ON THE SEPARATION OF HISTIDINE AND ARGININE.

II. THE SEPARATION OF THE SILVER COMPOUNDS AT pH 7.0.*

BY HUBERT BRADFORD VICKERY AND CHARLES S. LEAVENWORTH.

(From the Laboratory of the Connecticut Agricultural Experiment Station, New Haven.)

(Received for publication, December 28, 1926.)

In 1900 Kossel and Kutscher (1) showed that when barium hydroxide was added to a solution containing histidine and argi- nine together with an excess of a soluble silver salt, histidine silver was first precipitated completely and arginine silver began to precipitate only after the addition of a “‘somewhat greater excess” of the alkali. A few years ago Kossel and Edlbacher (2) sug- gested that the silver compound of histidine is completely precipitated at a faint alkaline reaction to phenolphthalein but that the silver compound of arginine does not come down until a still more alkaline reaction is reached. They imply that a separa- tion may thereby be effected. We recently (3) had occasion to examine the behavior of these silver compounds and found that at the reaction represented by the first faint pink of phenolphthalein, all of the histidine together with a large part of the arginine was precipitated. We drew attention, however, to the possibility that a separation of these bases might be effected if the hydrogen ion concentration at which the precipitation of histidine silver is complete were ascertained.

This has now been found to lie close to pH 7.0. A practically complete separation of these bases can be readily obtained by bringing the solution, containing an excess of a soluble silver salt, to this reaction by the careful addition of barium hydroxide.

Preliminary experiments were carried out with solutions of pure

* The expenses of this investigation were shared by the Connecticut Agricultural Experiment Station and the Carnegie Institution of Washing- ton, D. C.

403

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

ww

.jbc.org/D

ownloaded from

404 Histidine and Arginine. II

histidine and arginine in very dilute sulfuric acid. To a series of solutions of histidine sulfate, containing an excess of silver sulfate,

TABLE I.

Data of Experiments in Which Histidine Silver Was Remou@ at DiJerent Hydrogen Ion Concentrations and Arginine Silver Subsequently

Precipitated.

In Experiments 1 to 4 a single precipitation and in Nos. 5 and 6 a double precipitation of the silver compound was employed.

E;;rti-

NO.

(1)

-

Laction.

(2)

PH

7.2 7.0 6.8 6.6 7.0 7.0

I

.-

-

listidim N taken.

(3)

gm. gm. gm. gm. Bm.

0.158 0.357 0.165 0.014 0.343 0.210 0.357 0.216 0.017 0.345 0.237 0.469 0.243 0.019 0.435 0.207 0.488 0.168 0.028 0.504 0.206 0.485 0.195 0.013 0.468 0.232 0.447 0.233 0.007 0.447

. _

A$ggknl

(4)

1 I

-

N in histidine fraction.

(5)

1 1

-

4$np

Cstidine ‘raction.

(6)

N in arginine

fraction.

(7)

His$llne 1. argm,ne

fraction.

(8)

gm.

Trace. “

0.0094t

* The data in Experiments 3 to 6 were obtained by Koehler’s modifica- tion of the Van Slyke method and are corrected for the ammonia set free by the partial decomposition of histidine.

t Owing to the accidental loss of a part of the histidine mercury precipi- tate this figure represents only a fraction, probably about one-half, of the histidine actually present in this solution.

TABLE II.

Data Showing Recovery of Arginine and Hislidine as Salts of Dinitronaphthol- sulfonic Acid.

Experi- ment NO.

Hi$i$ne

di-salt.

gm. per cent

0.143 87.0 0.204 94.6 0.212 87.2 0.184 94.4 0.228 97.8

;;;;e;; N of

histidine fraction.

gm. per cent per cent

0.324 94.3 90.7 0.308 88.9 84.3 0.395 90.6 84.1 0.449 95.9 92.6 0.427 95.6 95.6

increasing quantities of dilute barium hydroxide solution were added, the precipit,ates centrifuged off, and their nitrogen content

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

ww

.jbc.org/D

ownloaded from

H. B. Vickery and C. S. Leavenworth 405

determined. In this way the conditions under which all of the histidine present was precipitated were readily found. The hydrogen ion concentration of the filtrates from the histidine silver was estimated colorimetricallyl by comparison with stand- ard buffer solutions. The reaction of the solution from which the histidine had been completely precipitated and which contained the least quantity of barium hydroxide was in the vicinity of pH 7.2. Similar experiments in which dilute barium hydroxide was added to a solution of arginine and an excess of silver sulfate, showed that no nitrogen appeared in the precipitate until pH 7.9 was exceeded. Thus there is a considerable range of hydrogen ion concentration within which a separation of the bases may apparent’ly be made.

