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Further studies on β-hydroxyaspartic acid
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 104, 79--83
(1964)
Further Studies on fl-Hydroxyaspartic Acid I M . L. K O R N G U T H
AND H. J. S A L L A C H
From the Department of Physiological Chemistry, The University of Wisconsin, Madison, Wisconsin Received June 12, 1963 Stability studies with the two diastereoisomers of ~-hydroxyaspartic acid have demonstrated that heating of either the erythro or the threo isomer in 6 N HCl at 120~ leads to an equilibrium mixture of the two forms. After 96 hours a ratio of 5:2 of the erythro to the threo isomer is established. When either diastereoisomer is heated in water at 120~ partial deamination and cleavage to glycine is observed as well as the interconversion of both compounds. The threo- predominates over erythro-~hydroxyaspartic acid in a ratio of 2:1 after 50 hours of heating. The pessible occurrence of fl-hydroxyaspartic acid in several proteins has been investigated. Even though a component having the same chromatographic properties as erythro-B-hydroxyaspartic acid has been found in the protein hydrolysates, it has failed to react positively in other tests characteristic of fl-hydroxyaspartic acid. INTRODUCTION H y d r o x y a s p a r t i c acid has been shown to participate in several enzymatic reactions. I t serves as a substrate in m a m m a l i a n transaminase (1-3) and t r a n s c a r b a m y l a s e systems (4). K o r n b e r g has recently established it as a key intermediate in the metabolism of Micrecoccus nitrfiicans grown on glycollate as the sole source of c a r b o n (5, 6). I n each of the above biological systems the erythro form of ~-hydroxyaspartic acid, rather t h a n the threo isomer, has been shown to be the active substrate. I n addition, the a m i n o acid has been found to occur in a v a r i e t y of biological materials. T h e erythro isomer of ~-hydroxyaspa1%ic acid has been isolated in trace a m o u n t s f r o m enzymatic digests of casein in this laboratory (7). T h e presence of the amino acid has been reported in the m u s h r o o m poison, phallacidin (8), in h u m a n cerebrospinal fluid (9), and in a peptide derived f r o m Azotobacter agile (10). T h e B-amide, ~-hydroxyasparagine, has been
crystallized f r o m n o r m a l h u m a n urine (11). I n view of the interest in the biological role and the n a t u r a l occurrence of ~-hydroxyaspartic acid, additional information regarding the chemical properties of this c o m p o u n d and its derivatives was deemed essential. T h e earlier work of D a k i n indicated t h a t the erythro form of the amino acid was unstable in HC1 at 125~ (12). T h e following studies have been carried out to investigate the stability and the possible interconversion of the diastereoisomers of ~-hydroxyaspartic acid, and to gain information a b o u t the properties of some of its derivatives. T h e n a t u r a l occurrence of the amino acid in several proteins has also been investigated. MATERIALS AND METHODS ]~-~-~u
DL-Erythro- and
ACIDS
DL-threo-~-hydroxyaspartic
acids were synthesized as described (13). L-
Erythro-fl-hydroxyaspartic acid was obtained enzymatically from oxaloglycollate and glutamate by transamination as reported earlier (1).
1 This investigation was supported in part by research grant AM 00922-09 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, U. S. Public Health Service; by the American Cancer Society; and by the Wisconsin Alumni Research Foundation.
STABILITY STUDIES Ten ~moles of threo- or erythro-f~-hydroxyaspartic acid were dissolved in 2 ml. of 6 N HC1 or distilled water. The solutions were sealed in 79
80
K O R N G U T H A N D SALLACH
ampoules and autoclaved at 120~ Samples were removed from the autoclave at the desired time intervals, quantitatively transferred to a roundbottom flask, and evaporated in vacuo. Excess HC1 was removed by repeated evaporation. The residue was taken up in 10 ml. of sodium citrate buffer, 0.2 M, pH 2.2, and a 2-ml. aliquot was analyzed by means of a Beckman-Spinco amino acid analyzer, nmdel MS, according to the procedure of Moore et al. (14). In this system both diastereoisomers of hydroxyaspartic acid are separated (13). A second aliquot was used for the determination of free ammonia (15).
DL-THREO-~-HYDROXYASPARTYL-GLYCINE This dipeptide was synthesized by a condensation of glycyl-glycine (0.008 mole) with glyoxylate (0.01 mole) according to the general procedure described for ~-hydroxyaspartic acid (13). To minimize the hydrolysis of the peptide the reaction was carried out for only 1.5 hours and the concentration of base was reduced to 0.1 N. The reaction mixture was neutralized with HC1 and applied to a 2.5 X 30 cm. column of Dowex 1-formate. The column was washed with water and eluted with 0.5 N formic acid. The fractions that contained threo-~-hydroxyaspartyl-glycine, the major product of condensation, were located by paper chromatography. The compound gave the same Rs as erythro-~-hydroxyaspartic acid in butanol:acetic acid:water (2:2:1), but was distinguished from the free amino acid by its initial yellow color with ninhydrin. In addition to the peptide, the same fractions also contained some free erythro-fl-hydroxyaspartie acid, as determined by analysis with the automatic amino acid analyzer. The contents of the fractions that contained the peptide, obtained from two different experiments, were pooled and lyophilized. After two recrystallizations from water, 0.43 g. (13% yield) of the crystalline dipeptide was obtained. When analyzed by means of the automatic amino acid analyzer, the compound was eluted as a single peak at 115 ml. effluent volume (column length: 156 cm.; jacket temp. : 30~ Hydrolysis in 6 N HC1 for 3 hours yielded equimolar amounts of threo/~-hydroxyaspartic acid and glycine. End group analysis according to the method of Sanger (16) indicated that /~-hydroxyaspartic acid occupies the N-terminal position. The analytical data for the compound were as follows: C~H~0N~O~, Calcd.: C 35.0, H 4.89, N 13.6 Found: C 35.0, H 4.92, N 13.1 The automatic amino acid analyzer was employed to determine the extent of hydrolysis of the dipeptide and of o-phosphoryl-~-hydroxyaspartie acid in further experiments.
