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CARBOXYMETHYLAMINO ACIDS AND PEPTIDES”

BY SAMUEL KORMANl’ AND HANS T. CLARKE

(From the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York)

(Received for publication, December 16, 1955)

The valuable method of Sanger (1) for the labeling of terminal amino groups in peptides by means of dinitrofluorobenzene suffers from the dis- advant’age that acid hydrolysis of t,he products is frequent,ly attended by extensive decomposition of the resulting dinitrophenylamino acids (2). The study here reported was undertaken in order to explore the stability, under conditions of acid hydrolysis, of N-carboxymet’hyl (Cm-)* derivatives of amino acids, and the feasibility of their separation from natural amino acids. In both respects the results offer promise.

In spite of our failure to include all natural amino acids in the survey and to obtain every product in crystalline form, we now present the results in the hope that they may find application in other laboratories.

Cm-sarcosine (3) and several a,a’-iminodicarboxylic acids such as oc- topin (4) and mono-Cm-alanine (5) and its homologues (G-9) have been described in the literature, but di-Cm-amino acids other than di-Cm-glycine (10) have received comparatively little attention. Michaelis and Schubert (11) prepared acid potassium salts of di-Cm-alanine, tetra-Cm-cystine, and N-di-Cm-cysteine. They also described the preparation of X-Cm-cysteine by brief treatment of an alkaline solution of cysteine with potassium chloroacetate; analogous reactions of cysteine with neutral iodoacet’ate had pre- viously been recorded by Dickens (12) and Rapkine (13).

Cm-amino Acids

The carboxymethyl group is conveniently introduced by treating amino acids with magnesium bromoacetate in an aqueous suspension of magnesium oxide. In most instances, when an excess of the reagent is employed, two Cm- groups attach themselves to the a-amino nitrogen atom, the chief exception, besides proline, being aspartic acid, from which the mono-Cm- derivative was preponderantly formed under the conditions employed. Glutamic acid and phenylalanine yielded some mono-Cm- as well as di- Cm- derivatives. Tyrosine gave two derivatives, in both of which the

* From a dissertation submitted by S. Korman in partial fulfilment of the require- ments for the degree of Doctor of Philosophy in the Faculty of Pure Science, Colum- bia University.

t Atomic Energy Commission Predoctoral Fellow, 1949-52. 1 Hereafter referred to as Cm-.

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114 CARBOXYMETHYLAMINO ACIDS AND PEPTIDES

phenolic hydroxyl group had also been carboxymethylated. The substitution of the hydroxylic hydrogen atom of tyrosine was accompanied (Fig. 1) by abolition of the shift in the ultraviolet absorption spectrum caused by alkali, charact’eristic of phenols (14). However, alkali induces a general increase in absorption in the region of the maximum and at shorter wave- lengths. A similar effect was observed (Fig. 2) with phenylalanine.

As was to be expected, the guanidino group of arginine does not react with bromoacetate; lysine, on the other hand, yields a tetra-Cm- derivative.

260 286 m.u

260 280 300

FIG. 1 FIG. 2

FIG. 1. Absorption spectra of tri-Cm-tyrosine. Curve 1, solvent, 0.1 N

Curve 2, solvent, 0.1 N NaOH. HCI ;

FIG. 2. Absorption spectra of phenylalanine. Curve 1, solvent, 0.1 N HCI; Curve 2, solvent, 0.1 N NaOH.

Attempts were made to prepare c-di-Cm-lysine by the action of bromoacetate upon the copper complex of lysine in a procedure analogous to that devised by Kurtz (15) for the synthesis of t-carbobenzyloxylysine. These were unsuccessful; substitution occurred at the a-amino group as well as at the terminal nitrogen atom. The desired compound, however, was secured, in the form of its mercuric derivative, from alanyllysylalanine.

When treated with bromoacetate in the presence of MgO, cysteine and histidine, like tyrosine, react in their specific functional groups. The reaction with the sulfhydryl group of cysteine is very rapid, as was found by Huggins and Jensen (16) in the case of iodoacetate; with histidine and


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S. KORMAN AND H. T. CLARKE 115

tyrosine the ability to yield color with diazobenzenesulfonic acid disap- peared at measurable rates (Table I). Iodoacetate can be employed in place of bromoacetate for the carboxymethylation of amino acids, but its action is less rapid; chloroacetate is even less effective.

The products obtained from all of the amino acids investigated have been found, irrespective of the number of carboxymethyl groups introduced, to be precipitable by means of mercuric nitrate under conditions which lead to the precipitation of none of the natural amino acids except cysteine, cystine, methionine, histidine, tryptophan, and aspartic acid; these, however, unlike the a-cm-amino acids, are precipitable by mercuric acetate

TABLE I

Reaction of Amino, Phenolic, and Imidazole Groups with Haloacetates

Chloroacetate Bromoacetate Iodoacetate

Time Tyrosine Histidine Tyrosine Histidine Tyrosine Histidine

NH2 Phenol Imidazole NH2 Phenol Imidazole NHz Phenol Imidazole ____--~~~~~~~

hrs.

3 6 0 1 72 8 18 59 2 14 7 9 0 2 100 95

16 44 60 24 35 0 6 55 74 11 50 48 56 0 II 76 95 23 74 72 76 0 14 87 100 26 86

The reaction mixtures, 0.025 1% in amino acid and 0.215 M in haloacetate, were gently agitated at 35-37” with an excess of MgO. The values shown indicate the per cent disappearance of amino nitrogen and groups which yield color with diazobenzenesulfonic acid.

in the presence of 0.1 M sodium chloride. The mercuric derivatives of the a-cm-amino acids melt, with decomposition, at definite temperatures (Table II). They readily dissolve in dilute solutions of sodium chloride or hydrochloric acid containing at least one chlorine ion for every atom of mercury; the resulting soluble complexes presumably contain the grouping -COOHgCl, in analogy with the formulation suggested by Toennies and Kolb (17) in their study of the action of mercuric chloride on methionine. On the addition of excess of mercuric nitrate to such solutions, the derivatives are reprecipitated. In general, they appear to contain 1 atom of mercury for every two carboxyl groups.