A series of separations was next carried out at different hydro- gen ion concentrations upon solutions containing sufficient known quantities of the two bases to permit recovery as crystalline deriv- atives. The data obtained are presented in Tables I and II. Several modifications in detail were introduced as the work pro- gressed. The method, as finally adopted (Experiments 5 and 6), was as follows.

Method of Separation.

To the solution containing the bases and a small amount of sulfuric acid, used to dissolve the arginine carbonate, at a volume of approximately 500 cc., boiling saturated silver sulfate solution was added until a spot test with barium hydroxide showed the presence of excess silver. This required about 800 cc. The solu- tion was cooled and cold saturated barium hydroxide solution was added from a graduated cylinder until, on stirring, the precipitate took on a faint buff color. At this point the solution was practi- cally neutral to litmus paper and the precipitate flocculated

1 We wish to express our thanks to Dr. Florence Fenwick, National Research Council Fellow in Chemistry at Yale University, for attempts made, at our request, to develop an electrometric method for the measure- ment of the hydrogen ion activity of these solutions. Owing to the pres- ence of a noble metal the hydrogen electrode could not be used and the oxygen electrode, employed on the assumption that the silver compounds of the bases might be hydroxides, was likewise found to be useless. The development of the simple colorrmetric technique rendered further experi- ments unnecessary.

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

ww

.jbc.org/D

ownloaded from

406 Histidine and Arginine. II

sharply. A 5 cc. sample was withdrawn, centrifuged, the clear fluid treated with a few drops of 0.04 per cent brom-thymol blue indicator solution prepared according to Clark’s directions, and the color compared with that of sealed tubes containing appro- priate buffer solutions and the same indicator.2 The test solution and precipitate were then returned to the main solution and addi- tion of barium hydroxide continued until a sample, tested in this way, matched the color of the buffer at pH 7.0. It is evident from the data of Table I that some latitude is permissible in adjusting this reaction, and that our selection of pH 7.0 is somewhat arbi- trary. Experiment 4, however, indicates that the acidity must be somewhat less than pH 6.6 as at this reaction some histidine es- caped precipitation.

After about 15 minutes of careful stirring the precipitate was centrifuged and the clear supernatant fluid poured off and acidi- fied to litmus with sulfuric acid. The precipitate was suspended in water to which a minimal amount of hydrochloric acid was added, and boiled to ensure complete decomposition of the his- tidine silver. Excess of boiling saturated silver sulfate was then added, the solution cooled and again brought to pH 7.0 by the cautious addition of barium hydroxide solution, again carefully stirred, and the precipitate of silver chloride, barium sulfate, and histidine silver centrifuged off. The supernatant fluid was re- moved, acidified, added to that from the first precipitation, and the whole concentrated in vacua to about 800 cc.

Ry this double precipitation the difficult washing of the first histidine silver precipitate was avoided and the quantity of argi- nine in the histidine fraction materially reduced, as is clear when Experiments 3 and 4, in which a single precipitation was employed, are compared with Experiment 6 (Table I, Column 6).

The second histidine silver precipitate was suspended in water, treated with a small excess of hydrochloric acid, and boiled, the precipitate of silver chloride, etc., centrifuged off and thoroughly washed with hot water. The solution containing the histidine was then evaporated to dryness in vacua to remove the excess of hydrochloric acid, made to standard volume, and nitrogen de- termined in an aliquot part.

2 The buffer solutions prepared by the LaMotte Chemical Products Company are satisfactory.

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

ww

.jbc.org/D

ownloaded from

H. B. Vickery and C. S. Leavenworth 407

The remaining portion of the solution was evaporated to approximately 30 cc. and histidine recovered as d&salt of dinitro- naphtholsulfonic acid in two successive crops (4).