~)L-Erythro-o-PHOSPHORYL-~I-IYDROXYASPARTIC ACID
DL-Erythro-~-hydroxyaspartic acid (0.034 mole) and POCI~ were reacted according to the method for o-phosphoryl-serine described by Neuhaus and Korkes (17). The product, obtained in 53% yield, was recrystallized from water and gave the following analytical data: C4HsNOsP, Calcd.: C 21.0, H 3.52, N 6.11, P 13.5 Found: C 21.7, H 4.62, N 5.95, P 13.2 The compound gave a positive ninhydrin reaction, but, as expected, did not yield ammonia upon treatment with periodate. Equimolar amounts of ~-hydroxyaspartic acid (1) and inorganic phosphate (18) were liberated during hydrolysis in 1 N H2SO4 (100~ over a time period of 5 hours. The t~-hydroxyaspartic acid moiety formed upon the complete hydrolysis of the phosphorylated compound in 6 N HC1 was determined to be of the erythro configuration. ISOLATION OF ACIDIC AMINO ACID FRACTIONS FROM PROTEIN HYDROLYSATES Five grams of each of the following proteins were hydrolyzed in 6 N HC1 for 48 hours at 100~ : casein, enzymatic digest (Mead Johnson and Co.), soybean, enzymatic digest (Nutritional Biochemicals Co.), recrystallized ovalbumin (generously provided by Dr. P. P. Cohen), and silk fibroin (U. S. Testing Laboratory, Hoboken, N. J.). The hydrolysates were treated once with charcoal to remove humin. The bulk of the HC1 was removed by repeated evaporation, and the remaining traces by extraction with tri-n-heptylamine in chloroform (19). Each hydrolysate, adjusted to pH 6.5, was applied to a 1.5 X 45 cm. column of Dowex 1-formate. The column was washed with water (200 ml.) and then eluted with 0.1 N formic acid. In a control experiment, C ~4erythro-~-hydroxyaspartic acid was added to the same amount of protein hydrolysate and the mixture was chromatographed as above. It was found that the radioactive material was not separated completely from the trailing edge of the aspartic acid peak. Therefore, in the experimental procedure the contents of the final tubes of the eluted aspartic acid (assayed by paper chromatography) were combined with those corresponding to the remainder of the f~-hydroxyaspartic acid area and lyophilized. A 20-40 rag. sample was obtained from each of the above proteins. Portions of this sample w e r e analyzed for amino acids, treated with periodate (pH 5.0), and heated in alkali or in water.
fl-HYDROXYASPARTIC ACID
81
RESULTS AND DISCUSSION
Since fl-hydroxyaspartic acid possesses an asymmetric center at both the a- and STABILITY IN ACID /3-carbons, the optical inversion at either The heating of either threo- or erythro-~- one of these would lead to the formation of a hydroxyaspartic acid in 6 N HC1 results in diastereoisomer. The most likely site for the an interconversion of the diastereoisomers inversion in the strongly acidic solution (Figs. 1 and 2). After 24 hours 20 % of the appears to be the a-carbon, as postulated erythro isomer is converted to the threo b y Neuberger for a-amino acids in general form, and 35 % of the threo is converted to (20). The enolization of the carboxyl group the erythro isomer. An equilibrium between could lead to the labilization of the a-hythe two diastereoisomers, in which the drogen and thus to the inversion of optical erythro predominates over the threo form configuration. That the effect of the fl-hyin a ratio of 5:3, is established after apdroxyl group in fl-hydroxyaspartic acid proximately 96 hours. During this time the may be minimal is suggested by the fact total amount of both forms decreases by that the rate of isomerization of this amino only 20 %. Of interest is the observation acid is similar to that observed with 7-hYthat in 6 N HC1 negligible decomposition droxylysine (21). of the amino acid occurs during the time period usually employed for the hydrolysis STABILITY IN BASE of proteins. This is in contrast to Dakin's As previously reported (13), the treatment earlier conclusion that fl-hydroxyaspartic of either diastereoisomer with 5 N KOH at acid is unstable to acid (12). 120~ leads to complete decomposition of B-hydroxyaspartic acid within 24 hours, 100V,. yielding glycine as one of the products. I ~ DL-ERYTHRO-pAdditional experiments for shorter time periods have shown that the ratio of the erythro to the threo isomer in the reaction mixture remains at 1:2. U STABILITY IN AQUEOUS SOLUTION o
20
o
HYDROXYASPARTIC ACID I
I
24
I
f
48 72 TIME (HRS.)