The calcium salts of Cm-amino acids are, like calcium aspartate and glutamate, readily soluble in water but insoluble in alcohol. The same is true of the magnesium salts.


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Titration curves, typified by Figs. 3 and 4, indicate that the introduction of carboxymethyl groups has, as was shown by Michaelis and Schubert in the case of di-Cm-glycine (II), little effect on the basic function of the

TABLE II

Melting Decomposition Points of Hg Derivatives 01 Carboxymethylamino Acids Precipitated by Mercuric Nitrate

Di-Cm-glycine Di-Cm-alanine Di-Cm-valine Di-Cm-leucine Di-Cm-isoleucine Mono-Cm-phenylalanine

“C.

186-187 177-17s 163-164 169-171 157-158 NO-182

Di-Cm-phenylalanine 174-175 Cm-proline 178-180 Mono-Cm-aspartic acid 181-184 Mono-Cm-glutamic acid 174-176

Di-Cm-glutamic acid Di-Cm-serine O,N-Di-Cm-tyrosine Tri-Cm-tyrosine Tetra-Cm-lysine Di-Cm-arginine Di-Cm-histidine Tri-Cm-histidine N, S-Di-Cm-cysteine Di-Cm-methionine

“C.

174-175 168-170 192-194 186-189 160-162 185-188 203 213-218 165-173 128

PH 10.0 -

8.0 -

6.0 -

4.0 -

.5 I.0 1.5 2.0 2.5

PH IO.0

9.0

80

70

6.0

50

40

3.0

20

I 0

EQUIVALENTS NaOH HCI-EQUlVALENTS+NaOH

FIG. 3 FIG. 4

FIG. 3. Titration curve of di-Cm-leucine FIG. 4. Titration curve of tetra-Cm-lysine

nitrogen. Over the pH range 4 to 8, the compounds exhibit little buffering power and must therefore exist in essentially dipolar ionic form.

The optical rotations of the Cm-amino acids in water, acid, and alkali, three of which are recorded in Table III, suggest that dextrorotational minima exist, as is the case (18) with natural amino acids, in their non- buffering regions.


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In many instances, individual Cm-amino acids can be separated by paper chromatography. The RF values, obtained by the conventional method of upward migration, with a solvent consisting of a mixture of butanol, ethanol, and aqueous ammonia, are shown in Table IV. Identi- cal results were obtained wi-ith saline solutions of the mercury derivat’ives, the metallic constituent of which is detached, presumably by the ammonia, and remains immobile. The di-a-Cm-amino acids yielded dark purple spots with the reduced ninhydrin reagent of Long, Quayle, and Stedman (19), whereas the colors given by mono-c-r-Cm-amino acids had a pinkish purple hue.

TABLE III

Specific Rotations [cx]~ in Water, Acid, and Alkali

Di-Cm-leucine. Di-Cm-phenylalanine. Mono-Cm-aspartic acid..

water N-HCl N-NEtOH

degrees degrees degrees

$2.6 +19 fl5 +3.5 $24 f28 +4.1 +11 +11

TABLE IV RF Values (~1~0.0s) of Carboxymethylamino Acids

Di-Cm-glycine 0.37 Di-Cm-alanine 0.48 Di-Cm-valine 0.48 Di-Cm-leucine 0.73 Di-Cm-isoleucime 0.54 Mono-Cm-phenylalanine 0.62 Di-Cm-phenylalanine 0.62 Cm-proline 0.45 Mono-Cm-aspartic acid 0.35 Mono-Cm-glutamic acid 0.38

Di-Cm-serine 0.33 0-Mono-Cm-tyrosine 0.44 Tri-Cm-tyrosine 0.36 Di-Cm-lysine 0.32 Tetra-Cm-lysine 0.27 Di-Cm-arginine 0.28 Di-Cm-histidine 0.27 Tri-Cm-histidine 0.32 Di-Cm-methionine 0.37

Some Cm-amino acids can be extracted from solution in 0.1 N hydrochloric acid by ether or ethyl acetate. When a group of markedly hydro- phobic character is present in the molecule, as in the derivatives of leucine and phenylalanine (Table V), the distribution coefficient may exceed unity.

In this admittedly incomplete study of the carboxymethylation of amino acids, we were unable to secure demonstrably homogeneous products from serine, threonine, cystine, and tryptophan. Further work on these is nec- essary.

Cm- Peptides

A few typical synthetic peptides have been carboxymethylated under similar conditions. The products, which likewise formed insoluble com-


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plexes on treatment with mercuric nitrate, after acid hydrolysis gave the Cm- derivatives of the N-terminal amino acids in yields, in most cases, of over 90 per cent.

The isolation of the Cm- derivatives of the terminal amino acids is com- plicated by the fact that a few natural amino acids (see above) yield insoluble mercuric salts with mercuric nitrate. The same is true of compounds such as E-di-Cm-lysine or 0-Cm-tyrosine, in which the a-amino groups are unsubstituted. However, all of these, with the exception of histidine carboxymethylated exclusively in the imidazole ring, are precipitated by the addition of mercuric acetate to their solutions in 0.1 to 0.5 M NaCl. Under these conditions, none of the a-cm-amino acids hitherto examined is precipitated by mercuric acetate. On the other hand, aspartic acid and glu-

TABLE V

Distribution Ratios, (N per Ml. of Solvent)/(N per Ml. of 0.1 N HC

Compound Ether Ethyl acetate

Di-Cm-glycine 0.0 0.0

Di-Cm-alanine 0.01 0.04 Di-Cm-valine 0.29 1.25 Di-Cm-leucine 0.61 1.88 Di-Cm-isoleucine 0.67 1.75 Mono-Cm-phenylalanine 0.0 0.01 Di-Cm-phenylalanine 1.02 3.85 Cm-proline 0.0 0.0 Mono-Cm-aspartic acid 0.0 0.01 Mono-Cm-glutamic acid 0 0

Di-Cm-glutamic acid Di-Cm-serine 0-Mono-Cm-tyrosine 0, N-Di-Cm-tyrosine Tri-Cm-tyrosine Tetra-Cm-lysine Di-Cm-arginine Tri-Cm-histidine Di-Cm-methionine

Ether

0.029

0 0.0 0.05 0.00 0.00 0.00 0

Ethyl acetate

0.074 0.02 0.007 0.01 1.00 0.00 0.00 0.01 0

tamic acid are precipitated by mercuric acetate when the chloride concen- tration is less than 0.2 M, and the mercuric salt of imidazole-di-Cm-histidine is quantitatively precipitated on the addition of an equal volume of ethanol.