The concentration of the acidified filtrates from the histidine silver permits the precipitation of arginine silver from a relatively small volume of solution and avoids much of the uncertainty aroused by the possibility that this substance is not wholly insol- uble. We have found that a little silver sulfate usually separates from this solution on concentration, and, when necessary, have removed it by titration.

Arginine silver was precipitated by the addition of warm satu- rated barium hydroxide solution until the reaction was strongly alkaline to phenolphthalein, centrifuged off, suspended in water, and decomposed by warming with a small excess of dilute hy- drochloric acid. Since a small amount of barium was always present, dilute sulfuric acid was added drop by drop until a sample, removed and centrifuged, showed the presence of a trace of sul- fate ion. The mixture of silver chloride and barium sulfate, which also contained a trace of adsorbed indicator from the previous tests, was centrifuged off and washed, the solution concentrated to dryness in vczcuo, and made to standard volume. An aliquot part was removed for total nitrogen determination and the arginine in the remainder recovered by precipitation with a small excess of dinitronaphtholsulfonic acid.

Saturation of the solution with powdered barium hydroxide in order to precipitate arginine silver, as recommended by Kossel, is wholly unnecessary. While we have not ascertained the exact hydrogen ion concentration at which arginine silver is completely precipitated, it probably lies near or somewhat beyond pH 10. Provided the solution was made strongly alkaline to phenolphtha- lein, we have encountered no case in which appreciable amounts of arginine escaped precipitation.

In order to obtain information regarding the completeness of the separation, it was necessary to analyze the histidine solution for arginine and the arginine solution for histidine. The deter- mination of small amounts of arginine in a solution containing relatively large amounts of histidine is, however, difficult and some- what uncertain. We have employed Van Slyke’s method of decomposition with sodium hydroxide and estimation of the

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

ww

.jbc.org/D

ownloaded from

Histidine and Arginine. II

ammonia thereby produced. In the earlier experiments (Nos. 1 and 2, Table I) Plimmer’s (5) modification of this method was used, in which, at the end of the 6 hour digestion, the water is withdrawn from the condenser and the ammonia distilled up into the absorption apparatus at the top. This method was later abandoned, as difficulty was experienced in completing the dis- tillation, and the convenient modification suggested by Koehler (6) was employed, in which a slow stream of air, passing through the flask, sweeps the ammonia into an absorption apparatus.

The fundamental difficulty with this estimation in the present case is, however, the instability of histidine. Plimmer (5) has published some experiments which show that 1.5 to 3 per cent of the nitrogen of histidine is split off as ammonia by a 6 hour diges- tion with strong sodium hydroxide solution (50 or 20 per cent). We have verified these findings using our purest preparation of free histidine. Our experiments indicate that a somewhat vari- able decomposition occurs with the evolution of an average pro- portion of approximately 2 per cent of the histidine nitrogen as ammonia. While this decomposition is probably not a serious matter in ordinary determinations of arginine, it involves a large correction in our application of the method to the determination of traces of arginine in the presence of much histidine. We there- fore submit the data (Table I, Column 6) we have obtained with some reservation.

To illustrate the calculation of the arginine content, the data from Experiment 5 may be given. 25 cc. of this solution con- tained 0.01945 gm. of nitrogen. Duplicate determinations of the ammonia nitrogen evolved on 6 hours digestion with 30 per cent sodium hydroxide solution gave 0.0011 and 0.0010 gm. of nitrogen. 2 per cent of the total nitrogen in each aliquot is 0.0004 gm. of nitrogen. Deducting this we have 0.00065 gm. of nitrogen as ammonia, presumably derived from the arginine in 25 cc., or 0.0065 in the whole 250 cc. of solution. Twice this or 0.013 gm. is therefore the arginine nitrogen present in the solution.

In order to test for the presence of histidine in the arginine frac- tion, Hopkins’ mercuric sulfate reagent was employed. As the presence of chlorides is inadmissible when this reagent is used, in the first four experiments the arginine silver precipitate was decomposed with hydrogen sulfide in the presence of dilute sul-

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

ww

.jbc.org/D

ownloaded from

H. B. Vickery and C. S. Leavenworth 409

furic acid, silver sulfide removed, the solution brought to a stand- ard volume, and an aliquot removed for nitrogen determination. Hopkins’ reagent was then added in large excess to the remainder of the solution. Only in the experiment in which the separation was effected at pH 6.6 (Table I, Column 8, Experiment 4) was there evidence of more than a trace of precipitate of the histidine compound. In this case filtration and determination of nitrogen was possible, but unfortunately a part of the precipitate was lost through an accident. The nitrogen determination on the re- mainder indicates the presence of an appreciable quantity of histidine. In the other experiments only the slightest turbidity developed on standing 48 hours.

