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Radioactive methionine: determination, and distribution of radioactivity in the sulfur, methyl and 4-carbon moieties
Journal of Biochemical and Btophyszcal Methods, 11 (1985) 1-11
1
Elsevier BBM 00466
Radioactive methionine" determination, and distribution of radioactivity in the sulfur, methyl and 4-carbon moieties John Giovanelli and S. Harvey Mudd Laboratory of General and Comparative Biochemtstry, National Institute of Mental Health, Bethesda, MD 20205, U.S.A.
(Received 10 July 1984) (Accepted 2 January 1985)
Summary A simple and inexpensive method is described for isoIation and determinauon of [14C]methionine in the non-protein fraction of tissues extenstvely labeled with 14C, The effectiveness of the method was demonstrated by isolation of non-protein [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from the plant Lemna which had been grown for a number of generations on [U-14C]sucrose and contained a 2000-fold excess of 14C in undefined non-protein compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. This method also offers an attractive alternative to the use of more expensive and sophisticated equipment for assay of radioactive methionine under less demanding conditions. An advantage is that the isolated methiomnecarboxymethylsulfonmm salt is readily degraded to permit separate determination of radioactivity in the 4-carbon, methyl and sulfur moieties of methionine. During this work, a facale labilization of 3H attached to the (carboxy)methylene carbon of methiomnecarboxymethylsulfoniumsalt was observed. This labilizauon is ascribed to formation of a sulfur ylid. Key words: methiomne; methionine sulfoxide; methiomnecarboxymethylsulfonium salt; paper chromatography; Lemna; sulfur ylid.
Introduction Determination of [14C]methionine in protein of tissues extensively labeled with ~4C is relatively straightforward, since other 14C compounds in protein are limited to known amino acids. A variety of methods for such determinations, including Address for correspondence: John Giovanelli, Bmlding 32, Room 101, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A.
automated amino acid analysis [1] and high-performance liquid chromatography [2], has been described. By contrast, comparable assay of non-protein [14C]methionine requires its resolution from large excesses of 14C in complex mixtures containing undefined compounds. Such an assay is especially difficult in plants since, in addition to the complement of carbon compounds common to all organisms, they also contain many unusual secondary products in the non-protein fraction [3,41. For example, over 200 non-protein amino acids, some of which comprise the major portion of soluble nitrogen, have been found in the plant kingdom [41. As illustrated by Becker [1] and the results of this work, currently available methods should be applied to the assay of non-protein [t4C]methionine only with caution, since they do not provide proof of the radiopurity of the amino acid. Here we describe the isolation and determination of non-protein [14C]methionine of established radiopurity from a plant extensively labeled with ~4C by long-term growth on [U-14Ctsucrose. The procedure is based on successive paper chromatographic purification first of methionine, then of the sulfoxide and carboxymethylsulfonium salt derivatives. Degradation of the latter derivative further provides, for the first time, a method for determination of radioactivity in each of the three moieties of methionine.
Materials and Methods
Chemicals Unlabeled methioninecarboxymethylsulfoniumsalt and methylthioacetic acid were prepared and isolated by Dowex 50-H + chromatography as described below for the radioactive compounds. Other radioactive compounds were obtained from Amersham, New England Nuclear, or ICN.
Chromatography Chromatography with Dowex 50-H + was performed as described [5]. Paper chromatograms on Whatman No. 1 paper were developed with the following solvents: solvent 1, 1-butanol/propionic acid/31.4 mM aqueous mercaptoethanol (250:124: 175, v/v); solvent 2, 2-propano1/88% formic acid/50 mM aqueous 2-mercaptoethanol (7 : 1 : 2, v/v).
Growth of plants Lemna paucicostata Hegelm. 6746 was grown for 2.1 generations mixotrophically with 20 /xM inorganic sulfate [61 and [U-14C]sucrose of specific activity 0.761 nCi/nmol. Plants grew with a normal doubling time of 36 h.
Harvesting and extraction of plants Plants (219 colonies) were harvested, washed to remove external radioactivity, frozen in liquid nitrogen, and homogenized in 10% trichloroacetic acid containing 50 nmol (181 000 dpm) of authentic [2-3H]methionine. The homogenate was separated into trichloroacetic acid-soluble and -insoluble fractions by centrifugation. Radioac-
tivity in the latter fraction was determined on an aliquot dissolved in 0.4 ml of Protosol (New England Nuclear).