96
FIG. 1. The stability of DL-erythro-~-hydroxyaspartic acid in 6 N HC1, 120~ I00
When either of the diastereoisomers of /~-hydroxyaspartic acid is heated in water (pH 3.3) it not only undergoes isomerization but also decomposes extensively (Figs. 3 and 4). An equilibrium between the two forms is established after 50 hours, and in this case the threo- to erythro-fl-hydroxyas-
I00, [~ DL-ERYTHRO-[B8 0 ~ _ HYDROXYASPARTIC ACID
X DL-THREO-13X • 1-80 zLLI 60
4op
L) rr
tO 4C fl,.
I
24
I
I
48 72 TIME (HRS.)
I
96
FIG. 2. The stability of DL-threo-~-hydroxyaspartic acid in 6 N ttC1, 120~
24
48 72 TIME (HRS.)
96
FIG. 3. The stability of DL-erythro-~-hydroxyaspartic acid in water, pH 3.3,120~
82
KORNGUTH AND SALLACH
IOO
that the hydrolysis of this derivative in 2 N HC1 was complete after 4 hours at 120~ \ DL-THREO-pThe phosphorylated derivative of serine is 20% hydrolyzed after 3 hours in 2.5 N HC1 at 100~ (23). The lability of the ester L~ , bond in o-phosphoryl-f~-hydroxyasparticacid m 401 was indicated also by its complete hydrolysis n _ in water at 120~ after 13 hours. Since both of the above derivatives of I GLY~ClNE ~ ~ l ~-hydroxyaspartic acid exhibit a greater lability than the same derivatives of other 24 48 72 96 TIME (HRS.) ~-hydroxy-a-amino acids, it appears that FIG. 4. The stability of DL-threo-~-hydroxy- the ~-carboxyl group of ~-hydroxyaspartic aspartic acid in water, pH 3.3,120~ acid may actively assist in the hydrolysis of adjacent bonds. The free B- and ~/-carboxyl partic acid ratio is 2:1. The appearance of groups of e-(a-aspartyl)-lysine and e-(afree glycine in the reaction mixture indicates glutamyl)-lysine, respectively, have been a cleavage of the amino acid between carbon shown to be reactive, since the presence of atoms 2 and 3. A 50 % loss of ninhydrin- the corresponding cyclic imides has been reacting groups occurs after 96 hours and is demonstrated in the partial acid hydrolyaccompanied by a corresponding increase sates of these compounds (24, 25). Furtherin the free NH3 level of the reaction mixture. more, the rate of hydrolysis of e-(a-gluSince a different ionic species is present in tamyl)-lysine has been found to increase with the weakly acidic solution as compared with time, presumably as a result of the cylic 6 N HC1, and the equilibrium is shifted imide formation and its subsequent contoward the threo isomer, it appears that a version to the less stable ~,-glutamyl peptide different mechanism is operative in the (25). isomerization reaction. STABILITY OF THE DERIVATIVES OF ~-HYDROXYASPARTIC ACID
STUDIES ON THE NATURAL OCCURRENCE OF ~-HYDROXYASPARTIC ACID
In order to obtain information about the properties of some derivatives of ~-hydroxyaspartic acid, which also might be of biological significance, threo-~-hydroxyaspartylglycine and erythro-o-phosphoryl-B-hydroxyaspartic acid were synthesized. Hydrolytic studies with the dipeptide indicated that the peptide bond was rapidly cleaved in acid solution. In 6 N HC1 at 120~ as measured by the appearance of the free amino acids, threo-~-hydroxyaspartyl-glycine was 50% hydrolyzed in 1 hour. The lability of this compound appears to be greater than that of threonyl-glycine, which is reported to be 66% hydrolyzed after 8 hours in 6 N HC1 at l l0~ (22). The amount of free erythro-~-hydroxyaspartic acid formed during this time period was consistent with that expected from the rate of isomerization of free threo-~-hydroxyaspartic acid. Experiments carried out with erythro-ophosphoryl-f~-hydroxyasparticacid indicated
The behavior of /~-hydroxyaspartic acid in acidic and alkaline solutions, together with other available data, suggested several experiments that may be employed for the preliminary identification of the amino acid in biological materials. If an isolated substance were f~-hydroxyaspartic acid, in addition to consistent chromatographic properties, it should (a) decompose upon treatment with periodate, (b) yield glycine in an alkaline solution, and (c) form the corresponding diastereoisomer at the observed rate when heated in 6 N HC1 or water. The sample of f~-hydroxyaspartic acid previously isolated from a pancreatic digest of casein (supplied by Mead Johnson and Co.) (7), reacted as synthetic erythro/~-hydroxyaspartic acid in these three reactions. Attempts to isolate larger amounts of this material by the identical procedure, however, did not prove successful. The most likely explanation of this result is that different batches of the casein digest have a
