ANALYTICAL BIOCHEMISTRY 70, 1-17 (1976)

Deuterium Oxide as a Tool for the Study of Amino Acid Metabolism I R.

MITRA, 2 J. BURTON, AND

J. E. VARNER~

Department of Biology, Washington University, St. Louis, Missouri 63130 Received September 19, 1974; accepted June 4, 1975 We have used deuterium oxide in nontoxic concentrations to study, in intact seedlings, the biosynthesis of amino acids. The extent and pattern of deuteration, as determined by a gas-liquid chromatograph-mass spectrometer system, permits conclusions about the biosynthesis of individual amino acids and also about their exposure to transaminases and other enzymes that might introduce deuterium into specific positions of the amino acid by exchange. This method could be used to study amino acid biogenesis in any organism that can tolerate 20-40% deuterium oxide for a period of a few hours to a few days.

Deuterium oxide in nontoxic concentrations has been used for the in vivo labeling of macromolecules such as nucleic acids and proteins. This is possible because of the nonexchangeability of the deuterium of the deuterium-carbon bonds that are formed during the biosynthesis of the monomeric precursors of such macromolecules. Deuterium oxide has the advantage as an in vivo label of freely and quickly entering all subcellular compartments. We here report a study of the biosynthesis in intact barley tissue of a number of amino acids using deuterium oxide as a tracer and the g l c - M S system to determine the labeling patterns of the individual protein amino acids. The extent and pattern of deuteration of each amino acid allow conclusions not only about the biosynthesis of the individual amino acids but also about their exposure to aminotransferases (transaminases), dehydratases, dehydrogenases, and other enzymes that might introduce deuterium into specific positions of the amino acids. We grew barley seedlings in the dark in 40% deuterium oxide under two conditions (Fig. 1): (i) germination and growth with the endosperm attached and (ii) germination and growth of the excised embryo (no endosperm) on nutrient medium either containing nitrate (+N) or lacking nitrate ( - N ) . Under the first condition we might expect the biosynthesis of at least some of the amino acids to be repressed or inhibited i Supported in part by the National Science Foundation (GB-39944). 2 An International Atomic Energy Agency Research Fellow while on deputation to the United States from the Biology and Agriculture Division, Bhabha Atomic Research Center, Bombay 400085, India. n To whom inquiries and reprint requests should be addressed.

Copyright© 1976by AcademicPress, Inc. All rightsof reproductionm any formreserved.

2

MITRA, BURTON AND VARNER Normal Embryos

Nutrients.

Excised Embryos

D~O

Nutrients,

D20

Nutrients, D20

N

Normal Excised

Root Coleoptde

| (

*N -N

( ~ I

Recovery of TCA-msoluble Protein, Derlvatlzatlon with T M.S.

+N

~ GLC

Stored Data

~ Mass Spectrometer

~

Print Out

FIG. 1. Experimental procedure. by the amino acids resulting from endosperm hydrolysis. Under the second condition, with added nitrate, we might expect all amino acid biosynthetic pathways to be functional. Excised embryos with no exogenous nitrate ( - N ) obviously could not accumulate much protein containing newly synthesized amino acids. Those embryos supplied with an exogenous nitrate supply (+N) could accumulate protein with newly synthesized amino acids. Thus, we anticipated that the difference between the (+N) and ( - N ) experiments would reflect the extent of de novo synthesis, since the ( - N ) embryo would obtain amino acids predominantly from turnover of preexisting protein. METHODS

Germination of Seedlings Barley seeds (Hordeum vulgare L. Cv. Himalaya) were treated with 20% Clorox (commercially available bleach) for 20 min, rinsed with sterilized water three or four times, placed in sterilized Petri dishes lined with two disks of Whatman No. 3 filter paper in 0 or 40% deuterium oxide and were allowed to germinate in the dark at room temperature. Roots and coleoptiles (containing the first leaf) were dissected after 6 days of growth.

Culture of Excised Embryos Barley seeds, after Clorox treatment and washing with water as above, were soaked in sterilized water for 12-15 hr. Embryos were dissected away from the endosperm and placed on 1% agar medium containing 200 mM sucrose (with or without 10 mM KNO~) and either water or 40% deuterium oxide (1). They were allowed to grow in the dark for 6 days. In 40% D20 the growth of the seedlings from both the normally germinating and excised embryos was about 18% inhibited on the basis of

D20 IN A M I N O ACID M E T A B O L I S M

3

fresh and dry weight measurements. Otherwise the pattern of growth appeared normal.