In the di-Cm- derivative of histidine mentioned above, produced from glycyl- and alanylhistidine with subsequent hydrolysis, one of the Cm- groups replaces the hydrogen atom of the nuclear imino group; the other contributes to the formation of a betaine-like structure, analogous to that described by Rung and Behrend (20).

CHEOO- CHzCOOH

I I +N=CH--N NH2

I I I CH- -----C-CH&HCOOH


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Histidine itself appears, under the experimental conditions, to accept the second carboxymethyl group on the imidazole nucleus with greater diffi- culty, if at all.

EXPERIMENTAL

Carboxymethylation of Amino Acids

In general, the reaction mixtures, consisting of an amino acid (0.025 M),

magnesium bromoacetate (0.2 M), and an excess of MgO were gently agitated at 35” for 3 days. Unless otherwise indicated, the solutions were filtered and acidified to Congo red; saturated mercuric nitrate solution was then added until no further precipitation occurred. Purification of the mercuric derivatives was effected by solution in 0.05 M NaCl and reprecipitation with mercuric nitrate. The precipitates were washed with water until free of nitrate, and decomposed with HZS. The resulting solutions were concentrated under reduced pressure to approximately 1 M, adjusted to pH 1 to 1.5 with NaOH, and the products isolated by the procedures described below.

Analytical estimations of mercury and nitrogen in the mercuric derivatives were conveniently carried out as follows. A weighed sample (30 to 50 mg.) was heated with 0.2 ml. of concentrated sulfuric acid in a tared centrifuge tube. When the mixture was nearly colorless (generally after 20 minutes), it was allowed to cool, diluted with 10 ml. of water, and saturated with hydrogen sulfide. The precipitate of HgS was well washed and dried to constant weight at 105’. The supernatant solution and washings were united, concentrated, and subjected to Kjeldahl analysis.

Di-Cm-alanine-The solution (5 ml.) from a 3 mmole preparation was adjusted to pH 1.3 and shaken with three 100 ml. portions of ethyl acetate. The extract, which contained 2.2 m.eq. of N, was taken to dryness under reduced pressure, 20 ml. of absolute alcohol were added, and the suspension was again evaporated in vacua. The residue was dissolved in 20 ml. of hot 95 per cent ethanol, the solution was allowed to cool, and the 258 mg. of product (1.2 m.eq. of N) which separated were recrystallized from 95 per cent ethanol. It melted at 208” with decomposition.

C?HIIOsN. Calculated, C 41.0, H 5.4, N 6.8; found, C 41.0, H 5.4, N 7.2

Di-Cm-valine-The solution (3 ml.) from a 3 mmole preparation at pH 1.6 was shaken with five 12 ml. portions of ethyl acetate. The extract (2.0 m.eq. of N) was concentrated to a syrup and dissolved in 1 ml. of hot ethyl acetate. The solution, which crystallized on cooling, was treated with 2 ml. of ether and allowed to stand for a few days. The dried product weighed 217 mg. (0.93 m.eq. of N). After purification by solution in


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1 ml. of wat’er, repetition of the extraction, and recrystallization from ethyl acetate, it melted at 176”.

CgH1506N. Calculated, C 46.4, H 6.4, N 6.0; found, C 46.6, H 6.6, N 6.1

Di-Cm-Zeucine-The solution (7 ml.) from a 9 mmole preparation lvas allowed to stand for several days at O-5”. The solid which separated was recrystallized three times from water. The product weighed 1.285 gm. (58 per cent of the theoretical amount), melted at 178”, and had [cy], +lS” (1.3 per cent in 0.5 N HCl) .

C~OHI~O~N. Calculated, C 48.6, H 6.9, N 5.7; found, C 48.4, H 6.7, N 5.6

The titration curve is shown in Fig. 3. The mercury derivative gave satisfactory analytical values for N and Hg,

but contained a small amount of Cl.

CmHsOmNiHg;. Calculated, N 2.6, Hg 55.4, Cl 0.0; found, N 2.5, Hg 55.2, Cl 1.9

The precipitability of the calcium salt is indicated by the following experiment. To a solution of 50 mg. (0.20 mmole) of di-Cm-leucine in 5 ml. of water, calcium hydroxide was added in excess. The alkaline suspension was centrifuged, the clear solution treated with 25 ml. of ethanol, and the mixture allowed to stand overnight in the refrigerator. After centrifuga- tion, the supernatant solution was found to contain 0.018 m.eq. of N.

Di-Cm-isoleucine-The solution from a 3 mmole preparation was shaken with four 20 ml. portions of ethyl acetate; the extract (2.4 m.eq. of N) was taken to dryness in vacua; the residue was dissolved in 3 ml. of warm ethyl acetate and the product precipitated by the addition of 3 ml. of light petroleum. After a second such reprecipitation, the yield was 251 mg. of an amorphous product melting at 121”. After three more such precipitations the melting point was 123-124”. This product was not obtained in crystalline condition.

CIOH1,OGN. Calculated, C 48.6, H 6.9, N 5.7; found, C 48.3, H 7.0, N 5.7

Mono-Cm-phenylalanine-The carboxymethylation mixture from a 3 mmole preparation was filtered and allowed to stand for a month in the refrigerator. The magnesium salt of the monosubstitution product, which separated in crystalline form, was washed with ice-cold water and dried at 120”. The yield was 150 mg.

C22H240sN2Mg.4Hz0. Calculated. C 48.8, H 5.9, N 5.2, Mg 4.5 Found. “ 48.6, (‘ 5.7, ‘( 5.2, “ 4.1

A 25 mg. portion of this salt was dissolved in 2.5 ml. of 0.1 N HCI. Crys- tallization of the free acid began almost immediately. After 24 hours at


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O-5”, the product was collected, washed, and dried over PzO6 in vacua at room temperature. Yield, 17 mg.; m.p. 228-233”, with decomposition.