~-HYDROXYASPARTIC ACID
83
ACKNOWLEDGMENTS variable composition since the only variable We are indebted to Mr. Edward H. Kmiotek in these and earlier experiments was the lot for his technical assistance and to Mr. Myron B. number of the preparation. In order to exclude the possibility that the Ziegler for his help in carrying out the amino later samples were not digested to the same acid analyses. REFERENCES extent as the earlier ones, and that/~-hydrox1. SALLACH, S . J., AND PETERSON, T. H., J. yaspartic acid was present as a derivative, Biol. Chem. 223, 629 (1956). the pancreatic digests were further hy2. SALLACH,H. J., J. Biol. Chem. 229,437 (1957). drolyzed in 6 N HC1. The partially purified 3. GARCIA-HERNANDEZ, M., AND KUN, E., acidic amino acid fraction obtained from the Biochim. Biophys. Acta 24, 78, (1957). hydrolysute (see Materials and Methods), 4. SALLACH,~-I. J., J . Biol. Chem. 234, 900 (1959). 5. KORNBERG,H. L., ANDMORRIS,J. G., Biochim. contained small amounts of a ninhydrinBiophys. Acta 65, 537 (1962). positive compound that migrated identically 6. KORNBERG,H. L., ANDMORRIS,J. G., Biochim. with erythro-fl-hydroxyaspartic acid on the Biophys. Acta 65,378 (1962). amino acid analyzer. However, this material 7. SALLACH, H. J., AND KORNGUTH,M. L., Biodid not decompose upon treatment with chim. Biophys. Acta 34, 582 (1959). periodate. On heating in alkali or in water it 8. WIELAND, W., Angew. Chem. 72, 892 (1960). yielded two components that migrated as 9. PERRY, T. L., AND JONES, R. W., J. Clin. serine and glutamic acid. Similar results were Invest. 40, 1363 (1961). obtained with the fractions isolated from 10. BULLEN,W. A., ANDLECoMTE,J. R., Biochem. the acid hydrolysates of the soybean prepBiophys. Res. Commun. 9,523 (1962). aration, ovalbumin, and silk fibroin. These 11. TOMINAGA, F., I-IIWAKI, C., MAEKAWA, T., AND YOSHIEA, 1-I., J. Biochem. (Tokyo) findings show that during the experimental 53, 227 (1963). procedure an acid stable adduct is formed which has chromatographic properties very 12. DAKIN, H. D., J. Biol. Chem. 48, 273 (1921). similar to those of erythro-~-hydroxyaspartic 13. KORN~UTH,M. L..aND SALLACH,H. J., Arch. Biochem. Biophys. 91, 39 (1960). acid. It is of interest that Ikawa and Snell 14. MOORE, S., SPACKlYIAN,D. ~-~., AND STEIN, have recently reported the formation of W. H., Anal. Chem. 30, 1185 (1958). o-(7-glutamyl)-serine from serine and glu- 15. CONWAY,E. J., AND O'MALLEY, H., Biochem. tamic acid in 3 N HC1 during evaporation J. 36, 655 (1942). (26). 16. SANGER,F., Biochem. J. 39, 507 (1945). Hydroxyaspartic acid has not been found 17. NEUnAVS, F. C., AND KORKES, S., in "Biochemical Preparations" (C. S. Vestling, ed.), in the different batches of casein digests Vol. 6, p. 75. Wiley, New York, 1958. even after additional hydrolysis with acid. 18. FISEE, C. H., AND SUBBAROW, Y., J. Biol. Therefore, it appears that either a chemical Chem. 66,375 (1925). or a biological variable was responsible for 19. HVeHES, D. E., AND WlLLIAMSON, D. H., its presence in the original casein preparaBiochem. J. 48, 487 (1951). tion. The amino acid could have been 20. NEUBERGER, A., in "Advances in Protein Chemistry" (M. L. ArisEn and J. T. Edsall, chemically formed during the digestion of eds.), Vol. IV, p. 297. Academic Press, this particular sample by a condensation of New York, 1948. glycine and glyoxylate. The reaction of these 21. HAMILTON, P. B. AND ANDERSON, R. A., two compounds in an alkaline medium J. Biol. Chem. 213, 249 (1955). constitutes one method of synthesis of 22. METZLER, O. E., LONGENECKER, J. B., AND SNELL E., E., J. Am. Chem. Soc. 76, 639 ~-hydroxyaspartic acid (13). The finding (1954). that f~-hydroxyaspartic acid is a contami- 23. LIPMANN, F., Biochem. Z. 262, 9 (1933). nant of some glycine samples (27) also 24. SW,~LLOW,D. L., AND ABRAHAM,E. P., Biopoints to the ease of its nonenzymatic formachem. J. 70,364 (1958). tion. On the other hand, variation in the 25. KORNGUTH,M. L., NEIDLE, n., AND WAELSCH, I-I., Biochemistry 2,740 (1963). amino acid composition of the protein itself 26. IXAWA,M., AND SNELL, E. E., J. Biol. Chem. can not be excluded, since, for example, 236, 1955 (1961). genetic differences in the proteins comprising 27. JENKINS,T. W., J. Biol. Chem. 236, 1121 (1961). casein have been reported (28). 28. ASCnAFFENBURG, R., Nature 192, 431 (1961).