Determination of [14C]methionine After removal of trichloroacetic acid by extraction with ether, the trichloroacetic acid-soluble fraction was adjusted to p H 5.0 with N H g O H , and boiled for 10 min to convert relatively unstable S-adenosylmethionine to 5'-methylthioadenosine and homoserine [7]. The boiled solution was chromatographed on Dowex 50-H +, and the N H 4 O H eluate evaporated to dryness. Any methionine sulfoxide was reduced to methionine by incubation of the eluted material at 100°C for 1 h in 0.2 ml of 0.7 M 2-mercaptoethanol in 10 mM potassium phosphate, p H 7.5. The incubation products were chromatographed with solvent 1. The peak localized with [3H]methionine was eluted, and chromatographed on Dowex 50-H +. The N H 4 O H eluate was evaporated to dryness, and the ehited material oxidized in 0.2 ml of 50% (v/v) dimethylsulfoxide in 3 N HC1 [8]. After 50-fold dilution with water, the reaction mixture was chromatographed on Dowex 50-H +. [14C]Methionine sulfoxide was recovered in the N H a O H ehiate and was chromatographed with solvent 1. The peak localized by [3H]methionine sulfoxide was eluted, reduced with 2-mercaptoethanol as described above, and evaporated to dryness. The residue was immediately dissolved in 1 ml of 0.215 M sodium iodoacetate in 20 mM formic acid (pH 5) and incubated at 40°C for 20 h to convert methionine to methioninecarboxymethylsulfonium salt [9]. The incubation mixture was diluted with 10 ml of water, and chromatographed on Dowex 50-H + to yield an N H 4 O H eluate. The latter was chromatographed with solvent 2 and the peak localized by [3H]methioninecarboxymethylsulfonium salt eluted for determination of the ratio of a4C/3H. This ratio was used to calculate the amount of [14C]methionine originally present in the plants.
Degradation of methioninecarboxymethylsulfonium salt Radioactive methioninecarboxymethylsulfonium salt was converted in 98% yield to homoserine (via the lactone) and methylthioacetate [9] by incubation for 80 min in 0.5 ml of 0.1 M Tris-HC1, p H 8.0, containing 100 nmol carrier methioninecarboxymethylsulfonium salt. The mixture was applied to a column of Dowex 50-H +, and radioactivity in methylthioacetate recovered in the effluent and water wash. These combined fractions were neutralized with Tris base, and radioactivity determined on an aliquot. Homoserine was eluted from the column with 5 ml of 3 N N H 4 O H and its radioactivity determined.
Wet combustion of methylthioacetate Conversion of methylthioacetate to inorganic sulfate (plus CO 2 and water) was based on the method of Toennies and Bakay [10]. The sample, together with Tris base (100 /~mol), Na2SO 4 (10 /~mol) and methylthioacetic acid (1 /~mol), was transferred to a vacuum hydrolysis tube (Kontes Glass, catalog No. K-896860-4015) equipped with an adjustable teflon seal. The solution was evaporated to dryness, and 0.4 ml of fuming H N O 3 and 0.05 ml of concentrated HC1 added to the residue. The contents of the tube were immediately frozen in a solid CO2-ethanol mixture, and
the tube evacuated. The bottom of the tube was immersed in a bath of silicone oil (HTF-100 Ucon fluid, Blue M Electric Co., Blue Island, Illinois), the surface of which was covered with a sheet (3 mm thick) of compressed asbestos ('Transite'). The major portion of the hydrolysis tube was allowed to protrude through a hole drilled in the cover. The cover helped to maintain a constant bath temperature, and was essential in preventing heat deformation of the seal and ensuing explosive release of the sample. After incubation at 250°C for 12 h, the contents of the tube were frozen, the seal removed, and 50 /xl of 1 M N a N O 3 added to the frozen contents. The contents were thawed, transferred quantitatively with water washes to a counting vial, and evaporated to dryness on a steam bath. To ensure complete removal of any 14CO2 formed by oxidation of the methyl moiety, 1 ml of 0.1 M N a H C O 3 was added to the residue, followed by 0.1 ml of 5 M HNO3, and the solution again evaporated before determination of radioactivity.
