ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Some Chemical
of Biochemistry,
(1975)
Properties of Carboxymethyl Amino Acids
CHRISTOPHER Department
167, 448-451
University
C. Q. CHIN’ of Minnesota,
Received
AND FINN Medical
October
School,
Derivatives
of
WOLD’ Minneapolis,
Minnesota
55108
15, 1974
Tritium-labeled carboxymethyl derivatives of various functional groups in proteins show ready exchange of the tritium label when exposed to standard protein hydrolysis conditions (6 N HCl, 21 h, 110°C). While N,-[3H]carboxymethyl lysine does not show any tritium exchange, the two N- [3H]carboxymethyl histidine derivatives lose their tritium with a half time of about 15 h, and S- [3H]carboxymethyl cysteine loses its tritium with a half time of about 1.5 h. The tritium exchange of S- [SH]carboxymethyl methionine was so fast that the derivative could not be prepared with any of the tritium label intact. The rate of exchange for this compound was consequently determined by following the disappearance of the methylene NMR signal when S-carboxymethyl methionine was dissolved in D,O. The half time for the exchange was about 12 min. Mechanisms involving either a sulfonium ion or enolization of the protonated conjugate carboxylic acid appear to give a satisfactory explanation of the relative stability of the different derivatives. The practical use of the differential rate of hydrogen exchange as a means of establishing rapidly and with small quantities of material the site of carboxymethylation in unknown proteins is suggested.
Haloacetates are among the most commonly used reagents in protein chemistry and enzymology. They react readily with the thiol group of cysteine, the thioether group of methionine, the imidazole nitrogens of histidine and the unprotonated t-amino group of lysine, and have been very useful in establishing the involvement of any of these groups in the activity function of proteins. The resulting carboxymethyl derivatives of cysteine, histidine and lysine are stable enough to withstand normal acid hydrolysis, but S-carboxymethyl methionine is completely destroyed during hydrolysis. The favored method for quantifying the extent of carboxymethylation of a protein is based on the use of the appropriately radio labeled reagent. In some recent studies on the inactivation of yeast alcohol dehydrogenase with “C- and 3H-labeled iodoacetate we found that the SH-label was readily lost from some of the protein derivatives. BeI Present address: Department University of Minnesota, St. Paul,
cause of the practical as well as more theoretical mechanistic significance of this finding, the phenomenon was studied further with the hope of establishing the chemical basis for the 3H-lo~~. The results of these studies are reported in this paper. EXPERIMENTAL
[3H]Iodoacetic acid (357 mCi/mmol and [3H]water (1 mCi/g) were obtained from New England Nuclear and diluted to appropriate specific radioactivity. The different [3H]carboxymethylated amino acid derivatives were prepared by standard procedures (see (1) for a review) by reacting the copper complexes of the amino acids with an excess of iodoacetate at pH 9.5. The carboxymethylated derivatives were separated from excess reagent and from copper by ion exchange chromatography on Dowex 50H+ eluted with 0.05 M pyridine acetate, pH 3.1. The products were collected by lyophilization, and their purity and specific activity were established via their elution position and quantification on the Beckman Model 120C Amino Acid Analyzer. Unlabeled S-carboxymethyl methionine was prepared by the method of Gundlach et al. (21, and the product was again purified by ion exchange chromatography. Radioactive counting was performed with a Beckman LS-133 liquid scintillation
of Biochemistry, MN 55108. 448
Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
SECTION
CARBOXYMETHYL
DERIVATIVES