(1964)
Further Studies on fl-Hydroxyaspartic Acid I M . L. K O R N G U T H
AND H. J. S A L L A C H
From the Department of Physiological Chemistry, The University of Wisconsin, Madison, Wisconsin Received June 12, 1963 Stability studies with the two diastereoisomers of ~-hydroxyaspartic acid have demonstrated that heating of either the erythro or the threo isomer in 6 N HCl at 120~ leads to an equilibrium mixture of the two forms. After 96 hours a ratio of 5:2 of the erythro to the threo isomer is established. When either diastereoisomer is heated in water at 120~ partial deamination and cleavage to glycine is observed as well as the interconversion of both compounds. The threo- predominates over erythro-~hydroxyaspartic acid in a ratio of 2:1 after 50 hours of heating. The pessible occurrence of fl-hydroxyaspartic acid in several proteins has been investigated. Even though a component having the same chromatographic properties as erythro-B-hydroxyaspartic acid has been found in the protein hydrolysates, it has failed to react positively in other tests characteristic of fl-hydroxyaspartic acid. INTRODUCTION H y d r o x y a s p a r t i c acid has been shown to participate in several enzymatic reactions. I t serves as a substrate in m a m m a l i a n transaminase (1-3) and t r a n s c a r b a m y l a s e systems (4). K o r n b e r g has recently established it as a key intermediate in the metabolism of Micrecoccus nitrfiicans grown on glycollate as the sole source of c a r b o n (5, 6). I n each of the above biological systems the erythro form of ~-hydroxyaspartic acid, rather t h a n the threo isomer, has been shown to be the active substrate. I n addition, the a m i n o acid has been found to occur in a v a r i e t y of biological materials. T h e erythro isomer of ~-hydroxyaspa1%ic acid has been isolated in trace a m o u n t s f r o m enzymatic digests of casein in this laboratory (7). T h e presence of the amino acid has been reported in the m u s h r o o m poison, phallacidin (8), in h u m a n cerebrospinal fluid (9), and in a peptide derived f r o m Azotobacter agile (10). T h e B-amide, ~-hydroxyasparagine, has been
crystallized f r o m n o r m a l h u m a n urine (11). I n view of the interest in the biological role and the n a t u r a l occurrence of ~-hydroxyaspartic acid, additional information regarding the chemical properties of this c o m p o u n d and its derivatives was deemed essential. T h e earlier work of D a k i n indicated t h a t the erythro form of the amino acid was unstable in HC1 at 125~ (12). T h e following studies have been carried out to investigate the stability and the possible interconversion of the diastereoisomers of ~-hydroxyaspartic acid, and to gain information a b o u t the properties of some of its derivatives. T h e n a t u r a l occurrence of the amino acid in several proteins has also been investigated. MATERIALS AND METHODS ]~-~-~u
DL-Erythro- and
ACIDS
DL-threo-~-hydroxyaspartic
acids were synthesized as described (13). L-
Erythro-fl-hydroxyaspartic acid was obtained enzymatically from oxaloglycollate and glutamate by transamination as reported earlier (1).
1 This investigation was supported in part by research grant AM 00922-09 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, U. S. Public Health Service; by the American Cancer Society; and by the Wisconsin Alumni Research Foundation.
STABILITY STUDIES Ten ~moles of threo- or erythro-f~-hydroxyaspartic acid were dissolved in 2 ml. of 6 N HC1 or distilled water. The solutions were sealed in 79
80
K O R N G U T H A N D SALLACH
ampoules and autoclaved at 120~ Samples were removed from the autoclave at the desired time intervals, quantitatively transferred to a roundbottom flask, and evaporated in vacuo. Excess HC1 was removed by repeated evaporation. The residue was taken up in 10 ml. of sodium citrate buffer, 0.2 M, pH 2.2, and a 2-ml. aliquot was analyzed by means of a Beckman-Spinco amino acid analyzer, nmdel MS, according to the procedure of Moore et al. (14). In this system both diastereoisomers of hydroxyaspartic acid are separated (13). A second aliquot was used for the determination of free ammonia (15).
DL-THREO-~-HYDROXYASPARTYL-GLYCINE This dipeptide was synthesized by a condensation of glycyl-glycine (0.008 mole) with glyoxylate (0.01 mole) according to the general procedure described for ~-hydroxyaspartic acid (13). To minimize the hydrolysis of the peptide the reaction was carried out for only 1.5 hours and the concentration of base was reduced to 0.1 N. The reaction mixture was neutralized with HC1 and applied to a 2.5 X 30 cm. column of Dowex 1-formate. The column was washed with water and eluted with 0.5 N formic acid. The fractions that contained threo-~-hydroxyaspartyl-glycine, the major product of condensation, were located by paper chromatography. The compound gave the same Rs as erythro-~-hydroxyaspartic acid in butanol:acetic acid:water (2:2:1), but was distinguished from the free amino acid by its initial yellow color with ninhydrin. In addition to the peptide, the same fractions also contained some free erythro-fl-hydroxyaspartie acid, as determined by analysis with the automatic amino acid analyzer. The contents of the fractions that contained the peptide, obtained from two different experiments, were pooled and lyophilized. After two recrystallizations from water, 0.43 g. (13% yield) of the crystalline dipeptide was obtained. When analyzed by means of the automatic amino acid analyzer, the compound was eluted as a single peak at 115 ml. effluent volume (column length: 156 cm.; jacket temp. : 30~ Hydrolysis in 6 N HC1 for 3 hours yielded equimolar amounts of threo/~-hydroxyaspartic acid and glycine. End group analysis according to the method of Sanger (16) indicated that /~-hydroxyaspartic acid occupies the N-terminal position. The analytical data for the compound were as follows: C~H~0N~O~, Calcd.: C 35.0, H 4.89, N 13.6 Found: C 35.0, H 4.92, N 13.1 The automatic amino acid analyzer was employed to determine the extent of hydrolysis of the dipeptide and of o-phosphoryl-~-hydroxyaspartie acid in further experiments.