TABLE III.

DataShowing Inapplicability of Correction for f?olubiEity of Arginine Silver in Saturated Barium Hydroxide to Conditions Employed in These

Experiments. -7

“:zl? NO.

(1)

1 2 3 5 6

A b

-

-

.rginim I taken

(2)

om. om.

0.357 0.343 0.357 0.345 0.469 0.435 0.485 0.468 0.447 0.447

Nin mginine ‘raction

(3)

VOlUIlX .rginine

in fi&te istidine from raction. am;

(4) (5) --

gm. cc.

0.014 1940 0.017 1920 0.019 2320 0.013 1280 0.007 1150

-

c (

--

T

Volubility :orrection 1.0116 gm.

2tr

Total :orrection (4) + (6)

(6) (7)

om. om.

0.0225 0.036 0.0222 0.039 0.0269 0.046 0.0148 0.028 0.0133 0.020

(

i I

,-

:orrectec arginino 1\T found. :3) + (7)

(8)

om. 0.379 0.384 0.481 0.496 0.467

p&l;

mrrected for

arginine in

histidine fraction. (3) + (4)

(9)

om.

0.357 0.362 0.454 0.481 0.454

The arginine fraction, in those experiments in which Hopkins’ reagent had been used, was freed from mercury with hydrogen sulfide, and barium hydroxide added until the solution was only faintly acid to litmus. The barium sulfate was then removed, the solution concentrated to approximately 200 cc., and the arginine precipitated with a slight excess of dinitronaphtholsulfonic acid. The somewhat low recoveries of arginine in the first three experi- ments recorded in Table II are partly due to the unavoidable losses involved in the removal of mercury and sulfuric acid.

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

ww

.jbc.org/D

ownloaded from

410 Hi&dine and Arginine. II

It has always been customary, in carrying out an analysis of the bases from proteins by Kossel’s method, to apply a correction for the solubility of arginine silver in the saturated solution of barium hydroxide from which this compound is precipitated. This correction (1) is founded upon Gulewitsch’s (7) determination of the solubility of arginine silver in saturated barium hydroxide which amounted to 0.036 gm. of arginine, or 0.0116 gm. of arginine nitrogen, per liter. When the precipitation of arginine silver is carried out at a strongly alkaline reaction to phenolphthalein, which is probably within the limits pH 10 to 11, and therefore not far from the isoelectric point of arginine (pH 10.97) (8), this cor- rection is not necessary. The data in support of this statement are contained inTable III. Comparison of Columns 8 and 2 of this table shows that the corrected arginine nitrogen is in every case materially higher than the arginine nitrogen taken. When, however, the correction for the arginine nitrogen found in the histidine fraction alone is added (Column 9) the nitrogen recoveries are within the error of such experiments.

Purijication of Arginine.-The arginine employed was a preparation of crude arginine carbonate that had been obtained from casein by the Kossel- Kutscher procedure. Analysis by the Van Slyke method indicated that it was approximately 95 per cent pure. We have found that the best salt for the further purification of arginine of this grade is the picrate which possesses convenient solubilities in water and is readily prepared by adding the theoretical amount of pure picric acid, dissolved in hot alcohol, to an aqueous solution of the carbonate. The picrate separates, on standing overnight, in large masses of yellow needles which decompose, when heated slowly, at 217-218°C. Slightly higher decomposition points could be ob- tained by rapid heating but this point was characteristic. Riesser (9), who also employed this method of purification, gave the decomposition point of arginine picrate as 205-206”. Our decomposition point was ob- tained with a short stem thermometer and was unchanged by recrystalliza- tion. The salt contains 2 molecules of water of crystallization, theory 8.20 per cent, found 8.18 per cent.