Recovery of TCA-Insoluble Protein After 6 days of germination the intact seedlings were dissected into roots and shoots for recovery of trichloroacetic acid-insoluble protein. The 6-day-old excised embryos were not dissected. The plant material was homogenized by hand with a mortar and pestle with 10% TCA, by using a volume 10 times that of the plant material. The homogenate was heated to 70°C for 30 min, cooled, and centrifuged at 10,000g for 15 rain, at 4°C. The residue was extracted with 10% TCA and centrifuged. The TCA-insoluble residue was extracted twice with 95% ethanol and twice with acetone. Each time the residue was recovered by centrifugation. The residue was dried under an infrared lamp.

Hydrolysis and Purification of the Hydrolyzate Amino acid hydrolyzates were prepared for analysis using the methods of Putter et al. (2) with the exception that the decolorization step was omitted. About 60-70 mg of TCA-insoluble material was hydrolyzed in a sealed tube in 5 ml 6 N HC1 at 110°C for 24-26 hr. The hydrolyzate was filtered through glass-fiber filter paper (Whatman GF/C) under suction and evaporated to dryness in a vacuum desiccator. The last trace of HC1 was removed by dissolving the hydrolyzate in water and repeating the evaporation to dryness. Dowex 50W, H ÷ form, mesh 50-100 (Sigma) resin was used to remove sugars and organic acids from the mixture of amino acids. The resin was regenerated with 1 N HCI and washed with water. A glass column of 1 × 30-cm dimensions was used with a 5.75-ml bed volume of resin. The hydrolyzate was dissolved in 5 ml of 0.1 N HCI and passed through the resin at a flow rate of 0.5 ml/min. An additional 10 ml of 0.1 N HC1 was also passed through the resin. Then three 10-ml portions of water were passed successively at a flow rate of 0.8 ml/min. The amino acids were eluted with 20 ml of 3 N NH4OH at a flow rate of 0.5 ml/min. The NH3 was evaporated in the presence of concentrated H2SO4 and Drierite in a vacuum desiccator. The residue was dissolved in 2 ml of 0.05 N HC1.

Derivatization The trimethylsilylation of the amino acids and their separation in the gas chromatography, system followed the methods of Gehrke and Leimer (3) with the important exception that the reaction time was 0.5 hr instead of 2.5 hr.

4

MITRA, BURTON AND VARNER

A 0.5-ml aliquot of the amino acid mixture was added to a silylation reaction tube (Corning Glassworks Co., No. 9826, 16 × 75 mm, screw cap culture tube with Teflon lined cap) and dried under N2 in a Temperature Block Module (Scientific Products, McGraw Park, I11. 60085) at 90°C. When the sample appeared completely dry, any remaining trace of water was removed by azeotropic evaporation with methylene chloride (CH2C12, dry, redistilled)three to six times. Acetonitrile (125/~l, Pierce Chemical Co.) was added, followed by 125/~1 of B S T F A (Regis Chemical Co.). The reaction vial was sonicated for 1 rain. The lid was tightly closed and the samples refluxed in an oil bath at 150°C for 30 rain. The reaction tube was dipped into the oil bath up to the level of solvent present in the tube.

Gas Chromatography Column: 10% OV-11 on Supelcoport, 100-120 mesh Dimension: 4.2 m × 2 mm i.d., spiral glass column Injector temperature: 250°C Detector temperature: 250°C Initial column temperature: 80°C for 3 rain Programming: 4°/min to 210°C, then hold at 210°C Flow rate of He: 30 ml/min Sample injection: 3-5/~1 Detector: flame detector Machine: Varian aerograph. The retention time for each amino acid derivative was determined by running each one separately through the glc column. The resolution of a known mixture of amino acids is shown in Fig. 2.

Gas-Liquid Chromatography-Mass Spectrometry Analysis o f Amino Acids The combined g l c - M S analysis was effected with and LKB Model 9000 instrument fitted with a PDP-12 computer for recording the scans and storing the data on tape. A detailed description of the device is given by Holmes et al, (4). Ten to fifteen micrograms of amino acid mixture in 3-5/,~1 were used for the analysis. Scans reported here were consistently made at the top of the peak. Ascending and descending sides of the peak were also scanned to determine the extent of isotope fractionation. It has been shown (5) that two peaks appear for both glycine and lysine when silylated in the presence of acetonitrile. The first glycine derivative which appears as an early peak in the glc scan is completely

D20 IN A M I N O ACID METABOLISM LEU

VAL

5

THR

ILE

SER

MET GLU

GLY-3

PHE

2 PRO

TYR

%

.J 18

~4o"

2,

1go°'

LYS - 4

30

;~co

36

2b0°

,?