CIIH,,OdN. Calculated, N 6.3; found, N 6.2

Di-Cm-phenylalanine-The original filtrate from the above magnesium salt was acidified to Congo red with KC1 and treated with 35 ml. of saturated mercuric nitrate solution. The precipitate was washed and dried; weight 1.935 gm. (2.3 m.eq. of N). The concentrated filtrate (8 ml.) obtained after removal of mercury as sulfide was adjusted to pH 1 and allowed to stand for a few days in the refrigerator. The crystalline di-cm-phenylalanine weighed 405 mg. After three recrystallizations from water it melted at 141” and had [aID +21” (1.4 per cent in 0.5 N HCl).

C13H1506N. Calculated, C 55.5, H 5.3, N 5.0; found, C 55.3, H 5.6, N 4.8

Cm-proline-The solution (2.5 ml.) from a 3 mmole preparation was treated with 15 ml. of acetone and cooled at O-5’ overnight. The syrupy precipitate was washed with 3 ml. of acetone, dissolved in 2 ml. of water, and treated with 1 ml. of methanol and 13 ml. of isopropanol. The crystalline product, weighing 231 mg. (1.3 m.eq. of N), was twice recrystallized from water with the addition of isopropanol. It melted at 220” (decomposition).

CTH1104N. Calculated, C 48.5, H 6.4, N 8.1; found, C 48.8, H 6.3, N 7.8

Mono-Cm-aspartic Acid-The reaction mixture from 1.000 gm. of aspartic acid (7.5 mmoles), 5.0 gm. of bromoacetic acid (36 mmoles), and excess of magnesium oxide in 75 ml. of water was filtered, concentrated at about 30” to 60 ml., and treated with 300 ml. of absolute ethanol. The precipitate was washed with 95 per cent ethanol. The filtrate and washings, which contained magnesium bromide, unchanged bromoacetate, and only 0.18 m.eq. of N, were discarded. The solid was dissolved in water, acidified to Congo red, and treated with an excess of mercuric nitrate solution. A portion of the precipitate was dissolved in NaCl, reprecipitated, and dried at 100’ for analysis.

C,,H1,012N2Hg3. Calculated, N 2.87, Hg 61.5; found, N 2.73, Hg 60.5

The remainder was suspended in water and decomposed with hydrogen sulfide. The filtrate, containing 6.4 m.eq. of N, was evaporated to dryness under reduced pressure, redissolved in 2 ml. of water, treated with 20 ml. of acetone, and allowed to stand overnight. The gummy precipitate, which had become microcrystalline and friable, was washed first with acetone containing 10 per cent of water, then with pure acetone. The filtrate and washings were evaporated to dryness and the residue was again treated


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with cold aqueous acetone, when a small second crop was secured. The combined product weighed 695 mg. (3.6 mmoles). On recrystallization by addition of isopropanol to a concentrated aqueous solution, it formed rec- tangular needles which melted at 198-199”.

CsH90sN. Calculated, C 37.7, H 4.7, N 7.3; found, C 37.8, H 4.7, N 7.2

A titration curve, determined in 0.1 M NaC1, showed the presence of three acid groups with apparent pK values of approximately 2, 2.4, and 3.9, respectively, and one basic group, pK 9.6.

In a similar preparation, the concentrated filtrate from the HgS was adjusted to pH 1.3 with sodium hydroxide before the addition of acetone. The product in this case was a crystalline, hydrated monosodium salt, m.p. 162”, [aID i-11.4” (1.2 per cent in 0.5 N HCl).

CsH806NNa.H20. Calculated. C 31.3, H 4.3, N 6.1, Na 10.0 Found. “ 31.8, “ 4.3, “ 6.1, (‘ 9.7

Di-Cm-aspartic acid appeared to be formed, as a minor product, when aspartic acid was treated with a larger excess of bromoacetate for a longer time and at a higher temperature: a mixture of 1.000 gm. of aspartic acid (7.5 mmoles), 10 gm. of bromoacetic acid (72 mmoles), and 5 gm. of magnesium oxide in 100 ml. of water was stirred for a week at 3742’, filtered, concentrated at 90” to a total weight of 30 gm., treated with 150 ml. of absolute ethanol, and allowed to stand at 3-5” for 3 days. The precipitate was collected and washed with ethanol. The filtrate and washings, which contained 0.13 m.eq. of N, were discarded; the solid product was dissolved in water, freed of alcohol by boiling, and acidified to pH 1 with 10 ml. of 5 N HzS04. This solution was subjected to continuous extraction with ethyl acetate for four successive 10 hour periods, during which 28, 4.3, 1.8, and 1.8 mg. of N, respectively, were extracted. The first extract, which weighed 2.7 gm. and contained much glycolic acid, was dissolved in water and treated with mercuric nitrate; 1.07 gm. of a mercuric derivative, m.p. 181-188”, were precipitated.

C8HTOsNHg,. Calculated, N 2.17, Hg 62.0; found, N 2.00, Hg 62.4

The acid solution remaining after the extraction yielded a mercuric salt which appeared to consist mainly of the mono-Cm- derivative (found, N 2.37, Hg 60.2).

Mono-Cm-glutamic Acid-The solution (5 ml.) from a 3 mmole preparation was adjusted to pH 1.2 and the product extracted with seven 20 ml. portions of ethyl acetate. To the aqueous phase, which contained 0.79 m.eq. of N, isopropanol (6 ml.) was added until a slight cloudiness appeared. The mixture, after a few days in the refrigerator, deposited crystals (30


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mg.) which were redissolved in water (2 ml.) and treated with isopropanol (7 ml.); crystallization occurred almost immediately; m.p. 155-156”.

C7Hl10eN. Calculated, C 41.0, H 5.4, N 6.8; found, C 41.2, II 5.4, N 6.9

Di-Cm-glutamic Acid-The above ethyl acetate extract, which contained 1.54 m.eq. of N, was evaporated to dryness under reduced pressure, redissolved in 5 ml. of hot ethyl acetate, and precipitated with light petroleum. This process was repeated. The resulting 270 mg. of amorphous product were crystallized from 3 ml. of hot ethyl acetate, when 15 mg. of crystalline, very hygroscopic di-Cm-glutamic acid were secured; m.p. 85”.

