Hydroxyaspartic acid from alfalfa and clover roots

Phytochemistry,1962, Vol. 1, pp. 241 to 244. PergamonPressLtd. Printedin England

HYDROXYASPARTIC ACID FROM ALFALFA CLOVER ROOTS M. DEAN Department

of Biochemistry,

WILDING

and

MARK

A.

AND

STAHMANN

University of Wisconsin, Madison 6, Wisconsin, U.S.A. (Received 26 May 1962)

Abstract-Hydroxyaspartic acid has been identified in extracts of the free amino acids from field grown alfalfa and clover root tissue. It was identified by chromatography and co-chromatography on ion exchange columns and the amount of ammonia liberated on periodate oxidation. It occurs in these legume root tissues in amounts ranging from 06-2 ,umoles/g of dry root tissue. This is much higher than the concentration of some of the more common amino acids. INTRODUCTION DURING the past ten years many

new amino acids have been isolated from plants by the use of paper or ion exchange chromatography. Consden, et a1.l in 1944 first described the use of paper chromatography in the separation of amino acids. Dent, et a1.2 demonstrated the potentialities of paper chromatography to plant biochemistry in a study of the amino acid complex of potato tubers and supplied definite evidence that other unknown ninhydrin positive substances (possibly amino acids) were present in green plants. Wilding, et ~1.~ have compared paper and ion exchange chromatography in a study of free amino acids in alfalfa and clover roots and have shown the advantages of the latter method. In 1921 Dakin4 synthesized the amino acid, hydroxyaspartic acid, and described its chemical and physical properties. Sallach and Peterson5 observed the formation of hydroxyaspartic acid by transamination between oxaloglycolate, the keto form of dihydroxyfumarate, and glutamic acid. Virtanen in 1957’j reported hydroxyaspartic acid in extracellular material produced by Azotobacter during nitrogen fixation by the organism. It appeared to be combined with other commonly occurring amino acids in Azotobacter. Sallach and Kornguth’ have reported the occurrence of hydroxyaspartic acid in pancreatic digests of casein. During recent work on the biochemistry of cold hardiness in legumes3v8 a very well resolved ninhydrin reacting component was observed preceding aspartic acid by approximately 36-37 ml of eluant in an ion exchange chromatogram of the free amino acid of both alfalfa and red clover root extracts. The recognition of this new component as hydroxyaspartic acid was facilitated by simultaneous collaborative work with Dr. H. J. Sallach and Mrs. M. L. Kornguth, for whom we were analyzing the amino acids in pancreatic digests of casein. A ninhydrin reacting peak which they obtained from this source, the one the authors had observed from legume extracts, and an authentic sample of synthetic hydroxyaspartic acid all occurred at an identical location on the elutograms and gave us strong evidence for believing that the 1 R. CONSDEN,A. H. GORDONand A. J. P. MARTIN,Biochem. J., 38, 224 (1944).

* C. E. DENT, E. STEPKAand F. C. STEWARD, Narure, 160,682 (1947). .s M. D. WILDING, M. A. STAHMANN and D. SMITH,PZant Physiol., 35, 726 (1960). 4 H. D. DAKIN, J. Biol. Chem., 48,273 (1921). 5 H. J. SALLACH and T. H. PETERSON,J. Biol. Chem., 223, 629 (1956). a A. I. VIRTANEN,Act. Chem. Fennicu, B, 30, 100 (1957). (Cited from Biol. Revs. 33, 393 (1958)). ’ H. J. SALLACHand M. L. KORNGUTH,Biochim. Biophys. Acta., 34, 582 (1959). 8 M. D. WILDING, M. A. STAHMANNand D. SMITH,Plant Physiol., 35, 733 (1960). 241

M. DEAN WILDING and MARK A. STAHMANN

242 component unknown results bined

from

the root

compound

presented in protein

tissue was indeed

at the same position

in this paper free extracts

provide

evidence

of alfalfa

acids

1 is typical

in alfalfa

root

of the results tissue

nHIS

AND

acid occurs

ninhydrin

acid analysis positive

peak

ARG

43

et al.g show an of rat liver.

The

uncom-

roots.

in the amino

the sharp

n

Moore,

DISCUSSION

obtained

showing

acid.

of an extract

that hydroxyaspartic

and clover

RESULTS Figure

hydroxyaspartic

in their elutogram

of the free amino (labelled

OH-Asp)

ASP-NH, GLU-NH-

E

Cystathionine

0.1

I

rn ’ AA-

P-ALA

001

570 50:pH

4.25.0.2N

Na c~+rofe -

1

FIG. 1. A TYPICAL ELUTOGRAM OF FREE AMINO ACIDS FROM A VERNAL ALFALFA ROOT EXTRACT. The upper right hand portion shows the position of the hydroxyaspartic acid peak in relation to the other acidic components. 1.0,

0.6

-

-

I.148

,~rn OH -osportic standard synthetic + olfolfo root sample

acid from mixture

0.142 alfalfa

acid

I pm

0.. -

O.D. 0.2 j II

0.1 -

I I-I I I I

pm root

OH - asportic samples

from

I

ml

eluted

from

column

FIG. 2. AN ELUTGGRAMOF A CALIVERDEALFALFAROOTEXTRACTSHOWINGTHE OF 1 .o THE

,uM

SYNTHETIC

ALFALFA

EXTRACT.