2;0"

FIG. 2. Separation of a mixture of TMS-amino acids by gas-liquid chromatography (time, minutes; temperature, °C).

converted in 40 hr of silylation time to the tri-TMS form. (The peak due to the tri-TMS glycine derivative appears later between isoleucine and proline.) In this paper we are concerned ofily with data for the di-TMS glycine derivative, the predominant derivative for our silylation time. Both derivatives of lysine (tri-TMS, tetra-TMS) were obtained in poor yields in 30 min.

Interpretation of Mass Spectra Four major fragmentation pathways were observed for the trimethylsilyl (TMS) derivatives of the amino acids as reported earlier (6,7). They are (a) the loss of a methyl group from the molecular ion M (M-15); (b) the loss of a methyl group and CO with retention of the silyloxy group (M-43); (c) the loss of carbotrimethyl silyloxy (M-117); and (d) loss of the amino acid side chain group R to yield a fragment consisting of the acarbon, trimethylsilylated carboxyl and amino groups of m/e 218. Signals occur at m/e 73 and m/e 147 due to the fragments [Si(CHa)a] + and [(CHs)2Si = OSi(CHa)a] ÷, respectively. For information regarding the incorporation of deuterium into the amino acids, we considered in particular the m/e (M-117) and m/e 218 fragments. The m/e 218 fragment provides information regarding the extent of transamination of the amino acid, because the ct-H can be replaced only through this mechanism. The replacement of hydrogen by deuterium will result in a mass increase of 1, and produce a strong signal at m/e 219.

6

MITRA, BURTON A N D V A R N E R

TYPICAL TMS FRAGMENTS R-group: (for alanine) m/e = 116 a-Hydrogen: m/e = 218

[ H,r"N~s~(cr")~] + /

L ""

u,

J

H

]+ Si(CHa)~

VandenHcuvel et al. (7) reported the occurrence of the substituted tropylium ion (CTH6-OTMSi) + in the spectra of tyrosine which results from lossof part of the sidcchain.Thus, the fragment we consideredfor tyrosinewas the tropylium ion,m/e 179,insteadof the (M-117) fragment at m/e 280. The presence of a strong signal at m/e 219 for threonine and methionine (without 2H labeling) also agrees with the observations of VandenHeuvel et al. (7) For glutamic acid, the ion of m/e 156 showed the strongest signal while the (M-117) fragment had low yield. The m/e 156 fragment probably came from a fragmentation pathway analagous to that of the N-trifluoroacetyl-O-alkyl ester (8) or the ethyl ester of glutamic acid (9). Baker et al. (10) have reported the occurrence of some common fragment ions of the TMS derivatives of amino acids. They have observed a fragment ion at (M+-90) due to [M+-HOSi (CH3)j. The (M-117) fragment ion of glutamic acid may be unstable and the exclusion of HOSi (CH3)3 may result in the m/e 156 fragment: O /C\ H2C-- C--H

Correction o f the R a w Data for the Natural Abundance o f 2H, 29Si, 15N, 180, and 13C The data printout from the PDP-12 computer attached to the LKB9000 g l c - M S consists of two columns listing the mass/charge (m/e) values and their corresponding relative intensities (R/I) (4). The abundance of a particular ion depends primarily on the activation energy for fragmentation, and it is therefore necessary to consider relative intensities of the peaks rather than their absolute intensities (11). Interpretation of the data is based on the assumption that the peak height ratios are proportional to the abundance of the molecular species from which they are derived (7). To determine the amount of deuterium incorporated into the various amino acids, all the components of each mass peak must be considered

D20 IN A M I N O A C I D METABOLISM

7

Relohve intensd/

n~I

+2

n+3

Moss number

/

No heoJy isotopes, no deuterrum Heavy isotopes only [H20 blank)

Deuteroled heovy isotope frogments

F--q

oeo,e,,om oo,y

FIG. 3. Fragment contributions to each mass peak.