CgH1BOsN. Calculated, C 41.1, H 4.9, N 5.3; found, C 40.8, H 5.1, N 5.5

A titration curve indicated the presence of five dissociable groups, of ap- proximate pK values 2, 2.4, 3, 4.5, and 8.5.

Di-Cm-se&e-The concentrated filtrate (3 ml.) from a 3 mmole preparation was brought to pH 1.3 with 0.15 ml. of 11 N NaOH, treated with 6 ml. of acetone, and allowed to stand overnight in the refrigerator. The precipitated syrup was washed with 3 ml. of acetone and dissolved in I ml. of water. Addition of a mixture of 1.5 ml. of methanol and 3 ml. of isopropanol caused the precipitation of a syrup which was redissolved in 1 ml. of water and treated with 9 ml. of methanol. The precipitate, which soon solidified when the mixture was cooled to 5”, was collected after the addition of 3 ml. of ethanol at -10”. After being washed with ethanol and ether, the amorphous product was dried over P205 at room temperature; m.p. 150-155”. Attempts to crystallize this product failed.

CrHllOrN. Calculated, N 6.3; found, N 5.7

0-Mono-Cm-tyrosine-A solution of 335 mg. (1.5 mmoles) of N-acetyl- tyrosine was carboxymethylated and the product precipitated with mercuric nitrate in the usual way. After decomposition with hydrogen sulfide, the filtrate was concentrated to 3 ml. and boiled under a reflux for 20 hours with an equal volume of concentrated HCl. The excess of HCl was removed by repeated evaporation under reduced pressure. The residue was dissolved in water (5 ml.), brought to pH 1.7, and chilled overnight in the refrigerator. The crystalline product was washed with ice-cold water, then with acetone, and dried at 110”; yield, 75 mg. After recrystallization from boiling water (9 ml.), it melted, with decomposition, at 260-261”.

CllH1305N. Calculated, C 55.3, H 5.4, N 5.8; found, C 55.4, H 5.6, N 5.7

The mercury derivative, precipitated on the addition of mercuric acetate, melted at 205-208”.

0, N-Di-Cm-tyrosine-The solution from a 3 mmole preparation was


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shaken with six 7 ml. portions of ethyl acetate. During the extraction, a crystalline precipitate formed in the aqueous layer. This was washed with water and dried over PZOS; it weighed 90 mg. and contained 0.29 m.eq. of N. After recrystallization from hot water (1.5 ml.), it melted with decomposition at 241-243”.

C13H160TN. Calculated, C 52.5, II 5.1, N 4.7; found, C 52.5, H 5.3, N 4.8

As this compound gave no color with Millon’s reagent, one of the carboxymethyl groups must have been linked to the phenolic oxygen atom.

Tri-Cm-tyrosine-The foregoing ethyl acetate extract xvas evaporated to dryness and the residue recrystallized from ethyl acetate; yield, 560 mg. After recrystallization from hot water (5 ml.) it melted with decomposition at 183-184”.

C15H1709N. Calculated, C 50.7, H 4.8, N 3.9; found, C 50.7, H 5.1, N 3.9

Tetra-Cm-Zysine-The solution (4 ml.) from a 3 mmole preparation was brought to pH 1.3, treated with 17 ml. of acetone, and chilled at O-5” for a day. The syrupy precipitate Teas dissolved in 2 ml. of water, reprecipitated with 5 ml. of isopropanol, and redissolved in 2 ml. of water. The hot solution was treated with 14 ml. of hot methanol and chilled; the solid product, weighing 741 mg. (60 per cent of the theoretical amount), was twice recrystallized from 7 ml. portions of hot water. It mehed with decomposition at 220”.

C~H22010N2. Calculated, C 44.4, H 5.8, N 7.4; found, C 44.4, H 6.0, N 7.4

The titration curve (Fig. 4) indicates the presence of three titratable and two compensated acid groups.

Formation of the copper complex of lysine (15) failed to prevent the reaction of bromoacetate with the a-amino group. A solution of 344 mg. of lysine dihydrochloride in 30 ml. of water was adjusted to pH 8 with sodium hydroxide and heated on the steam bath for 2 hours with excess of basic copper carbonate. The mixture was then filtered. Aliquot portions were treated with magnesium oxide and bromoacetate by the standard procedure, and with sodium bromoacetate in the presence of sodium carbonate at pH 9.5. Addition of 5 volumes of 95 per cent ethanol to the resulting solutions caused the precipitation of blue, amorphous solids or syrups which, after the removal of copper by hydrogen sulfide, gave no color with ninhydrin and no amino nitrogen in the Van Slyke test.

Di-Cm-arginine-The reaction mixture from 1.6 mmoles of arginine and 18 mmoles of bromoacetate in 50 ml. of water was filtered and allowed to stand for 3 weeks in the refrigerator. The crystals which separated weighed


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323 mg. and contained 3.6 m.eq. of N. This salt was twice recrystallized from hot water and dried at 130”.

C10H1606N4Mg. Calculated, N 17.9, Mg 7.8; found, N 17.8, Mg 7.7

A 250 mg. portion of the Mg salt was dissolved in 20 ml. of 0.05 N HCl and treated with an excess of mercuric nitrate. The washed precipitate was decomposed with H$ and the filtrate concentrated to 1.5 ml. under reduced pressure. On the addition of 3.5 ml. of isopropanol, crystallization soon began. After 20 hours in the refrigerator, the product was washed with 60 per cent aqueous isopropanol; dried at 110” after recrystallization from water-isopropanol, it weighed 120 mg. and melted at 160-161”.

C10H1806N4. Calculated, N 19.3; found, N 19.9

B-i-Cm-histidine-The solution from a 3 mmole preparation was evaporated under reduced pressure and the syrupy residue was dissolved in 10 ml. of acetone. Treat’ment of this solution with 10 ml. of ethyl ether caused the separation of a liquid which was no longer soluble in acetone. It was dissolved in 2 ml. of water, treated with 15 ml. of isopropanol, and chilled to -lo”, when a gummy solid separated. The supernatant solution contained 4.5 m.eq. of N, which corresponded to 50 per cent of the histidine employed. The gum was converted to an amorphous solid by dissolving it in 3 ml. of water and adding 15 ml. of methanol. After being dried over PZ05, this product, which gave no color with the Pauly reagent, weighed 124 mg.