spectrometer using glass vials containing 10 ml of Beckman fluoralloy TLA-toluene cocktail containing 10% (v/v) Bio Solv formula BBS-3 solubilizer. Nuclear magnetic resonance spectra were obtained with a Varian T60 Spectrometer. Specifically the following properties of the different carboxymethyl derivatives were used in their characterization. (The elution times from the long column of the amino acid analyzer are given relative to the following amino acid positions: cysteic acid, 19 min; aspartic acid, 43 min; proline, 68 min; alanine, 87 min; valine, 112 min.) S-carboxymethyl cysteine eluted as a single peak from the amino acid analyzer at 40 min. Upon oxidation with performic acid at 0°C the compound was converted primarily to the sulfone, eluting at 18-19 min and to a small amount of sulfoxide, eluting at 21 min. Acid Ihydrolysis (6 N HCl at 110°C for 21 hl of the oxidized derivative gave a 15% recovery of the sulfone and traces of cysteine. These observations are consistent with the data in the literature (3) on S-carboxymethyl cysteine and its oxidized derivatives. One additional bit of information gathered in this work relates to the products of the acid hydrolysis of S-carboxymethyl cysteine sulfone. As already stated, 85% of the starting material was destroyed by acid hydrolysis and no ninhydrin-positive product was recovered. When the hydrolysate was subjected to quantitative assays for pyruvate by the lactate dehydrogenase-NADH method, 80% of the starting sulfone was accounted for as pyruvate, or since the enzyme will act on several keto acids, a keto acid, most likely pyruvate. This suggests that the major degradation path for S-carboxymethyl cysteine sulfone under these conditions is through an elimination to yield dehydroalanine which in turn gives rise to pyruvate. The mechanism of this proposed reaction is not understood, but his under investigation. S-Carboxymethyl methionine eluted from the amino acid analyzer as the characteristic double peak of the two diastereoisomers at 45 and 47 min. Since this corresponds to the elution position of the isomer pair of methionine sulfoxides, the synthetic product was treated with 0.2 M phosphate buffer, pH 6.5 for 1 h at 100°C and was found to be converted essentially quantitatively to homoserine. This is a characteristic property of S-carboxymethyl methionine (2). The product obtained from ion exchange chromatography contained a small impurity of homoserine (58%). Carboxymethyl histidine. Both the N-l and the N-3 derivatives were characterized only by their elution positions (1) at 72 min and 100 min, respectively. The two derivatives were separated by ion exchange chromatography and studied individually. N,-Carboxymethyl lysine, eluted from the long column at 133 min (2-3 min after the buffer change); it was readily separated from about 10% of the by-product, dicarboxymethyl lysine.
OF AMINO
449
ACIDS
The hydrogen exchange reaction was followed either by exposing the pure [3H Jcarboxymethyl amino acid derivatives to 6 M HCl or by exposing the unlabeled derivative to [3H]H,0-6 M HCI for different lengths of time, transferring the content of the sealed reaction vial to a lyophilizer flask, and taking the sample to dryness by repeated lyophilization from water. The final, constant radioactive count obtained for the pure derivative, quantified on the amino acid analyser, gave the fraction of tritium remaining. In the case of S-carboxymethyl methionine, the exchange between unlabeled derivative and [2H]H,0 was followed by monitoring the methylene singlet nmr signal at T 5.8 (relative to tetramethylsilane at T 10). RESULTS
The rate of the exchange of the methylene hydrogens of N,- [3H]carboxymethyl lysine, N,- and N,- [3H]carboxymethyl histidine and S- [3H]carboxymethyl cysteine in 6 MHC~ and at 110°C is shown in Fig. 1. The lysine derivative loses tritium very slowly under these conditions, both of the histidine derivatives have a significant exchange, and the cysteine derivative exchanges quite readily. To establish whether the C-H bond labilization was restricted to the methylene of the carboxymethyl group, a reverse exchange was carried out with S-carboxymethyl cysteine. After incubation of S-carboxymethyl cysteine in 6 M HC1-[3H]H,0 at 110°C for 21 and 96 h, the incorporated radioactivity corresponds to 2.0 and 2.1 hydrogen atoms exhanged per mole of derivative, showing that a single methylene group is involved in the exchange. Performic acid oxidation at 0” did not cause any loss of tritium. It should be emphasized that the recovery of all three carboxymethyl derivatives after exposure to 6 M HCl at 110°C for up to 96 h was essentially lOO%, if proper precautions
0
5
IO
I5
20
TIME I hours I
FIG. 1. The rate of exchange of the carboxymethyl methylene hydrogens of different amino acid derivatives upon incubation at 110°C in 6 N HCl.