and corrections made for naturally occurring isotopes (see Fig. 3). At mass n, the total signal is due only to the background due to the normal fragment with neither heavy isotopes nor deuterium present. At n + 1, the additional mass unit may be due either to the presence of some naturally occurring heavy isotope (such as 13C or 15N) or the incorporation of a single deuterium. At n + 2 and successive mass values, the number of components increases as various combinations of heavyisotope and deuterated heavy-isotope fragments become possible. Thus the calculations on the raw data involve (i) the assumption that the water blank values represent the contributions of naturally occurring heavy isotopes (which can be verified by a comparison of actual data to predicted values using a binomial distribution for each fragment) and (ii) the estimation and subtraction of the contribution of deuterated fragments at mass nj + 1 coming from nondeuterated species at mass nj which contain some other heavy isotope and therefore do not represent a deuterium contribution to the n~ + 1 signal. (However, this deuterated fragment does represent a contribution to some lower m/e signal and must be added back to the appropriate m/e value.) The remainder of signal after the above two subtractions represents the contributions of those fragments of interest whose mass is greater than n due only to the incorporation of deuterium. The following is a sample calculation according to the above out-

8

MITRA, BURTON AND VARNER

lined procedure applied to the alanine mass-ll6-fragment. Mass spectral data are from excised embryos grown in 40% deuterium oxide nutrient without a source of nitrate ( - N ) . Water blank. The contribution of naturally occurring heavy isotopes of carbon, nitrogen, silicon, hydrogen, and oxygen to mass values greater than n may be calculated assuming a binomial distribution (12). These calculated values are then compared to experimental data as a check on the assumption that the water blank data give adequate representation of heavy isotope contributions to each signal. The alanine fragment of mass 116 is composed of 5 carbon, 14 hydrogen, 1 nitrogen, and 1 silicon atoms, each having a heavy isotope in the following abundance (13): Isotope

Natural abundance (%)

13C 2H I~N 29Si 3°Si

1.11 0.015 0.37 4.70 3.09

The calculated contributions of the above isotope to the n + I signal is as follows: + 1 Isotopes

Number possible

b (x ;n,p)

Value

13C

5

(51) (.0111)(.9889) 4

.0531

2H

14

(141) (.00015)(.9998) 13

.0021

15N

1

.0037

2asi

1

.0470 .1059

Percentage of n + 1 signal due to naturally occurring isotopes: 10.59. In the above, the notation

b(x;n,p)=(nx)pXqn-Xrepresentstheprobabil-

ity ofx successes in n trials, each with a probability p of success, probabilityq o f f a i l u r e ( q - - 1 - p ) , w h e r e ( ~ ) combinatorial form (11).

is the binomial coefficient in

9

D20 IN AMINO ACID METABOLISM

At n + 2 all possible combinations of heavy isotopes must be considered, e.g., 13C and ZH, lac and 15N, 13C + ~3C, etc. These probabilities are calculated and totaled to give a percentage of total signal appearing at n + 2 which is due to naturally occurring isotopes, i.e., for alanine, 3.5%. Expected values are the calculated percentages of total signal for the given fragment, i.e., 10.59 and 3.5% of 78.80 for alanine masses 117 and 118, respectively. Proportion of signal

m/e

Expected

Experimental

116 117 118 119

8.34 2.76

68.56 7.44 2.46 0.34 78.80

Relative intensity values, in the uncorrected data, less than 4.0 are unreliable (due to poor correlation between water blank samples and amino acid standards below this value) and have been discarded.

Deuterium-Heavy-Isotope Contributions In the discussion of the mass spectrometry data, the following notation will be used: For any amino acid fragment being considered, let n represent the m/e value for those species containing neither deuterium nor any naturally occurring heavy isotope, e.g., for alanine, n = 116 (Fig. 4). Then n + 1, n + 2, etc., will represent those fragments which have increased mass due to incorporation of some heavy isotope, e.g., alanine 117, 118, etc. For any given m/e value, the contributions will be designated as D j (deuterium only), Ij (naturally occurring isotopes), and I~D j (deuterated plus natural isotopes), where j represents the mass units of that contribution. Thus, for the alanine m/e fragment n + 1, 11 represents the natural isotope contribution and D I the deuterium contribution to that peak. The largest signal for any fragment usually occurs at mass n (116 for alanine), accounting for the preponderance of nondeuterated species. At higher D~O concentrations, with no isotope discrimination, one would expect smaller n values than observed here, because an increase in the probability of deuterium incorporation would produce larger signals at higher mass numbers. At n + 1 (117 for alanine) the signal (81.9) has only two components, the natural isotope contribution (I 1 = 10.8) and that due to singly deuterated fragments, i.e., m/e - 11 -- D 1 = 71.1. Thus, D 1 is easily found