CnHdhNa. Calculated, N 12.8; found, N 12.2

It was not quite homogeneous, for on subjection to paper chromatography it yielded two spots. The major, more rapidly moving component gave the dark purple color characteristic of the di-a-cm-amino acids. The mi- nor, slower moving component gave a pinkish color with reduced ninhydrin and therefore appeared to be the cr-mono-Cm derivative.

N , S-Di-Cm-cysteine-Carboxymethylation of 3 mmoles of cysteine with 36 mmoles of bromoacetate was carried out for 3 days at 37”. The filtrate from the decomposition of the mercuric derivative was concentrated to 3 ml. and adjusted to pH 1.4, when a crystalline product separated. After being washed with cold water and dried, it weighed 150 mg.; m.p. 237” (decomposition).

CTHllOsNS. Calculated, N 6.0; found, N 6.4

Extraction of the mother liquor with ethyl acetate yielded a syrup which could not be induced to crystallize.

Di-Cm-methionine-The solution (5 ml.) from a 1.5 mmole preparation


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was brought to pH 1.7, treated with 25 ml. of methanol and 25 ml. of isopropanol, and chilled overnight in the refrigerator. The precipitate, which contained 0.61 m.eq. of N, was recrystallized three times from water-isopropanol; yield, 75 mg.; m.p. 168”.

C9H1506NS. Calculated, C 40.8, H 5.7, N 5.3; found, C 40.8, H 6.1, N 5.3

Cm-amino Acids from Peptides

The carboxylation of peptides was carried out exactly as with the amino acids. The products likewise yielded insoluble salts on treatment with mercuric nitrate but, as the precipitates could not be completely freed from nitrate ion by washing, they were decomposed with H&S and the calcium salts, precipitated by addition of alcohol, were subjected to acid hydrolysis. After the removal of most of the excess hydrochloric acid and adjustment of the pH to 1.5, mercuric acetate was added; in most cases this precipitated the amino acids, but not the Cm-amino acids. These were then precipitated with mercuric nitrate.

Glycylglycine-The peptide, prepared by the method of Dunn, Butler, and Deakers (21), was carboxymethylated with 9 molar equivalents of bromoacetate for 3 days at 36”. After filtration to remove excess MgO, 5 volumes of ethanol were added. The amorphous product was redissolved in a small quantity of water and reprecipitated by adding 5 volumes of ethanol. It contained 7.0 per cent of N but was not further characterized.

A 150 mg. portion (0.75 m.eq. of N) was hydrolyzed in 4 ml. of 6 N HCl for 22 hours under a reflux. The solution was evaporated under reduced pressure, most of the excess of HCl being removed by repeated vacuum distillation after addition of water. The residue was then treated with 5 ml. of water, when di-Cm-glycine immediately crystallized. After 20 hours at O-5” the product was collected, washed with cold water, and dried. The yield was 65 mg. (91 per cent of the theoretical amount) of di-Cm-glycine. It melted with decomposition at 242-251”.

CsHsOsN. Calculated, N 7.3; found, N 7.3

The filtrate from the crude di-Cm-glycine was made alkaline, shaken with benzoyl chloride, and acidified. The precipitate, after being washed with ligroin and recrystallized, yielded 45 mg. of hippuric acid, m.p. 183” (64 per cent of the theoretical amount).

Leucylglycylglycine-The peptide, synthesized by the method of Schott, Larkin, Rockland, and Dunn (22), possessed the melting point and specific rotation recorded by these authors. A 0.75 mmole portion was carboxymethylated by the standard procedure. The filtrate from the excess MgO was acidified to Congo red and treated with an excess of saturated mercuric nitrate. The resulting precipitate was well washed with water and decom-


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posed with HGS. The filtrate was concentrated in vacua to 2 ml., made alkaline with calcium hydroxide, and treated with 7 volumes of ethanol. The precipitate was washed with ethanol, redissolved in water, filtered, reprecipitated with ethanol, and dried over PZ06 at room temperature. The yield was 317 mg. of a calcium salt containing 1.99 m.eq. of N (88 per cent of the theoretical amount).

A 307 mg. portion of this product (1.92 m.eq. of N) was hydrolyzed in 6 ml. of 6 N HCl under a reflux for 40 hours. After removal of excess HCl under reduced pressure, the residue was dissolved in 3 ml. of water and the solution, which gave no precipitate with mercuric acetate, was treated with saturated mercuric nitrate. The precipitate was well washed with water, dried (347 mg.), dissolved in 15 ml. of M NaCl, and reprecipitated with mercuric nitrate. After being washed and dried over CaC&, the product contained 0.55 m.eq. of N (86 per cent of the theoretical amount). It melted at 169-171” with decomposition.

CdLdhN&~. Calculated, N 2.6, Hg 55.2; found, N 2.2, Kg 56.1

Glutathione-A solution of 0.55 mmole of the tripeptide in 10 ml. of water was treated with MgO, aerated until it no longer reduced phosphotung- stic acid, and carboxymethylated with 6 mmoles of bromoacetate. The filtrate was acidified with concentrated HCI (0.2 ml.) and treated with saturated mercuric nitrate (11 ml.). The well washed precipitate was decomposed with H&l. The filtrate was made alkaline with calcium hydroxide and twice precipitated with ethanol. It weighed 254 mg. and contained 1.30 m.eq. of N (79 per cent of the theoretical amount).

A sample of the calcium salt containing 0.586 m.eq. of N was hydrolyzed in 3 ml. of 6 N HCl under a reflux for 24 hours; the excess of HCI was evaporated and the residue dissolved in 2.5 ml. of water. The solution was adjusted to pH 1 and treated with 2 ml. of 1 M mercuric acetate; the precipitate, presumably the mercuric complex of cystine, weighed 109 mg. and contained 0.154 m.eq. of N (79 per cent of the theoretical amount).