~)L-Erythro-o-PHOSPHORYL-~I-IYDROXYASPARTIC ACID
DL-Erythro-~-hydroxyaspartic acid (0.034 mole) and POCI~ were reacted according to the method for o-phosphoryl-serine described by Neuhaus and Korkes (17). The product, obtained in 53% yield, was recrystallized from water and gave the following analytical data: C4HsNOsP, Calcd.: C 21.0, H 3.52, N 6.11, P 13.5 Found: C 21.7, H 4.62, N 5.95, P 13.2 The compound gave a positive ninhydrin reaction, but, as expected, did not yield ammonia upon treatment with periodate. Equimolar amounts of ~-hydroxyaspartic acid (1) and inorganic phosphate (18) were liberated during hydrolysis in 1 N H2SO4 (100~ over a time period of 5 hours. The t~-hydroxyaspartic acid moiety formed upon the complete hydrolysis of the phosphorylated compound in 6 N HC1 was determined to be of the erythro configuration. ISOLATION OF ACIDIC AMINO ACID FRACTIONS FROM PROTEIN HYDROLYSATES Five grams of each of the following proteins were hydrolyzed in 6 N HC1 for 48 hours at 100~ : casein, enzymatic digest (Mead Johnson and Co.), soybean, enzymatic digest (Nutritional Biochemicals Co.), recrystallized ovalbumin (generously provided by Dr. P. P. Cohen), and silk fibroin (U. S. Testing Laboratory, Hoboken, N. J.). The hydrolysates were treated once with charcoal to remove humin. The bulk of the HC1 was removed by repeated evaporation, and the remaining traces by extraction with tri-n-heptylamine in chloroform (19). Each hydrolysate, adjusted to pH 6.5, was applied to a 1.5 X 45 cm. column of Dowex 1-formate. The column was washed with water (200 ml.) and then eluted with 0.1 N formic acid. In a control experiment, C ~4erythro-~-hydroxyaspartic acid was added to the same amount of protein hydrolysate and the mixture was chromatographed as above. It was found that the radioactive material was not separated completely from the trailing edge of the aspartic acid peak. Therefore, in the experimental procedure the contents of the final tubes of the eluted aspartic acid (assayed by paper chromatography) were combined with those corresponding to the remainder of the f~-hydroxyaspartic acid area and lyophilized. A 20-40 rag. sample was obtained from each of the above proteins. Portions of this sample w e r e analyzed for amino acids, treated with periodate (pH 5.0), and heated in alkali or in water.
fl-HYDROXYASPARTIC ACID
81
RESULTS AND DISCUSSION
Since fl-hydroxyaspartic acid possesses an asymmetric center at both the a- and STABILITY IN ACID /3-carbons, the optical inversion at either The heating of either threo- or erythro-~- one of these would lead to the formation of a hydroxyaspartic acid in 6 N HC1 results in diastereoisomer. The most likely site for the an interconversion of the diastereoisomers inversion in the strongly acidic solution (Figs. 1 and 2). After 24 hours 20 % of the appears to be the a-carbon, as postulated erythro isomer is converted to the threo b y Neuberger for a-amino acids in general form, and 35 % of the threo is converted to (20). The enolization of the carboxyl group the erythro isomer. An equilibrium between could lead to the labilization of the a-hythe two diastereoisomers, in which the drogen and thus to the inversion of optical erythro predominates over the threo form configuration. That the effect of the fl-hyin a ratio of 5:3, is established after apdroxyl group in fl-hydroxyaspartic acid proximately 96 hours. During this time the may be minimal is suggested by the fact total amount of both forms decreases by that the rate of isomerization of this amino only 20 %. Of interest is the observation acid is similar to that observed with 7-hYthat in 6 N HC1 negligible decomposition droxylysine (21). of the amino acid occurs during the time period usually employed for the hydrolysis STABILITY IN BASE of proteins. This is in contrast to Dakin's As previously reported (13), the treatment earlier conclusion that fl-hydroxyaspartic of either diastereoisomer with 5 N KOH at acid is unstable to acid (12). 120~ leads to complete decomposition of B-hydroxyaspartic acid within 24 hours, 100V,. yielding glycine as one of the products. I ~ DL-ERYTHRO-pAdditional experiments for shorter time periods have shown that the ratio of the erythro to the threo isomer in the reaction mixture remains at 1:2. U STABILITY IN AQUEOUS SOLUTION o
20
o
HYDROXYASPARTIC ACID I
I
24
I
f
48 72 TIME (HRS.)