10

MITRA,

Mass number

116 tt

Peak height a

100

Water blank a

117 n+l

BURTON

AND

118 n+2

VARNER 119 n+3

120 n+4

81.9

51.6

18.8

4.4

11=10.8 D1=71.1

12= 3.6 I1D 1 = 7.7 D 2=40.3

I a = 0.5 I2D I = 2.6 I1D 2 = 4.4 D z=11.3

14=0 I3D1=0.4 I2D 2 = 1 . 5 PD z=1.2 D ~ = 1.3

Sums D°=114.9 D 1 = 81.8 D R= 46.2 D z = 12.5 D4= 1.3

R a t e s of deuteration D1/D ° = 0.71 D2/D 1 = 0.57 D 3 / D z = 0.28 Notation Is = natural isotope contribution } m a s s j = 0,1,2,3 D ~= incorporated deuterium contribution a N o r m a l i z e d s u c h t h a t n = 100 for c o m p a r a t i v e p u r p o s e s . FIG. 4. P r o c e d u r e for c o r r e c t i n g m a s s s p e c t r o m e t r y data.

by subtraction and the ratio D1/D ° (in this case, 0.71), where D o is the signal at n , is calculated to give a rough approximation at subsequent mass numbers of the incorporation of a single deuterium into a nondeuterated fragment (see Fig. 4). At n + 2 and for successive masses, the contributions to the signals from the possible combinations of deuterated fragments already containing heavy isotopes must be considered. For alanine, the signal (51.6) at n + 2 can be accounted for by (i) the presence of naturally occurring isotopes which could add two mass units designated by 12, e.g., 3°Si or combinations of single isotopes such as lac and 15N present simultaneously and (ii) singly deuterated fragments containing an isotope which adds one mass unit (I1Da). The latter cannot be obtained experimentally, so is assumed to be the amount of P isotopes which are singly deuterated as calculated from signal at n + 1, i.e., the product of D1/D ° and the I ~ contribution, 10.8 × 0.71 = 7.7. The only remaining contribution, D 2, is that of those fragments which have increased mass by 2 units due solely to the incorporation of deuterium. Thus D 2 = m / e - (I 2 + IID a) -- 40.3. Now the addition of a second deuterium to a singly deuterated fragment may be approximated by the ratio D2/D 1 (40.3/71.1 ~ 0.57) for use in successive calculations. At n + 3, the components of the signal (18.8) are 13 (combinations such as 3°Si and lac); singly deuterated fragments which contain two additional mass units due to heavy isotope, I2D1; doubly deuterated fragments which contain a single heavy isotope, IID2; and finally the contribution

D20 IN AMINO ACID METABOLISM

11

of those fragments containing deuterium only, D z. The values of each of the above are determined as follows: Fragment type (n + 3)

Derivation

Value

13

Directly from water blank data

0.5

I2D 1

Product of I 2 from water blank and the ratio D1/D °

2.6

UD 2

Product of IID 1 (found at n + 2) and the ratio of D2/D 1

4.4

Da

m/e

-

(I z + I2D 1 + I I D 2)

11.3

The above procedure is repeated for successive mass numbers, i.e., n + 4, n + 5, etc., where the number of components will increase due to the various possible combinations of deuterated and natural isotope species. Because singly deuterated fragments appear at n + 2, n + 3, and n + 4 (I1D ~, I2D ~, and IaD 1, respectively) as well as at n + 1, it is necessary to sum all of these to obtain the total number of those species which have incorporated exactly one deuterium, in this case, 81.8. The total of doubly deuterated fragments, 46.2, is similarly a sum of D 2, UD 2, and I2D 2. For three deuterium, the sum (D z + UD a) is 12.5. Analogous sums would be taken to measure total deuterium incorporation for those fragments which have signals at n + 4 and higher. Table 1 shows the results of similar calculations for the three remaining experimental conditions for alanine and the II other amino acids examined. The signal from raw data at n (e.g., 116 for alanine) was initially normalized to I00. The amount by which n is greater than I00 represents the sum of the contributions of naturally occurring heavy isotopes as indicated by the water blank (and checked by calculating the expected abundance from a binomial distribution as explained above). Thus, the corrected n value for alanine, 115, represents the sum of I 1, 12, 13, and 14 (the nondeuterated species from the water blank data), i.e., D °. The sums indicated at the right of Fig. 4 have been calculated for each amino acid at n, n + 1, n + 2, etc., and are summarized in Table 1. The possibility that isotope fractionation during gas-liquid chromatography gives rise to misleading data may be reduced by scanning the amino acid samples on the leading and trailing sides as well as at exact glc peak height. No peak broadening was observed, however, scans taken on the upslope of the peak showed detectable enrichment of the fragments with deuterium relative to the fragments from scans taken on the downslope. This isotope fractionation would have to be taken into