450
CHIN AND WOLD
-0
IO
20 30 TIME (minutes)
40
FIG. 2. The rate of exchange of the carboxymethyl methylene hydrogens of S-carboxymethyl methionine, obtained by following the decrease in the methylene nmr signal (7 5.8 relative to tetramethylsilane at T 10.0) when S-carboxymethyl methionine acetate was dissolved in D,O.
were taken to eliminate oxygen from the reaction mixture. (Actual values varied from 85 to 100% in different experiments.) The results for S-carboxymethyl methionine are presented separately in Fig. 2. This derivative was found to be unique in its very rapid loss of tritium; so rapid in fact that the product obtained from the reaction of [3H]iodoacetate with methionine never contained more than traces of radioactivity after purification on the ion exchange column. (This was the earliest step at which an accurate tritium analysis could be carried out with any certainty that all side products had been removed.) In order to evaluate the rate of hydrogen exchange in this derivative, the rate of loss of the characteristic methylene hydrogen signal from S-carboxymethyl methionine in D,O was followed as indicated in Fig. 2. The half life of the exchange reaction was found to be about 12 min. The exchangeability in strong acid of methylene hydrogens adjacent to a carboxy1 group is well established, and based on the previous studies of hydrogen exchange in the y-methylene of glutamic acid (tM in 25% HCl at 100°C of 4 days) (4) and in the p-methylene of aspartic acid (complete exchange in 6 M (18%) HCl at 110°C in 22 h) (5), it has been suggested that the rate of exchange is dependent upon the ease with which the adjacent carboxyl group can be protonated (5):
\
z=z OH
\
OH
It is clear that another mechanism must be invoked for the S-carboxymethyl derivatives of methionine and cysteine. The extremely rapid exchange in the case of methionine suggests that the sulfonium ion is involved, and that the most likely mechanism is: 7H3 R-;-3H-COO,,
OH _c
R-CH=C’
\
OH
-
y3 R-S=CH-COOH
By analogy,‘it seems reasonable that the carboxymethyl hydrogens of S-carboxymethyl cysteine can be labilized in two distinct ways corresponding to both of the above mechanisms, ,-,J+,~” $H 4 R-S-Lc
OF BIOCHEMISTRY
AND
BIOPHYSICS
Some Chemical
of Biochemistry,
(1975)
Properties of Carboxymethyl Amino Acids
CHRISTOPHER Department
167, 448-451
University
C. Q. CHIN’ of Minnesota,
Received
AND FINN Medical
October
School,
Derivatives
of
WOLD’ Minneapolis,
Minnesota
55108
15, 1974
Tritium-labeled carboxymethyl derivatives of various functional groups in proteins show ready exchange of the tritium label when exposed to standard protein hydrolysis conditions (6 N HCl, 21 h, 110°C). While N,-[3H]carboxymethyl lysine does not show any tritium exchange, the two N- [3H]carboxymethyl histidine derivatives lose their tritium with a half time of about 15 h, and S- [3H]carboxymethyl cysteine loses its tritium with a half time of about 1.5 h. The tritium exchange of S- [SH]carboxymethyl methionine was so fast that the derivative could not be prepared with any of the tritium label intact. The rate of exchange for this compound was consequently determined by following the disappearance of the methylene NMR signal when S-carboxymethyl methionine was dissolved in D,O. The half time for the exchange was about 12 min. Mechanisms involving either a sulfonium ion or enolization of the protonated conjugate carboxylic acid appear to give a satisfactory explanation of the relative stability of the different derivatives. The practical use of the differential rate of hydrogen exchange as a means of establishing rapidly and with small quantities of material the site of carboxymethylation in unknown proteins is suggested.