The picrate was decomposed with hot 10 per cent sulfuric acid, picric acid removed with ether, and a faint trace of yellow color removed with norit. Sulfuric acid was exactly removed by the addition of the requisite amount of pure barium hydroxide, the barium sulfate centrifuged off and thoroughly washed with boiling water. On concentration in vacua to a thin sirup and scratching, free arginine slowly separated in short, thick, clear prisms. The crystals rapidly disintegrate into a white powder in the air with formation of the carbonate. Free arginine, rapidly dried between

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

ww

.jbc.org/D

ownloaded from

H. B. Vickery and C. S. Leavenworth 411

filter papers, darkened at 223’ and decomposed at 238“. This is much higher than the decomposition point given by Gulewitsch (7) of 207-207.5”. In order to make certain that our higher decomposition points were not due to racemization we determined the rotation. A 16.27 per cent solution gave [a]: = + 12.5” and the same solution diluted to 8.13 per cent gave [a]; = + 12.86”. Gulewitsch found that the specific rotation of a 9.6 per cent solution of the chloride which contained an excess of barium hy- droxide was +12.94”. Our product was therefore probably optically pure. The preparation was converted to the carbonate and dried over lime.

Purijication of Hi&dine.-A preparation of histidine dichloride ob- tained by the method of Hanke and Koessler (10) from blood cells was found to retain some impurity even after several recrystallizations. Since free histidine can be crystallized from water and even better from 50 per cent alcohol, the dichloride was treated, in 10 or 20 gm. lots, with excess of hot silver sulfate solution, silver chloride removed, excess silver pre- cipitated as sulfide and the solution concentrated slightly to remove hydrogen sulfide. It was then brought to pH 7.2 by the addition of pure barium hydroxide. Barium sulfate was then removed and washed, and the solution was evaporated in wcuo to crystallization. The addition of an equal volume of absolute alcohol at this stage materially increases the yield of pure substance.

The decomposition point of free histidine is somewhat unsatisfactory. One of our preparations darkened at 255’ and decomposed with efferves- cence at 265” on slow heating. When rapidly heated it darkened at 260” and decomposed at 280”. Abderhalden and Weil (11) described prepara- tions of free histidine which darkened from 255-260” and decomposed at 279-280” on rapid heating. We have prepared one specimen which was crystallized twice as chloride, once as di-salt of dinitronaphtholsulfonic acid, and twice as free base, which darkened from 250” and decomposed at 277” on slow heating.

The purified histidine when dissolved in water gave a solution, the hydrogen ion concentration of which lay at pH 7.2 as determined by means of brom-thymol blue or phenol red. Since the isoelectric point of this sub- stance is pH 7.15 (12) this furnishes a sensitive test of its purity. It was noted that samples obtained from the mother liquors of the purer material gave solutions which were distinctly more alkaline. Moreover a solution of pure histidine on boiling became slightly alkaline. Free histidine is therefore somewhat, unstable.

Recovery of DinitronaphthoZsulfonates.-The data obtained from Experi- ment 6 (Table II) may be given to illustrate the details of the recovery of the dinitronaphtholsulfonates of histidine and arginine.

The histidine solution contained 0.232 gm. of nitrogen in 250 cc. Of this 180 cc. were concentrated to about 30 cc. and 3.2 gm. (2.5 gm. = 2 mols) of dinitronaphtholsulfonic acid were added. On standing overnight 2.966 gm. (dried at 105”) of di-salt separated. This decomposed at 252” after darkening from 240”. On concentration the mother liquor yielded a fur- ther crop of 0.097 gm. These two crops contained histidine nitrogen equivalent to 0.225 gm. based on the whole solution.

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

ww

.jbc.org/D

ownloaded from

412 Histidine and Arginine. II

The arginine solution contained 0.447 gm. of nitrogen in 250 cc. Of this 240 cc. were concentrated slightly and 4.2 gm. (3.8 gm. = 1 mol) of dinitro- naphtholsulfonic acid added at boiling temperature. The salt promptly separated from the hot solution in orange plates with a golden yellow luster and after standing overnight 3.473 gm. (dried at 105”) were obtained, nitrogen, found 16.9 per cent, theory 17.2 per cent. The mother liquor was concentrated to 10 cc. and on standing yellow crystals separated. These were filtered off and boiled with a little water when a part dissolved and the insoluble residue became orange.3 On further standing 0.099 gm. of orange plates was obtained. These two crops contained arginine nitrogen equiva- lent to 0.427 gm. based on the whole solution.