96
FIG. 1. The stability of DL-erythro-~-hydroxyaspartic acid in 6 N HC1, 120~ I00
When either of the diastereoisomers of /~-hydroxyaspartic acid is heated in water (pH 3.3) it not only undergoes isomerization but also decomposes extensively (Figs. 3 and 4). An equilibrium between the two forms is established after 50 hours, and in this case the threo- to erythro-fl-hydroxyas-
I00, [~ DL-ERYTHRO-[B8 0 ~ _ HYDROXYASPARTIC ACID
X DL-THREO-13X • 1-80 zLLI 60
4op
L) rr
tO 4C fl,.
I
24
I
I
48 72 TIME (HRS.)
I
96
FIG. 2. The stability of DL-threo-~-hydroxyaspartic acid in 6 N ttC1, 120~
24
48 72 TIME (HRS.)
96
FIG. 3. The stability of DL-erythro-~-hydroxyaspartic acid in water, pH 3.3,120~
82
KORNGUTH AND SALLACH
IOO
that the hydrolysis of this derivative in 2 N HC1 was complete after 4 hours at 120~ \ DL-THREO-pThe phosphorylated derivative of serine is 20% hydrolyzed after 3 hours in 2.5 N HC1 at 100~ (23). The lability of the ester L~ , bond in o-phosphoryl-f~-hydroxyasparticacid m 401 was indicated also by its complete hydrolysis n _ in water at 120~ after 13 hours. Since both of the above derivatives of I GLY~ClNE ~ ~ l ~-hydroxyaspartic acid exhibit a greater lability than the same derivatives of other 24 48 72 96 TIME (HRS.) ~-hydroxy-a-amino acids, it appears that FIG. 4. The stability of DL-threo-~-hydroxy- the ~-carboxyl group of ~-hydroxyaspartic aspartic acid in water, pH 3.3,120~ acid may actively assist in the hydrolysis of adjacent bonds. The free B- and ~/-carboxyl partic acid ratio is 2:1. The appearance of groups of e-(a-aspartyl)-lysine and e-(afree glycine in the reaction mixture indicates glutamyl)-lysine, respectively, have been a cleavage of the amino acid between carbon shown to be reactive, since the presence of atoms 2 and 3. A 50 % loss of ninhydrin- the corresponding cyclic imides has been reacting groups occurs after 96 hours and is demonstrated in the partial acid hydrolyaccompanied by a corresponding increase sates of these compounds (24, 25). Furtherin the free NH3 level of the reaction mixture. more, the rate of hydrolysis of e-(a-gluSince a different ionic species is present in tamyl)-lysine has been found to increase with the weakly acidic solution as compared with time, presumably as a result of the cylic 6 N HC1, and the equilibrium is shifted imide formation and its subsequent contoward the threo isomer, it appears that a version to the less stable ~,-glutamyl peptide different mechanism is operative in the (25). isomerization reaction. STABILITY OF THE DERIVATIVES OF ~-HYDROXYASPARTIC ACID
STUDIES ON THE NATURAL OCCURRENCE OF ~-HYDROXYASPARTIC ACID
In order to obtain information about the properties of some derivatives of ~-hydroxyaspartic acid, which also might be of biological significance, threo-~-hydroxyaspartylglycine and erythro-o-phosphoryl-B-hydroxyaspartic acid were synthesized. Hydrolytic studies with the dipeptide indicated that the peptide bond was rapidly cleaved in acid solution. In 6 N HC1 at 120~ as measured by the appearance of the free amino acids, threo-~-hydroxyaspartyl-glycine was 50% hydrolyzed in 1 hour. The lability of this compound appears to be greater than that of threonyl-glycine, which is reported to be 66% hydrolyzed after 8 hours in 6 N HC1 at l l0~ (22). The amount of free erythro-~-hydroxyaspartic acid formed during this time period was consistent with that expected from the rate of isomerization of free threo-~-hydroxyaspartic acid. Experiments carried out with erythro-ophosphoryl-f~-hydroxyasparticacid indicated
The behavior of /~-hydroxyaspartic acid in acidic and alkaline solutions, together with other available data, suggested several experiments that may be employed for the preliminary identification of the amino acid in biological materials. If an isolated substance were f~-hydroxyaspartic acid, in addition to consistent chromatographic properties, it should (a) decompose upon treatment with periodate, (b) yield glycine in an alkaline solution, and (c) form the corresponding diastereoisomer at the observed rate when heated in 6 N HC1 or water. The sample of f~-hydroxyaspartic acid previously isolated from a pancreatic digest of casein (supplied by Mead Johnson and Co.) (7), reacted as synthetic erythro/~-hydroxyaspartic acid in these three reactions. Attempts to isolate larger amounts of this material by the identical procedure, however, did not prove successful. The most likely explanation of this result is that different batches of the casein digest have a