Haloacetates are among the most commonly used reagents in protein chemistry and enzymology. They react readily with the thiol group of cysteine, the thioether group of methionine, the imidazole nitrogens of histidine and the unprotonated t-amino group of lysine, and have been very useful in establishing the involvement of any of these groups in the activity function of proteins. The resulting carboxymethyl derivatives of cysteine, histidine and lysine are stable enough to withstand normal acid hydrolysis, but S-carboxymethyl methionine is completely destroyed during hydrolysis. The favored method for quantifying the extent of carboxymethylation of a protein is based on the use of the appropriately radio labeled reagent. In some recent studies on the inactivation of yeast alcohol dehydrogenase with “C- and 3H-labeled iodoacetate we found that the SH-label was readily lost from some of the protein derivatives. BeI Present address: Department University of Minnesota, St. Paul,
cause of the practical as well as more theoretical mechanistic significance of this finding, the phenomenon was studied further with the hope of establishing the chemical basis for the 3H-lo~~. The results of these studies are reported in this paper. EXPERIMENTAL
[3H]Iodoacetic acid (357 mCi/mmol and [3H]water (1 mCi/g) were obtained from New England Nuclear and diluted to appropriate specific radioactivity. The different [3H]carboxymethylated amino acid derivatives were prepared by standard procedures (see (1) for a review) by reacting the copper complexes of the amino acids with an excess of iodoacetate at pH 9.5. The carboxymethylated derivatives were separated from excess reagent and from copper by ion exchange chromatography on Dowex 50H+ eluted with 0.05 M pyridine acetate, pH 3.1. The products were collected by lyophilization, and their purity and specific activity were established via their elution position and quantification on the Beckman Model 120C Amino Acid Analyzer. Unlabeled S-carboxymethyl methionine was prepared by the method of Gundlach et al. (21, and the product was again purified by ion exchange chromatography. Radioactive counting was performed with a Beckman LS-133 liquid scintillation
of Biochemistry, MN 55108. 448
Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
SECTION
CARBOXYMETHYL
DERIVATIVES
spectrometer using glass vials containing 10 ml of Beckman fluoralloy TLA-toluene cocktail containing 10% (v/v) Bio Solv formula BBS-3 solubilizer. Nuclear magnetic resonance spectra were obtained with a Varian T60 Spectrometer. Specifically the following properties of the different carboxymethyl derivatives were used in their characterization. (The elution times from the long column of the amino acid analyzer are given relative to the following amino acid positions: cysteic acid, 19 min; aspartic acid, 43 min; proline, 68 min; alanine, 87 min; valine, 112 min.) S-carboxymethyl cysteine eluted as a single peak from the amino acid analyzer at 40 min. Upon oxidation with performic acid at 0°C the compound was converted primarily to the sulfone, eluting at 18-19 min and to a small amount of sulfoxide, eluting at 21 min. Acid Ihydrolysis (6 N HCl at 110°C for 21 hl of the oxidized derivative gave a 15% recovery of the sulfone and traces of cysteine. These observations are consistent with the data in the literature (3) on S-carboxymethyl cysteine and its oxidized derivatives. One additional bit of information gathered in this work relates to the products of the acid hydrolysis of S-carboxymethyl cysteine sulfone. As already stated, 85% of the starting material was destroyed by acid hydrolysis and no ninhydrin-positive product was recovered. When the hydrolysate was subjected to quantitative assays for pyruvate by the lactate dehydrogenase-NADH method, 80% of the starting sulfone was accounted for as pyruvate, or since the enzyme will act on several keto acids, a keto acid, most likely pyruvate. This suggests that the major degradation path for S-carboxymethyl cysteine sulfone under these conditions is through an elimination to yield dehydroalanine which in turn gives rise to pyruvate. The mechanism of this proposed reaction is not understood, but his under investigation. S-Carboxymethyl methionine eluted from the amino acid analyzer as the characteristic double peak of the two diastereoisomers at 45 and 47 min. Since this corresponds to the elution position of the isomer pair of methionine sulfoxides, the synthetic product was treated with 0.2 M phosphate buffer, pH 6.5 for 1 h at 100°C and was found to be converted essentially quantitatively to homoserine. This is a characteristic property of S-carboxymethyl methionine (2). The product obtained from ion exchange chromatography contained a small impurity of homoserine (58%). Carboxymethyl histidine. Both the N-l and the N-3 derivatives were characterized only by their elution positions (1) at 72 min and 100 min, respectively. The two derivatives were separated by ion exchange chromatography and studied individually. N,-Carboxymethyl lysine, eluted from the long column at 133 min (2-3 min after the buffer change); it was readily separated from about 10% of the by-product, dicarboxymethyl lysine.