Results
Validation of [3H]methionine as internal marker Addition to the sample of a known amount of radioactivity in [3H]methionine provides a convenient marker during the chromatography of methionine and its derivatives, and allows corrections for losses during the purification of these compounds. Use of [3H]methionine for these purposes requires that no labilization of 3H occurs during the overall procedure. No significant change in 14C/3H was detected when mixtures of authentic [3H]- and [14C]methionine were carried through the procedures for isolation of methioninecarboxymethylsulfonium salt. These mixtures contained [methyl-3H]methionine or [2-3 H]methionine, each in the presence of either [methyl-14C] - or [1-14Clmethionine. This finding establishes the stability of 3H in [methyl-3H] - and [2-3Hlmethionine, and validates the use of these 3H compounds in the assay.
Lability of 3H on (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt A facile labilization of 3H on the (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt was observed during the synthesis of this compound from iodo[3H]acetate and [35S]methionine. 35S was incorporated in good yield into methioninecarboxymethylsulfonium salt, but no incorporation of 3H was detected. Authenticity of the iodo[3H]acetate was established by the stoichiometric incorporation of 3H into carboxymethylcysteine following incubation of iodo[3H]acetate with [U -14 Clcysteine.
Purification and determination of [~ 4C]methionine Since cellular carbon of Lemna growing under the standard mixotrophic conditions is derived predominantly from sucrose [6], extensive labeling with 14C was observed. Thus the plants incorporated 5.223 × 107 dpm 14C into the trichloroacetic acid-insoluble fraction and 9.018 × 106 dpm into the trichloroacetic-soluble fraction. Table 1 summarizes the main steps in purification of [14C]methionine in the latter
fraction, together with the degree of purification and recovery of 3H and 14C obtained in each of these steps. Fig. 1A-C illustrates the paper chromatographic purification of [14C]methionine and its sulfoxide and carboxymethylsulfonium salt derivatives. Paper chromatography of fraction 2 of Table 1 revealed a peak of 14C localized by [3H]methionine that comprised about 6% of 14C in this fraction (Fig. 1A). Although at first sight the approximate correspondence of 14C and 3H in this peak might suggest that the ratio of these radioactivities would provide an estimate of [14C]methionine, the results presented below show that [14C]methionine comprised only 2% of 14C in this peak. Radioactivity localized by [3H]methionine was eluted from the chromatogram of Fig. 1A and recovered in the NH4OH eluate after Dowex 50-H + chromatography (fraction 4 of Table 1). During this step a 3.6-fold purification occurred, the reason for which was not clarified. The NH4OH eluate, after oxidation with dimethylsulfoxide, was rechromatographed in the same solvent as used for the chromatogram of Fig. 1A. Use of the same solvent system ensures that only 14C material sensitive to oxidation undergoes a change in migration rate. Fig. 1B shews that oxidation resulted in essentially complete conversion of [3H]methionine to a compound with an appreciably lower Rf corresponding to that of methionine sulfoxide; only 11% of the 14C was converted to material migrating with methionine sulfoxide. The ratio of 14C/3H in the area corresponding to methionine sulfoxide indicated that an overall 1465-fold purification of [14C]methionine had been achieved (Table 1), but the 14C shoulder on the leading edge showed that 14C impurity was still present. Radioactivity in the methionine sulfoxide peak was eluted and, after reduction, incubated with iodoacetate. 14C and 3H now co-migrated during paper chromatography as a major peak with a mobility corresponding to that of methioninecarboxy-
TABLE 1 PURIFICATION OF [14C]METHIONINE
14C
Fraction
3H
Number Description
dpm (X10 -3)
%
181
(100) 9018
1 2 3 4 5 6
Trichloroacetic acid-soluble fraction NH4OH eluate from Dowex 50-H + Methiomne peak (eluate) NH4OH eluate from Dowex 50-H + Methionine sulfoxide peak (eluate) Methioninecarboxymethylsulfonium salt peak (eluate)
164 132
91 73
145
80
125
69 38
68.7
dpm (X10 -3)
2682 146 44.7
14C/3H %
Purification (-fold) a
(100) 30 1.6
49.8
(1.0)
16.3 1.11
3.0 45
0.50
0.308
162
4.27
0.047
0.034
1465
1.67
0.019
0.024
2075
a Calculated by dividing 49.8 (14C/3H in fraction 1) by 14C/3H in fraction.
A.
B.
[~CD
150,000 [I
I
100/
~
~4000
I
I
2000~
:
~
,
j L;
5o
~20~
~____~1~ ~ - o
!