An investigation of the application of this method of separating histidine from arginine to the analysis of the bases derived from protein is in progress. Preliminary experiments have already shown that the method greatly facilit’ates this analysis and also indicates that it may be applied to the preparation of arginine and histidine on a large scale. Investigation of this is also planned. In this connection it may be well to draw attention to the recent suggestion of Kiesel (13) who avoids the large volumes required when much silver sulfate must be added to a solution by adding the silver in the form of a paste of the oxide, maintaining the acid- ity of the solution by the addition of sulfuric acid as needed.

The fact that both histidine and arginine are completely pre- cipitated as silver compounds in the vicinity of their respective isoelectric points, is very suggestive. While we are ignorant of the exact nature of these compounds it would seem that the reac- tion takes place between silver ion and ihe organic base in its undissociated form. This point deserves detailed study.

The general principle underlying this method of separation can probably be quite widely applied. We have already employed precipitation of silver compounds at acid, neutral, and alkaline reactions as a convenient and rapid means of preparing purine, histidine, arginine and lysine fractions from yeast extracts. Much investigation of the details of the procedure is, however, necessary in this more complicated case.

3 We suspect that arginine may, like histidine, form two compounds with dinitronaphtholsulfonic acid. This behavior will be investigated.

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

ww

.jbc.org/D

ownloaded from

H. B. Vickery and C. S. Leavenworth 413

SUMMARY.

Histidine can be approximately completely separated from arginine by bringing the reaction of a solution of the two bases, which also contains an excess of a soluble silver salt, to pH 7.0. By a second precipitation as silver compound a histidine fraction is obtained which contains no material quantity of arginine.

No appreciable amount of histidine escapes this precipitation, the arginine fraction being practically free from this base.

Arginine silver is subsequently precipitated at a strong alkaline reaction to phenolphthalein (pH 10 to 11). Saturation of the solution with barium hydroxide in order to precipitate arginine silver is shown to be not only unnecessary but inadvisable.

The silver precipitates can be conveniently decomposed with hydrochloric acid and the bases recovered very completely as salts of dinitronaphtholsulfonic acid.

The method offers many advantages over that previously em- ployed for the separation of these bases as the entire separation can be performed in a few hours. The application to the analysis of the bases from proteins and to the preparation of arginine and histidine in large quantities will be developed.

It is suggested that the general principle of silver precipitations at different hydrogen ion concentrations may receive a much wider application.

BIBLIOGRAPHY.

1. Kossel, A., and Kutscher, F., 2. physiol. Chem., 1900, xxxi, 165. 2. Kossel, A., and Edlbacher, S., 2. physiol. Chem., 1920, cx, 241. 3. Vickery, H. B., and Leavenworth, C. S., J. Biol. C&m., 1926, Ixviii, 225. 4. Vickery, H. B., J. Biol. Chem., 1926, lxxi, 303. 5. Plimmer, R. H. A., Biochem. J., 1916, x, 115. 6. Koehler, A. E., J. Biol. Chem., 1920, xiii, 267. 7. Gulewitsch, W., 2. physiol. Chem., 1899, xxvii, 178. 8. Hunter, A., and Borsook, H., Biochem. J., 1924, xviii, 833. 9. Riesser, O., 2. physiol. Chem., 1906, xlix, 210.

10. Hanke, M. T., and Koessler, K. K., J. BioZ. Chem., 1920, xliii, 521. 11. Abderhalden, E., and Weil, A., 2. physiol. Chem., 1912, lxxvii, 435. 12. Kanitz, A., 2. physiol. Chem., 1906, xlvii, 476. 13. Kiesel, A., Z. chysiol. Chem., 1926, clxi, 147.

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

ww

.jbc.org/D

ownloaded from

LeavenworthHubert Bradford Vickery and Charles S.

7.0OF THE SILVER COMPOUNDS AT pH

AND ARGININE: II. THE SEPARATION ON THE SEPARATION OF HISTIDINE

1927, 72:403-413.J. Biol. Chem. 

  http://www.jbc.org/content/72/1/403.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

  ml#ref-list-1

http://www.jbc.org/content/72/1/403.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

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

ww

.jbc.org/D

ownloaded from