OF AMINO
449
ACIDS
The hydrogen exchange reaction was followed either by exposing the pure [3H Jcarboxymethyl amino acid derivatives to 6 M HCl or by exposing the unlabeled derivative to [3H]H,0-6 M HCI for different lengths of time, transferring the content of the sealed reaction vial to a lyophilizer flask, and taking the sample to dryness by repeated lyophilization from water. The final, constant radioactive count obtained for the pure derivative, quantified on the amino acid analyser, gave the fraction of tritium remaining. In the case of S-carboxymethyl methionine, the exchange between unlabeled derivative and [2H]H,0 was followed by monitoring the methylene singlet nmr signal at T 5.8 (relative to tetramethylsilane at T 10). RESULTS
The rate of the exchange of the methylene hydrogens of N,- [3H]carboxymethyl lysine, N,- and N,- [3H]carboxymethyl histidine and S- [3H]carboxymethyl cysteine in 6 MHC~ and at 110°C is shown in Fig. 1. The lysine derivative loses tritium very slowly under these conditions, both of the histidine derivatives have a significant exchange, and the cysteine derivative exchanges quite readily. To establish whether the C-H bond labilization was restricted to the methylene of the carboxymethyl group, a reverse exchange was carried out with S-carboxymethyl cysteine. After incubation of S-carboxymethyl cysteine in 6 M HC1-[3H]H,0 at 110°C for 21 and 96 h, the incorporated radioactivity corresponds to 2.0 and 2.1 hydrogen atoms exhanged per mole of derivative, showing that a single methylene group is involved in the exchange. Performic acid oxidation at 0” did not cause any loss of tritium. It should be emphasized that the recovery of all three carboxymethyl derivatives after exposure to 6 M HCl at 110°C for up to 96 h was essentially lOO%, if proper precautions
0
5
IO
I5
20
TIME I hours I
FIG. 1. The rate of exchange of the carboxymethyl methylene hydrogens of different amino acid derivatives upon incubation at 110°C in 6 N HCl.
450
CHIN AND WOLD
-0
IO
20 30 TIME (minutes)
40
FIG. 2. The rate of exchange of the carboxymethyl methylene hydrogens of S-carboxymethyl methionine, obtained by following the decrease in the methylene nmr signal (7 5.8 relative to tetramethylsilane at T 10.0) when S-carboxymethyl methionine acetate was dissolved in D,O.
were taken to eliminate oxygen from the reaction mixture. (Actual values varied from 85 to 100% in different experiments.) The results for S-carboxymethyl methionine are presented separately in Fig. 2. This derivative was found to be unique in its very rapid loss of tritium; so rapid in fact that the product obtained from the reaction of [3H]iodoacetate with methionine never contained more than traces of radioactivity after purification on the ion exchange column. (This was the earliest step at which an accurate tritium analysis could be carried out with any certainty that all side products had been removed.) In order to evaluate the rate of hydrogen exchange in this derivative, the rate of loss of the characteristic methylene hydrogen signal from S-carboxymethyl methionine in D,O was followed as indicated in Fig. 2. The half life of the exchange reaction was found to be about 12 min. The exchangeability in strong acid of methylene hydrogens adjacent to a carboxy1 group is well established, and based on the previous studies of hydrogen exchange in the y-methylene of glutamic acid (tM in 25% HCl at 100°C of 4 days) (4) and in the p-methylene of aspartic acid (complete exchange in 6 M (18%) HCl at 110°C in 22 h) (5), it has been suggested that the rate of exchange is dependent upon the ease with which the adjacent carboxyl group can be protonated (5):
\
z=z OH
\
OH
It is clear that another mechanism must be invoked for the S-carboxymethyl derivatives of methionine and cysteine. The extremely rapid exchange in the case of methionine suggests that the sulfonium ion is involved, and that the most likely mechanism is: 7H3 R-;-3H-COO,,
OH _c
R-CH=C’
\
OH
-
y3 R-S=CH-COOH
By analogy,‘it seems reasonable that the carboxymethyl hydrogens of S-carboxymethyl cysteine can be labilized in two distinct ways corresponding to both of the above mechanisms, ,-,J+,~” $H 4 R-S-Lc
