that an aqueous emulsion is easily obtained.

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

J. FOLCH 445

Efect of Storage on Phosphatidyl Serine

On storage in vacua in the dark at room temperature phosphatidyl serine does not change its elementary composition, but some sort of re- arrangement takes place in the molecule, so that the concentration of carboxyl N drops sharply. NHZ-N decreases to a much lesser degree. The results of analyses on one preparation (Nov. III) stored under the conditions given above were as follows: COOH-N: original 1.62 per cent; after 3 months 1.39 per cent; after 5 months 1.32 per cent; after 10 months 1.25 per cent; after 19 months 1.20 per cent; after 26 months 1.14 per cent. On the other hand, NHz-N, which was 1.70 per cent originally, was 1.37 per cent after 26 months. These changes with storage are much less marked in the case of base-free preparations. For instance, base-free Preparation Nov. III, containing originally 1.65 per cent COOH-N and 1.80 per cent NH2-N was found to contain 1.50 per cent COOH-N and 1.70 per cent NH2-N after 26 months storage. The fact that the NH2-N values decrease to a much lesser degree than carboxyl N values suggests that the -COOH group is mainly involved in the observed change.

Storage in a CHC13 solution at -72” (dry ice box) appears to be more satisfactory. Preparations stored this way for 18 months have shown no change in composition.

Isolation of L-Serine from Phosphatidyl Serine

2.25 gm. of phosphatidyl serine containing 1.42 per cent carboxyl N were freed of base by emulsification in mater and precipitation of the phosphatide by addition of HCl up to 0.1 N concentration. The precipitate was washed once with 0.1 N HCl and next hydrolyzed by boiling 6,~ HCI under reflux for 3 hours. After cooling, the hydrolysate was freed of fatty acids by filtration, and the filtrate was concentrated to 4 cc. volume. To it wereeadded 800 mg. of p-hydroxyazobenzene-p-sulfonic acid, which was dissolved by heating in the boiling water bath. Crystals formed on stand- ing overnight in the ice box were centrifuged in the cold and washed twice with 3 cc. of ice-cold water. The washed crystals were recrystallized twice from 4 cc. of water.

After drying at 100” the crystals weighed 710 mg. On analysis they proved to be serine p-hydroxyazobenzene-p-sulfonate. They accounted for 80.7 per cent of the carboxyl N present in the starting material. The results were as follows:

CI;II,rOrN3S. Calculated. C 47.0, H 4.44, carboxyl N 3.66, base 0.00 Found. “ 46.7, “ 4.39, ‘I “ 3.62, “ 0.3

569 mg. of this compound were dissolved in water, lead acetate was added to the solution, the lead salt removed by filtration, and the filtrate freed of traces of p-hydroxyazobenzene-p-sulfonic acid with charcoal.

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

446 PIIOSPILQTIDYL SERINB

The filtrate from the charcoal was dried, the residue dissolved in 2 cc. of water, and 20 cc. of alcohol were added to the solution. The crystals formed on standing overnight in the ice box were collected and dissolved in 0.5 cc. of water, and 10 cc. of alcohol were added to the solution. After standing overnight in the ice box, the crystals that formed were collected, dried, and analyzed.

The crystals weighed 142 mg. On analysis they proved to be L-serine, and accounted for 91 per cent of the serine present in the serine p-hydroxy- azobenzene-p-sulfonate or 73.6 per cent of the carboxyl N present in the starting phosphatidyl serine. The results were as follows:

CzHHIOIN. Calculated. C 34.2, H 6.67, N 13.32, carboxyl N 13.32 Found (corrected “ 34.15, ” 6.55, “ t3.22, “ “ 13.22

for 0.95% base).

Rotation-A solution in 1 N HCl containing 47 mg. of crystals per cc showed in a 1 dm. tube a rotation of +0.67” with sodium light; crE” = 14.3”. Fischer and Jacobs give +14.5’ (22).

Isolation of Glycerophosphoric Acid from Phosphatidyl Se&e

3.35 gm. of phosphatidyl serine (Preparation May 111-41) were emul- sified in 150 cc. of HzO, and 15 cc. of N HCI were added to the emulsion. The resulting precipitate was centrifuged, washed once with 0.1 N HCl, and then hydrolyzed with 6 N HCl for 100 minutes in the boiling water bath. After cooling, the hydrolysate was filtered and the filtrate evaporated to dryness in a vacuum. The dry residue was taken up in 50 cc. of water and the solution treated with 1 gm. of Ag,O after the addition of 0.5 cc. of concentrated acetic acid. The precipitate was filtered off and the silver was removed with hydrogen sulfide. The silver sulfide was filtered off, after which the filtrate was evaporated to dryness. The residue was dissolved in 30 cc. of Hz0 and brought to pH 10 by the addition of 12 cc. of saturated Ba(OH)2. The precipitate that separated was centrifuged off and washed twice with water. To the clear solution an equal volume of alcohol was added and the precipitate that separated was centrifuged off and washed once with 50 per cent alcohol and dried in a vacuum at 142” to constant weight. It weighed 831.2 mg. On analysis it was found to contain 11.1 per cent P, 32.5 per cent glycerol, 32.76 per cent Ba, and 0.2 per cent N. It accounted for 81 per cent of the P present in the starting material. It appeared to be barium glycerophosphate mixed with acid barium glycerophosphate.