~-HYDROXYASPARTIC ACID
83
ACKNOWLEDGMENTS variable composition since the only variable We are indebted to Mr. Edward H. Kmiotek in these and earlier experiments was the lot for his technical assistance and to Mr. Myron B. number of the preparation. In order to exclude the possibility that the Ziegler for his help in carrying out the amino later samples were not digested to the same acid analyses. REFERENCES extent as the earlier ones, and that/~-hydrox1. SALLACH, S . J., AND PETERSON, T. H., J. yaspartic acid was present as a derivative, Biol. Chem. 223, 629 (1956). the pancreatic digests were further hy2. SALLACH,H. J., J. Biol. Chem. 229,437 (1957). drolyzed in 6 N HC1. The partially purified 3. GARCIA-HERNANDEZ, M., AND KUN, E., acidic amino acid fraction obtained from the Biochim. Biophys. Acta 24, 78, (1957). hydrolysute (see Materials and Methods), 4. SALLACH,~-I. J., J . Biol. Chem. 234, 900 (1959). 5. KORNBERG,H. L., ANDMORRIS,J. G., Biochim. contained small amounts of a ninhydrinBiophys. Acta 65, 537 (1962). positive compound that migrated identically 6. KORNBERG,H. L., ANDMORRIS,J. G., Biochim. with erythro-fl-hydroxyaspartic acid on the Biophys. Acta 65,378 (1962). amino acid analyzer. However, this material 7. SALLACH, H. J., AND KORNGUTH,M. L., Biodid not decompose upon treatment with chim. Biophys. Acta 34, 582 (1959). periodate. On heating in alkali or in water it 8. WIELAND, W., Angew. Chem. 72, 892 (1960). yielded two components that migrated as 9. PERRY, T. L., AND JONES, R. W., J. Clin. serine and glutamic acid. Similar results were Invest. 40, 1363 (1961). obtained with the fractions isolated from 10. BULLEN,W. A., ANDLECoMTE,J. R., Biochem. the acid hydrolysates of the soybean prepBiophys. Res. Commun. 9,523 (1962). aration, ovalbumin, and silk fibroin. These 11. TOMINAGA, F., I-IIWAKI, C., MAEKAWA, T., AND YOSHIEA, 1-I., J. Biochem. (Tokyo) findings show that during the experimental 53, 227 (1963). procedure an acid stable adduct is formed which has chromatographic properties very 12. DAKIN, H. D., J. Biol. Chem. 48, 273 (1921). similar to those of erythro-~-hydroxyaspartic 13. KORN~UTH,M. L..aND SALLACH,H. J., Arch. Biochem. Biophys. 91, 39 (1960). acid. It is of interest that Ikawa and Snell 14. MOORE, S., SPACKlYIAN,D. ~-~., AND STEIN, have recently reported the formation of W. H., Anal. Chem. 30, 1185 (1958). o-(7-glutamyl)-serine from serine and glu- 15. CONWAY,E. J., AND O'MALLEY, H., Biochem. tamic acid in 3 N HC1 during evaporation J. 36, 655 (1942). (26). 16. SANGER,F., Biochem. J. 39, 507 (1945). Hydroxyaspartic acid has not been found 17. NEUnAVS, F. C., AND KORKES, S., in "Biochemical Preparations" (C. S. Vestling, ed.), in the different batches of casein digests Vol. 6, p. 75. Wiley, New York, 1958. even after additional hydrolysis with acid. 18. FISEE, C. H., AND SUBBAROW, Y., J. Biol. Therefore, it appears that either a chemical Chem. 66,375 (1925). or a biological variable was responsible for 19. HVeHES, D. E., AND WlLLIAMSON, D. H., its presence in the original casein preparaBiochem. J. 48, 487 (1951). tion. The amino acid could have been 20. NEUBERGER, A., in "Advances in Protein Chemistry" (M. L. ArisEn and J. T. Edsall, chemically formed during the digestion of eds.), Vol. IV, p. 297. Academic Press, this particular sample by a condensation of New York, 1948. glycine and glyoxylate. The reaction of these 21. HAMILTON, P. B. AND ANDERSON, R. A., two compounds in an alkaline medium J. Biol. Chem. 213, 249 (1955). constitutes one method of synthesis of 22. METZLER, O. E., LONGENECKER, J. B., AND SNELL E., E., J. Am. Chem. Soc. 76, 639 ~-hydroxyaspartic acid (13). The finding (1954). that f~-hydroxyaspartic acid is a contami- 23. LIPMANN, F., Biochem. Z. 262, 9 (1933). nant of some glycine samples (27) also 24. SW,~LLOW,D. L., AND ABRAHAM,E. P., Biopoints to the ease of its nonenzymatic formachem. J. 70,364 (1958). tion. On the other hand, variation in the 25. KORNGUTH,M. L., NEIDLE, n., AND WAELSCH, I-I., Biochemistry 2,740 (1963). amino acid composition of the protein itself 26. IXAWA,M., AND SNELL, E. E., J. Biol. Chem. can not be excluded, since, for example, 236, 1955 (1961). genetic differences in the proteins comprising 27. JENKINS,T. W., J. Biol. Chem. 236, 1121 (1961). casein have been reported (28). 28. ASCnAFFENBURG, R., Nature 192, 431 (1961).