12

MITRA, BURTON AND VARNER TABLE 1 INCORPORATION OF DEUTERIUM INTO AMINO ACIDSa Excised

Normal embryo Amino acid fragment (+ ion)

Mass number of the fragment

Root

Coleoptile

115 e 153 100 26 3

n n + 1 n + 2 n + 3

embryo -N

+N

115 120 58 11 I

115 82 46 13 2

115 112 100 38 0

132 95 32 2

132 120 39 2

132 75 28 4

132 123 18 1

n n + 1

136 49

136 49

136 51

136 55

n n + 1 n + 2 n + 3

132 53 21 4

132 73 13 2

132 51 16 5

132 52 13 0

n n + 1

133 30

133 25

133 23

133 28

n n + 1 n + 2

115 44 5

115 35 3

115 36 6

115 78 11

n n + 1 n +2 n +3 n+4 n+5

131 107 80 23 1 0

131 96 31 7 1 0

131 106 80 34 10 2

131 110 109 68 24 5

n n + n + n + n + n+5

118 118 96 60 4 1

118 163 53 12 2 0

118 80 71 36 9

118 78 55 20 7

1

1

Alanine nb n + n + n + n +

NHSI(CH3) 3

CH3-- C--I

H

1 2 3 4

Aspartic acid

(CH~)~sio/

~,

~HSi(CH~)~

N H S I(C H 3)3 I --- C - - C O O S I ( C H 3 ) I H

z

Serine H NHSi(CHz)3 (CHs)sSiO-- C-- C - - I I H H NHSi(CH3)3 I

--- c - COOSi(CH.). H Glycine NHSi(CHz)3 H-- C--l

H

Methionine

H

H H NHSi(CHs)3

H

H H H

i i i [ H - C--S--C-- C--C---

Glutamic acid oII /c\ H2C

/NSI(CHz)3

H2C--C--H

1 2 3 4

13

DzO I N A M I N O A C I D M E T A B O L I S M T A B L E 1 (Continued)

Normal embryo A m i n o acid fragment (+ ion)

Mass number of the fragment

R oot

Coleoptile

n n + 1

118 22

Excised embryo -N

+N

118 20

118 16

6 6 1

3 3 1

5 8 1

118 23 11 8 4

n n + 1

133 20

133 20

133 20

133 19

n n n n n

1 2 3 4

122 54 8 6 3

122 52 19 11 5

122 36 15 9 5

122 40 28 20 12

n n + 1

131 45

131 37

131 34

131 38

n n + 1 n +2

119 36 6 2

119 45 6 2

119 45 20 10

Valine H--C--C--/ \ HsC H

n+2 n+3 n+4

NHSi(CHs)z t

---C-- COOSi(CHs)a i

H Leucine H3C~ /NHSi(CH~)s H--C--CH~-- C--H3C NHS*(CHz)z

+ + + +

I

--- C-- COOSi(CHz) 3 i

H I so leucine

c~-cH~c-

c--H

H3C

---C --COOSi(CHs)3 I tt Tyrosine H\ /tt

c=c

/c-os~(CH~)~

/C--C \ H tt Phenylalanine H tt ~C=C~

~- % H

H

NHSi(CH3)z I

- - - C-- COOSi(CH3)z [

H

NHSI(CH3) z

,c-cn,--c---

/C--C \

0

4

n n + 1

129 26

129 38

129 31

129 30

n n n n n

1 2 3 4

130 48 13 1 0

No scan taken

130 38 15 0 0

130 44 14 1 0

n n + 1 n + 2 n + 3

126 48 34 18

126 34 18 10

126 43 38 18

5 0

4 2

7 2

126 66 72 42 13 6

134 25

134 22

134 21

134 31

n+3 n+4

NHSI(CH3) 3

---c~,-c

0

119 39 16 8 3

H

+ + + +

n+4 n+5 n n + 1

14

MITRA, B U R T O N A N D V A R N E R TABLE 1 (Cont&ued)