Four precipitations from mater solution, by addition of an equal amount of alcohol, brought the nitrogen concentration down to 0.03 per cent. Finally, the Ba salt was dissolved in 50 cc. of HzO, the solution was made

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

J. FOLCH 447

strongly alkaline by addition of 10 cc. of saturated aqueous Ba(OH)S solution, and an equal volume of alcohol added to it. The alcohol-insoluble precipitate was centrifuged, washed twice with cold 50 per cent alcohol, dried, redissolved in 30 cc. of mater, and COZ bubbled through the solution. A slight precipitate was removed by centrifugation and to the supernatant was added an equal volume of methyl alcohol. The precipitate that formed was centrifuged, washed with 50 per cent methyl alcohol, and dried to constant weight at 142’.

575 mg. of material were thus obtained. On analysis it proved to be Ba glycerophosphate. The analytical results were as follows:

C&I~OSP Ba. Calculated. C 11.73, P 10. 1, Ba 44.5, glycerol 29.3 Found. “ 11.69, “ 10.05, “ 44.6, “ 28.7

Estimation of Fatty Acids

Phosphatidyl serine was saponified with 8 per cent alcoholic NaOH by refluxing for 4 hours. After cooling, the solution was nearly neutralized with hydrochloric acid and concentrated to dryness in vacua. The residue was acidified with N HCl and the fatty acids were extracted with four successive portions of ether. The combined ether extracts were washed three times with equal volumes of water, and the washed ethereal extract was evaporated to dryness. By this method, saponification appeared to be complete in 4 hours, the same results being obtained for periods of saponification of 4 and 8 hours.

A number of preparations were thus analyzed. The purest ones (con- taining 97 per cent of total N as COOH-N) yielded 68.6 per cent of the weight of the starting material as fatty acids, neutral equivalent 283; (moles of fatty acid)/(atoms of P) = 2.00. The theory for the K salt of phosphatidyl serine assumed to contain oleic and stearic acid radicals is 68.8 per cent of the starting material as fatty acids and the neutral equiva- lent for an equimolar mixture of oleic and stearic acids is 283. Preparations of lesser purity yielded fatty acids of higher neutral equivalent (up to 291).

Isolation of Component Fatty Acic&-4 gm. of phosphatidyl serine were emulsified in 300 cc. of HzO, and 25 cc. of 19.2 N NaOH were added to the emulsion. The solution was immersed in a boiling water bath for 6 hours. After cooling, the solution was acidified with HCl to pH 1.5 and allowed to stand overnight in the ice box. The next morning it was extracted four times in succession with an equal volume of ether each time. The ethereal extracts were combined and washed twice with an equal volume of mater, after which the solution was evaporated to dryness. The residue weighed 2.7 gm.

The fatty acids were separated into saturated and unsaturated acids by the method of Twitchell (23) as follows: The residue was dissolved in 80 cc.

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

443 PHOSPHATIDYL SERINE

of alcohol in a 100 cc. centrifuge tube immersed in a water bath at 75”, and to the hot solution were added in succession 0.5 cc. of acetic acid and 7 cc. of a 25 per cent aqueous neutral lead acetate solution. A small amount of precipitate settled to the bottom. The clear supernatant solution was transferred to another centrifuge tube in the same bath and then the bath was allowed to cool. Next morning the clear supernatant was decanted and the precipitate was washed with alcohol. The combined washing and supernatant solutions were evaporated to dryness. The residue was dissolved in 30 cc. of ether and the ethereal solution allowed to stand over- night in the ice box. The small amount of precipitate formed was filtered off and discarded. The filtrate was evaporated to dryness and the residue dissolved in 40 cc. of methyl alcohol, after which the lead was removed with hydrogen sulfide. The filtrate from the lead sulfide was evaporated to dryness. The residue was an oil which contained some white crystals. It weighed 1250 mg.

The oily residue was treated with 30 cc. of petroleum ether. Most of it went into solution. After standing overnight in the ice box, the petroleum ether solution was filtered and the filtrate evaporated to dryness. The residue was a clear oily liquid and weighed 1100 mg. The neutral equiva- lent was 283 and the iodine number 87.0. It appeared to be slightly impure oleic acid. Attempts to isolate analytically pure oleic acid from it have failed. The amount of impure oleic acid obtained amounted to 79.5 per cent of the amount present in the starting material, according to the postulated formula.

The alcohol-insoluble lead soaps were reprecipitated from 60 cc. of hot alcohol (to which 0.5 cc. of acetic acid had been added), the solution being allowed to cool over a period of 2 hours. After standing overnight at room temperature, the precipitate formed was collected by centrifugation and washed with 95 per cent alcohol.