On addition of 3 ml. of saturated mercuric nitrate to the supernatant solution and washings, the mercury derivative of the di-Cm-glutamic acid was collected. After reprecipitation, this weighed 99 mg. and contained 0.144 m.eq. of N (74 per cent of the theoretical amount). It melted with decomposition at 174-175”.

CJMhNHgz. Calculated, N 2.1, Kg 60.7; found, N 2.0, Hg 58.8

Alanyllysylalanine-An aqueous solution (kindly furnished by the late Dr. Erwin Brand of this laboratory) of 0.43 mmole of the peptide (23) was carboxymethylated with 4.3 mmoles of bromoacetate; the mercuric derivative of the product was prepared and decomposed with H2S. The car-


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boxymethylated tripeptide, precipitated as its calcium salt with the aid of ethanol, weighed 161 mg. and contained 0.96 m.eq. of N (56 per cent of the theoretical amount).

A solution of 149 mg. of this calcium salt, containing 0.89 m.eq. of N, in 3 ml. of 6 N HCl was boiled under a reflux for 18 hours. The excess of HCl was removed under reduced pressure and the residue dissolved in 3 ml. of water. To this solution, mercuric acetate solution (1 molar in 1 per cent acetic acid) was added until precipitation was complete (2 ml.). The supernatant solution and washings were concentrated in vacua to 2 ml. and a second small quantity of precipitate was collected. There were thus obtained 224 mg. of a mercury salt of E-di-Cm-lysine containing 0.44 m.eq. of N (99 per cent of the theoretical amount). The compound was purified by solution in dilute sodium chloride and reprecipitation with mercuric acetate. It melted with decomposition at 165-167’.

C10H1506N&1Hg2. Calculated. N 4.0, Cl 5.1, Hg 57.6 Found. “ 4.0, ‘I 5.4, “ 56.2

The product obtained after removal of mercury failed to crystallize. The supernatant solution from the mercuric c-di-Cm-lysine was treated

with 1.3 ml. of saturated mercuric nitrate, which precipitated 151 mg. of mercuric di-Cm-alanine containing 0.22 m.eq. of N (99 per cent of the theoretical amount). After two reprecipitations the product melted with decomposition at 177-178”.

C14H16012N2Hg3. Calculated, N 2.8, Hg 59.8; found, N 2.7, Hg 56.2

Glycylhistidine-Samples of this peptide (24), kindly provided by Dr. du Vigneaud, were carboxymethylated with 12 equivalents of bromoacetate under the standard conditions for 6 and for 25 hours. The Pauly react,ion then indicated that 66 and 92 per cent, respectively, of the imidazole group had reacted. The reaction mixture from the 25 hour sample was acidified and treated with mercuric nitrate. The precipitate was mashed and decomposed with hydrogen sulfide; the filtrate was rendered alkaline with calcium hydroxide, filtered, and treated with 2 volumes of ethanol. The precipitate contained 76 per cent of the nitrogen in the starting material.

A portion containing 0.54 m.eq. of N was hydrolyzed wit’h boiling 6 N

HCl for 20 hours. The excess of HCl was removed by distillation under reduced pressure, and the residue was dissolved in 3 to 4 ml. of water and adjusted t’o pH 2 with sodium hydroxide, when di-Cm-glycine rapidly crystallized. After a few days in the refrigerator the crystals were collected, washed with water, then with alcohol, and dried; yield, 19 mg.; m.p. 245- 247” (decomposition).

CsHoOaN. Calculated, N 7.3; found, N 7.4


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The mother liquor was treated with mercuric acetate and an equal volume of ethanol, whereby a mercuric salt of imidazole-di-Cm-histidine was precipitated. After being washed with 50 per cent ethanol and dried, it weighed 119 mg.; m.p. 256-257” (decomposition).

‘&HdhN~CLHg~. Calculated. N 4.5, Cl 7.6, Hg 64.0 Found. “ 4.8, “ 7.9, “ 66.4

The filtrate was treated with mercuric nitrate, which precipitated the mercuric salt of a little di-Cm-glycine which had not crystallized. This product after reprecipitation weighed 15 mg.; m.p. 180-183” (decomposition).

ClzH1201zNaHg8. Calculated, N 2.9; found, N 3.2

The total recoveries of di-Cm-glycine and di-Cm-histidine were accord- ingly 100 and 101 per cent of the theoretical amounts, respectively, on the basis of nitrogen content.

The 6 hour run yielded products with identical properties, but only 52 per cent of the peptide nitrogen was obtained as the Ca salt, from which the hydrolytic products were obtained in respective yields of 98 and 90 per cent. In this case, a small amount of precipitate appeared on the addition of mercuric acetate prior to the addition of alcohol. This was probably the imidazole mono-Cm- derivative but was not investigated.

Alanylhistidine-This peptide, synthesized by Dr. J. Polatnick of this laboratory by the method of Hunt and du Vigneaud (25), was employed in the form of its dihydrochloride, 450 mg. (1.5 mmoles) of which were carboxymethylated with 18 mmoles of bromoacetate for 24 hours at 35-37”. The product was precipitated with mercuric nitrate, regenerated with hydrogen sulfide, and isolated as the alcohol-insoluble calcium salt. This weighed 470 mg. and contained 2.85 m.eq. of N, corresponding to a yield of 48 per cent of the theoretical amount.

Hydrolysis of 213 mg. of this (1.29 m.eq. of N) was carried out in 5 ml. of boiling 6 N HCI. The excess of acid was volatilized; the residue was dissolved in water and the pH raised from 0.3 to 1.6 in a final volume of 5 ml. No precipitate was formed on the addition of mercuric acetate.2

Mercuric nitrate gave 425 mg. of a precipitate which contained 1.18 m.eq. of N (91 per cent of total) and was therefore a mixture. After reprecipitation of 406 mg., the weight fell to 298 mg.; the product still contained three times as much nitrogen (0.95 m.eq.) as could have been present as di-Cm-alanine. A 282 mg. portion (0.90 m.eq. of N) was decomposed

2 When this experiment was performed, it had not yet been recognized that precipitation of mercuric imidazole-di-Cm-histidine could be elicited by addition of alcohol.