HYDROXYASPARTIC

ACID

WITH

AN UNKNOWN

NINHYDRIN

CO-CHROMATOGRAPHY REACTING

PEAK

FROM

This extract was analyzed on the 150 cm column of the Beckman/Spinco amino acid analyzer.

DS. MOORE, D. H. SPACKMAN and W. H. STEIN, Federation Proc., 17, 1107 (1958).

Hydroxyaspartic

acid from alfalfa

243

that came off the column before aspartic acid. Figure 2 shows a portion of the elutogram obtained by co-chromatography of 1 ,uM of authentic hydroxyaspartic acid and 2 ml of the alfalfa root extract in relation to the position of this peak. From a quantitative determination using the ninhydrin colour value of the authentic sample, we estimated that the alfalfa extract contained 0.142 pmoles of hydroxyaspartic acid. The results obtained on co-chromatography show that the authentic hydroxyaspartic came off the column at exactly the same position as the unknown and that it produced the expected theoretical increase in ninhydrin colour. Nicolet and ShinnlO showed that ammonia is quantitatively liberated from molecules having vicinal amino and hydroxyl groups upon oxidation with periodate. The ammonia release following periodate oxidation of an authentic sample of synthetic hydroxyaspartic acid and of an aliquot of the effluent containing the unknown peak thought to be hydroxyaspartic was determined. Based upon an equal ninhydrin colour value for the amino acid in the effluent and for the synthetic hydroxyaspartic acid; the ammonia released by the isolated amino acid was 100.7 per cent of that recovered from the authentic synthetic sample of hydroxyaspartic acid. The amount of free hydroxyaspartic acid found in alfalfa root tissue varied from 0.58-1.17 ,uM/g of dry root tissue .3 There was some slight differences between the two alfalfa varieties. The lowest values were found in those root samples collected in October; both the August and December values were somewhat higher. Clover roots contained a little less; values from 0.42-0.58 PM/g of clover root tissues were obtained with samples collected in November.s It is interesting to note that the concentration of free hydroxyaspartic acid in alfalfa or clover roots was often considerably higher than that of the common amino acids, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, or lysine. Our results show that these alfalfa and clover roots contained free hydroxyaspartic acid. Further work is needed to determine whether this amino acid is formed by the green plant or if it is produced solely by the nitrogen-fixing bacteria that may have been present in the roots of these legumes. EXPERIMENTAL

Extraction

and chromatography

of free amino acids

Alfalfa (Medicago sativa L., var. Caliverde and Vernal) and Red Clover (Trifolium pratense L., var. Wisconsin common) roots were harvested in the fall from field grown plants, washed free of soil and the upper 4 in. frozen immediately in liquid air to prevent enzymatic change. The root tissue was ground while frozen, lyophilized and the free amino acids extracted with acetone-water as previously described.3fs Protein in the extracts was removed with picric acid according to the method of Stein and Moore.ll The free amino acids in the extracts then were separated by ion exchange chromatography according to the method described by Spackman, Stein and Moore.12 Two ml of each extract, which was equivalent to 120 mg of dry tissue was chromatographed on a Beckman/Spinco amino acid analyzer. The 150 cm column was employed for acidic and neutral amino acids using sodium citrate buffer at pH 3.25 at 30”-50” as the eluting agent. A typical elutogram is shown in Fig. 1. lo B. H. NICOLET and L. A. !&INN, J. Am. Gem. Sot., 61, 1615 (1939). I1 W. H. STEIN and S. MOORE, J. Bioi. Chem., 211, 915 (1954). I* D. H. SPACKMAN, W. H. STEIN and S. MOORE, Anal. Gem., 30, 1190 (1958). L

244

M. DEAN WILDING and MARK A. STAHMANN

Other chromatograms were run using 2 ml of an extract from alfalfa to which was added 1 ,uM of authentic, synthetic hydroxyaspartic acid4 kindly supplied by Dr. Sallach. The results of the co-chromatography are shown in Fig. 2. Periodate oxidation

An amount of extract equivalent to 4.8 g of dry root tissue was chromatographed in 4 runs and the effluent containing the ninhydrin positive material thought to be hydroxyaspartic acid was collected and concentrated to 4 ml. Two ml of the concentrated effluent was placed in the outer well of a Conway diffusion dish containing 2 ml of 0.2 M periodate and O-1 ml of 5 per cent glycine solution to bind the formaldehyde released. The center well contained 2 ml of 2 per cent boric acid solution with indicator. The contents were stirred gently, incubated 3 hr at room temperature and the ammonia released then titrated. Standards containing 1,uM of authentic synthetic hydroxyaspartic acid and controls without periodate were run simultaneously. Acknowledgemenrished with the approval of the Director of the Wisconsin Agricultural Research Station. Baaed on a portion of a Ph.D. thesis by M. Dean Wilding. Research supported by grants from the Herman Frasch Foundation and the Research Committee of the Graduate School, University of Wisconsin, from funds supplied by the Wisconsin Alumni Research Foundation.