Normal embryo Amino acid fragment (+ ion)

Excised embryo

Mass number o f the fragment

Root

Coleoptile

- N

+N

116 7 3 1 0 0

116 5 3 2 l 0

116 13 15 12 5 4

116 22 35 33 20 12

Proline

/ ttoC--CH H~C\'/\/NSi(CH~) 3 CH2

n n n n n

+ 1 + 2 + 3 +4

n+5

a D a t a are given for R-group fragments and the a-H fragment (if available) obtained from normally germinating and excised embryo tissues grown 6 days in 40% deuterium oxide medium. n is the ion fragment containing only hydrogen; n + 1, n + 2, etc. are the fragments containing one, two, etc. deuterium atoms. c The value o f n represents the sum o f all nondeuterated species. After normalizing the original signal at n to 100, the contributions of nondeuterated fragments containing heavy isotopes ( D ° P , D ° I 2, etc) have been determined and added to 100 (see Fig. 4). Similar calculations give values for n + 1, n + 2, etc.

account in any attempt to assess quantitatively various biosynthetic pathways to a given amino acid. RESULTS AND DISCUSSION

We examined the extent of deuterium incorporation into alanine, glycine, valine, leucine, isoleucine, proline, serine, aspartic acid, glutamic acid, methionine, phenylalanine, and tyrosine (other amino acids were destroyed during hydrolysis or recovered in poor yields after derivatization). All of these amino acids became deuterated to some extent both in the normally germinating seedlings and in the excised embryos. The extent of deuteration provides information regarding biosynthesis and exposure to enzymes which introduce hydrogen into specific positions on the carbon skeleton. The differences in deuterium incorporation into the + N and - N excised embryos ( + N ) - ( - N ) were positive for most amino acids as expected. The significant exceptions, aspartate, glutamate, and isoleucine, might be indicative of stress conditions (due to lack of nitrogen) in the - N embryos, which possibly cause more rapid cycling of these amino acids through intermediate pathways. Alanine, aspartic acid, serine, glycine, methionine, and glutamic acid were the most heavily deuterated. Thus, although the shoots and roots of the normally germinating seedlings may have had access to

D20 IN AMINO ACID METABOLISM

15

these preformed amino acids, they were not incorporated directly into protein, but at the least underwent extensive transamination. The introduction of 2H into the n + I , n + 2 , n + 3 , and n + 4 positions in alanine may not involve any enzyme other than glutamatepyruvate transaminase because G P T apparently catalyzes exchange with the medium of the fl-H's as well as the a-H of alanine (14). Therefore we cannot draw conclusions about the biosynthesis of the carbon skeleton of alanine under our experimental conditions. The signals at n + ! for the serine and aspartic acid amino acid fragments are substantially greater than the a-carbon n + 1 signals in the 218 fragments, implying participation of serine and aspartic acid in reactions other than transaminase. However if, for these amino acids, transaminases (or serine dehydratase) equilibrate the fl-H's as well as the a-H, we can conclude nothing about the synthesis of serine and aspartic acid from examination of data from the amino acid fragments only. Because transaminase can exchange only one of the H's of glycine (14) the appearance of any signal at the n + 2 level is indicative of the involvement of the carbon skeleton of glycine in some metabolic reaction in addition to transamination. The signal at the n + 4 and n + 5 positions of methionine may be due to the presence of 2H on either the methyl carbon or methylene carbons. Because the MS cannot distinguish between these two possibilities on the fragment examined, we can conclude little about the biosynthesis of the carbon skeleton of methionine. The pattern of deuteration could be determined by examination of various fragments which can be obtained by use of other derivatives. Appearance of a signal at the n + 2, n + 3, n + 4, and n + 5 positions of glutamic acid indicates involvement in metabolic reactions other than transamination (15) (since G O T and G P T do n o t exchange the fl-H's in glutamate). Valine, leucine, and isoleucine were not extensively deuterated. Incorporation of 2H into the 218 fragment at the a-carbon provides overlapping evidence for the extent of transamination at the n + I position of these amino acids. Significant differences between the amino acid fragment n + 1 signal and the n + 1 from the 218 fragment indicate participation in metabolism other than transamination. This is corroborated by substantial values for leucine at n + 3 and n + 4. Because tyrosine and phenylalanine are formed via two independent pathways from prephenic acid, we would expect similar deuteration patterns in the amino acid fragments. However, the p a r a - h y d r o g e n of phenylalanine is not present in tyrosine and the a-carbon moiety is removed from tyrosine during derivatization and fragmentation; thus, the two amino acid fragments are not comparable at corresponding signals.