After drying in a vacuum, the lead soaps were transferred to a separatory funnel with ether and the ethereal suspension washed with dilute nitric acid and then four times with water, after which the ethereal solution was evaporated to dryness. The residue was a crystalline mass which weighed 1100 mg., m.p. 65”. Neutral equivalent 279; iodine value <l.O. It was assumed that the material was impure stearic acid. The residue was dissolved in 80 cc. of hot alcohol. On cooling, a small amount of precipitate separated which was filtered off and the filtrate evaporated to dryness, after which the residue was dissolved in 40 cc. of hot alcohol. On cooling, a small precipitate formed. The solution was filtered, evaporated to dryness, and the residue dissolved in hot alcohol. No precipitate formed on cooling. The solution was evaporated to dryness.

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

J. FOLCH 449

The crystalline residue weighed 950 mg. On analysis it proved to be pure stearic acid. The results were as follows:

GsHasOz. Calculated. C 76.0, H 12.72, Base Found. “ 75.8, (‘ 12.65, “ 0.1

1M.p. 69.3”; mixed m.p. with stearic acid, 69.3”; neutral equivalent 284.0. The amount of pure stearic acid obtained represents 69 per cent of the

amount present in the starting material, according to the postulated formula.

SUMMARY

1. A method is described for the isolation of phosphatidyl serine of at least 92 per cent purity.

2. As cleavage products, glycerophosphoric acid, L-serine, and fatty acids have been isolated in molecular proportions of 1: 1: 2. Fatty acids present appear to be mainly stearic acid and oleic acid.

3. Phosphatidyl serine reacts with HNOz and ninhydrin as an cY-amino acid, which shows that both the -COOH and the --NH2 groups are free. It does not react with HIO+ which shows that either its -NH2 or its -OH group is combined. Therefore, it appears that the combination of serine in the phosphatidyl serine molecule is through its -OH group.

4. It is concluded that the structure of phosphatidyl serine is that of stearyloleylglycerylphosphoryl serine. The results of analyses are found to agree with the values calculated from the postulated formula.

5. As isolated by neutral solvents, phosphatidyl serine is obtained as a K and Na (the former being the most abundant) salt, (equivalence of base)/- (atoms of P) = 1.00. The inorganic cations can be removed by treatment with 0.1 N HCl.

6. Phosphatidyl serine studied in this paper represents 60 per cent of total lipide carboxyl N in brain. Other preparations of phosphatidyl serine can be obtained in which Na is the most abundant base.

BIBLIOGRAPHY

1. Folch, J., J. Biol. Chem., 139, 973 (1941). 2. Folch, J., J. Biol. Chem., 146, 35 (1942). 3. Folch, J., J. Biol. Chem., 146, 31 (1942). 4. Chibnall, A. C., and Channon, II. J., Biochem. J., 23, 176 (1929). 5. Van Slyke, D. D., Dillon, R. T., MacFadyen, D. A., and Hamilton, P., J. Biol.

Chem., 141, 627 (1941). 6. Van Slyke, D. D., J. BioZ. Chem., 83, 425 (1929). 7. Nicolet, B. H., and Shinn, L. A., J. Am. Chem. Sot., 61, 1615 (1939). 8. Stein, W. H., Moore, S., Stamm, G., Chou, C.-Y., and Bergmann, M., J. BioZ.

Chem., 143, 121 (1942).

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

450 PHOSPBATIIKL SERINE

9. Folch, J., Federation Proc., 6, 134 (1946). 10. Van Slyke, D. D., and Folch, J., J. Biol. Chem., 136, 509 (1940). 11. Kirk, E., J. Biol. Chem., 106, 191 (1934). 12. Van Slyke, D. D., J. Biol. Chem., 71, 235 (1926-27). 13. Sperry, W. M., Ind. and Eng. Chem., Anal. Ed., 14, 33 (1942). 14. Yasuda, M., J. Biol. Chem., 94, 401 (1931-32). 15. Folch, J., Schneider, H. A., and Van Slyke, D. D., Proc. Am. Sot. Biol. Chem.,

J. Biol. Chem., 133, p. xxxiii (1940). 16. Folch, J., and Lauren, hf., J. Biol. Chem., 169, 539 (1947). 17. Salit, P. W., J. Biol. Chem., 96, 059 (1932). 18. Peters, J. P., and Van Slyke, D. D., Quantitative clinical chemistry; Methods,

Baltimore (1932). 19. Blix, G., Mikrochim. a&, 1, 75 (1937). 20. Christensen, H. N., and Hastings, A. B., J. Biol. Chem., 136, 387 (1940). 21. Folch, J., and Schneider, H. A., J. Biol. Chem., 137, 51 (1941). 22. Fischer, E., and Jacobs, W. A., Ber. them. Ges., 39, 2942 (1906). 23. Twitchell, E., J. Znd. and Eng. Chem., 13, 806 (1921).

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Jordi FolchPHOSPHATIDYL SERINE

THE CHEMICAL STRUCTURE OF

1948, 174:439-450.J. Biol. Chem. 

  http://www.jbc.org/content/174/2/439.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/174/2/439.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on July 15, 2018http://w

ww

.jbc.org/D

ownloaded from