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with HzS; the filtrate was concentrated to 13 ml. and shaken with five 100 ml. portions of ethyl acetate. The evaporated extract was dissolved in 3 ml. of ethanol. By addition of 8 ml. of ethyl acetate, followed by 30 ml. of light petroleum, 15 mg. of pure di-Cm-alanine (m.p. 207-208’; N, 6.8) were secured.

The aqueous solution remaining after the extraction with ethyl acetate was diluted with 3 volumes of isopropanol and chilled overnight in the refrigerator. The solid which separated contained 0.64 m.eq. (95 per cent of the theoretical amount) of N. After two recrystallizations from water- isopropanol it melted at 263-264” (decomposition) and gave analytical values for imidazole-di-Cm-histidine.

C10H1306N3. Calculated. C 44.3, H 4.8, N 15.5, NH,-N 5.2 Found. “ 44.3, ‘I 5.0, “ 15.5, “ 5.1

On treatment with mercuric nitrate, this compound yielded a precipitate which melted with decomposition at 203” after darkening at 196-198”. Mercuric acetate yielded a precipitate (m.p. 256-257”, decomposition) in 50 per cent alcoholic, but not in aqueous, solution.

Chromatography of Cm-amino Acids-The solvent employed was a mixture of n-butanol (2 volumes), ethanol (1 volume of 95 per cent), and aqueous ammonia (2 volumes of 3 N). The paper was No. 1 Whatman, 15 to 20 cm. in width. After at least 4 hours for equilibration with the atmos- phere within the vessel, upward chromatographic migration was allowed to proceed for about 20 hours at room temperature (29-31’). The solvent front was marked; the paper was dried at 70-80” for 15 to 20 minutes, sprayed with the reduced ninhydrin reagent of Long, Quayle, and Stedman (19), heated at 110-115” for 1 to 3 minutes, and allowed to stand at room temperature overnight. The background color had then faded; the spots of di-Cm-amino acids were purple, those of mono-Cm-amino acids pink, and those of native amino acids dark bluish brown.

Distribution Coeficients-Solutions of about 20 mg. of each Cm-amino acid in 3 ml. of 0.1 N HCl were shaken for 90 minutes at room temperature with appropriate volumes (7 ml. or 12 ml.) of ethyl ether or ethyl ace- t’ate. When the layers had separated, measured samples of each werr withdrawn and subject,ed to Kjeldahl analysis. The results are presented in Table V.

SUMMARY

1. Simple a-amino acids react with bromoacetate at, pH 9, with replace- ment of the amino hydrogen atoms by carboxymethyl groups. In most cases, two such groups enter the molecule, but monosubstitution products can be isolated from phenylalanine and glutamic acid, and with aspartic acid the second group enters hardly at all.


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2. Under the conditions adopted, the hydroxyl group of tyrosine, the c-amino group of lysine, and the imidaxole group of histidine also accept carboxymethyl groups. The guanidino group of arginine does not react.

3. Carboxymethylamino acids form insoluble mercuric salts on treatment with mercuric nitrate, but not with mercuric acetate.

4. On treatment with bromoacetate, peptides form products from which, after acid hydrolysis, the carboxymethylated N-terminal amino acids can be isolated in high yields.

BIBLIOGRAPHY

1. Sanger, F., B&hem. J., 39,507 (1945). 2. Desnuelle, P., Rovery, M., and Fabre, C., Compt. rend. tIcad., 233, 987 (1951).

Schroeder, W.A., andLeGette, J., J. Am. Chem. Sot., 76,4612 (1953). Fletcher, C. M., Lowther, A. G., and Reith, W. S., Biochem. J., 66, 106 (1954).

3. Eschweiler, W., Ann. Chem.., 279,39 (1894). 4. Irvin, J. L., and Wilson, D. W., J. Biol. Chem., 127, 555 (1939). 5. Karrer, P., and Appenzeller, R., Helv. chim. acta, 26, 1149 (1942). 6. Karrer, P., Koenig, H., and Legler, R., Helv. chim. acta, 24, 127, 861 (1941). 7. Karrer, P., and Appenzeller, R., Helv. chim. acta, 26, 595 (1942). 8. Karrer, P., and Brandenberger, H., Helv. chim. acta, 34, 82 (1951). 9. Kogl, P., and de Flines, J., Rec. trav. dim. Pays-Bus, 72, 1009 (1953).

10. Heintz, W., Ann. Chem. u. Pharm., 122, 257 (1862). 11. Michaelis, L., and Schubert, M. P., J. Biol. Chem., 106,331 (1934). 12. Dickens, F., Biochem. J., 27,114l (1933). 13. Rapkine, L., Compt. rend. Sot. biol., 112,790 (1933). 14. Holiday, E. R., Biochem. J., 32, 1166 (1938). 15. Kurtz, A. C., J. BioZ. Chem., 140,705 (1941). 16. Huggins, C., and Jensen, E. V., J. Biol. Chem., 179, 645 (1949). 17. Toennies, G., and Kolb, J. J., J. Biol. Chem., 126, 367 (1938). 18. Litz, O., and Jirgensons, B., Ber. them. Ges., 63, 448 (1930). 19. Long, A. G., Quayle, J. R., and Stedman, R. J., b. Chem. Xoc., 2197 (1951). 20. Rung, F., and Behrend, M., Ann. Chem., 271,28 (1892). 21. Dunn, M. S., Butler, A. W., and Deakers, T., J. BioZ. Chem., 99, 217 (1932-33). 22. Schott, H. F., Larkin, J. B., Rockland, L. B., and Dunn, M. S., J. Org. Chem., 12,

490 (1947). 23. Brand, E., Erlanger, B. F., Polatnick, J., Sachs, H., and Kirschenbaum, D., J.

Am. Chem. Sot., 73, 4027 (1951). 24. Hunt, M., and du Vigneaud, V., J. BioZ. Chem., 127,43 (1939). 25. Hunt, M., and du Vignenud, V., J. Biol. Chem., 124.699 (1938).


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Samuel Korman and Hans T. ClarkePEPTIDES

CARBOXYMETHYLAMINO ACIDS AND

1956, 221:113-132.J. Biol. Chem.

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