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MITRA, B U R T O N A N D V A R N E R

Biosynthesis of phenylalanine is suggested by the low 219 value and the appearance of significant signal at n + 2, n + 3, n + 4, and n + 5 in the amino acid fragment. Although the tyrosine amino acid fragment does not show deuterium incorporation at n + 3 and n + 4, we might a priori expect about the same extent of biosynthesis as that of phenylalanine. The strong signals at n + 1 and n + 2 (where there is no significant transaminase contribution) is consistent with this supposition. The difference in the extent and pattern of deuteration of proline from the excised seedlings as compared to proline from the normally germinating seedlings makes it clear that the presence of the endosperm almost entirely prevents proline biosynthesis and that removal of the endosperm permits extensive synthesis of proline, even in the absence of added nitrate. Preliminary results indicate that 5 mM proline inhibits the biosynthesis of proline by excised seedlings growing on mineral salts, sucrose, and nitrate (Mitra, unpublished observations). In principle much more information than we have obtained could be extracted from the protein hydrolysate. By preparing other derivatives of the amino acids, by using different ionization conditions (either procedure could lead to a variety of fragments different from those examined in our experiments) and by using a high resolution mass spectrometer the position of each deuterium in each amino acid could be found. Nor is the application of this technique limited to plant tissues. Amino acid biosynthesis could be studied in any organism that could tolerate 20-40% deuterium oxide. For example, it should be possible to determine the essential amino acids for a small animal in a few hours to a few days by feeding or injecting the animal with deuterium oxide to a final concentration of 20-40% and withdrawing samples of blood for isolation and hydrolysis of blood proteins and derivatization and analysis of the hydrolysate. Essential amino acids would contain deuterium only in those positions subject to exchange by transaminases, dehydrogenases, etc., while nonessential amino acids would be deuterated more extensively. ACKNOWLEDGMENTS We thank Mr. W. H. Holland, Dr. W. F. Holmes, and Dr. W. R. Sherman, who together develop, manage, and operate the Washington University Mass Spectrometry Facility, for their help in the isotope analysis and gas chromatography used in this study. The Facility is housed in the laboratories of the Department of Psychiatry of the Washington University School of Medicine.

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3. Gehrke, C. W., and Leimer, K. (1971). J.'Chromatogr. 57, 219. 4. Holmes, W. F., Holland, W. H., Shore, B. L., Bier, D. M., and Sherman, W. R. (1973). Anal. Chem. 45, 2063. 5. Bergstrom, K., Gurtler, J., and Blomstrand, R. (1970). Anal. Biochem. 34, 74. 6. VandenHeuvel, W. J., Smith, J. L., Putter, I., and Cohen, J. S. (1970) J. Chromatogr. 50, 405. 7. VandenHeuvel, W. J., Smith, J. L., and Cohen, J. S. (1970) J. Chromatogr. Sci. 8, 567. 8. Gelpi, E., Koenig, W. A., Gibert, J., and Oro, J. (1969) J. Chromatogr. Sci. 7, 604. 9. Blackburn, S. (1968) in Amino Acid Determination Methods and Techniques, p. 205, Marcel Dekker, New York. 10. Baker, K. M., Shaw, M. A., and Williams, D. H. (1969) Chem. Commun. 1015, 1108.

11. Waller, G. R. (ed.) (1972) Biochemical Applications of Mass Spectrometry, WileyInterscience, New York. 12. Mosteller, F., Rourke, R. E., and Thomas, G. B. (1972) in Probability with Statistical Applications, Ch. 4, Addison-Wesley, Reading, Mass. 13. Weast, R. C., and Setby, S. M. (eds.) (1967) Handbook of Chemistry and Physics, p. B-6. Chemical Rubber Co.. Cleveland, Ohio. 14. Babu, U. M., and Johnston, R. B. (1974) Biochem. Biophys. Res. Commun. 58, 460. 15. Gadal, P., and Varner, J. E. unpublished observations.