"
1~0 i
,
4000
,
3000
~
20~
s ~ ~ ~ o
IO®~
o
4~
~
72000
~ 0
10
20
30
,~
Ioo
(2~)
@
J1000
50 )
0
40
~
0
~ 10
20
30
40
DISTANCE FROM ORIGIN(cm)
Fig. 1. Paper c h r o m a t o g r a p h i c purification of n o n - p r o t e i n [14C]metb~omne f r o m Lemna g r o w n u p o n
[U-14C]sucrose. The positions of authentic methmmne (1), methionine sulfoxide (2), methiomnecarboxymethylsulfomum salt (3), and homoserine (4) chromatographed in parallel were visualized with nimhydnn. Brackets indicate those sections of chromatograms that were eluted for further study. (A) Fraction 2 (see Tabte 1) chromatographed with solvent 1. (B) Peak localized by [3H]methiomne (indicated by bracket) from A after oxidation with dimethylsulfoxade, and chromatography as in A. (C) Peak locahzed by [3H]methiomne sulfoxlde (indicated by bracket) from B after reduction and incubation with iodoacetate Chromatography was with solvent 2. (D) Peak localized by [3H]methioninecarboxymethylsulfomum salt (indicated by bracket) from C after incubation at pH 8.0. The NH4OH eluate obtamed by Dowex 50-H+ chromatography of the incubation products was mixed with authentic homoserine and chromatographed with solvent 2. The shaded area shows the &stnbution of homoserine detected with
ninhydrin.
m e t h y l s u l f o n i u m salt (Fig. 1C). R a d i o a c t i v i t y localized b y a u t h e n t i c [3H]methioninecarboxymethylsulfonium salt (indicated by bracket in Fig. 1C) was eluted, and the ratio of 1 4 C / 3 H in this eluate (fraction 6 of Table 1) used to calculate [14 C]methionine originally present in the tissue. The radiopurity of 14C was confirmed by the finding that incubation of the eluate at p H 8.0 resulted in quantitative conversion of 14C to homoserine and methylthioacetate (see Table 2). Paper c h r o m a t o g r a p h y of the radioactive products of this incubation retained b y D o w e x 50-H + is illustrated in Fig. 1D. One major peak of 1 4 C w a s observed that migrated appreciably faster than the parent c o m p o u n d of Fig. 1C. This change in migration rate results specifically from incubation at p H 8.0, since identical solvent systems were used for the chromatograms of Fig. 1C, D. The 14C p r o d u c t co-migrated with authentic [3 H]homoserine produced during the degradation, and with authentic carrier homoserine visualized with ninhydrin. One minor peak of radioactivity was also detected, and was ascribed to a trace of undegraded methioninecarboxymethylsulfonium salt. Radioactive methylthioacetic acid, the other expected p r o d u c t of the
TABLE 2 DETERMINATION OF 14C IN METHYL AND 4-CARBON GROUPS OF METHIONINE Experiment No.
Ortgin of methioninecarboxymethylsulfonium salt
Relative 14C in: Methylthioacetate (%) a
Homoserine (%) a
1
[methyl-14C]methionine
2 3 4 5
[2-14 C]methionine [U-14C]methionine [35S, U-~4C]methionine non-protein [14C]methionine
99.2 (100) 0.8 (0) 20.5 b (20.0) 21.0 cd (20.0) 36.8 e
0.8 (0) 99.2 (100) 79.5 b ( 8 0 . 0 ) 79.0 ° (80.0) 63.2 e
The same procedures (including paper chromatography steps) as outlined in Fig. 1 were followed for conversion of authentic radioactive methionine to radioactwe methioninecarboxymethylsulfoniumsalt. a Values calculated from the amounts of 14C in each fraction relative to the total recovery of 14C, which was essentially quantitative. Expected values are shown in parentheses. No corrections were made for the small proportions (2% or less) in undegraded methioninecarboxymethylsulfoniumsalt, smce these correctmns would have decreased the relative 14C in homoserine by less than 1 percentage unit. b Mean of 3 values (SE, 0.5). ° Mean of 11 values (SE, 0.4). d The amount of 14C in methylthioacetate was calculated by subtracting the amount of 3Ss, determined by wet combustion of methylthioacetate, from the total radioactivity in methylthioacetate. In this experiment there was an approximately 2-fold excess of 3Ss over 14C in methylthioacetate. e Mean value (SE, 2.0) obtained for fraction 6 (Table 1) and an additional sample isolated from Lemna growing under identical conditions. degradation, was characterized b y its r e t e n t i o n b y Dowex 1-formate at p H 5.5, b u t n o t b y Dowex 50-H +.
Determination of relative radioactivity in three moieties of methionine D e g r a d a t i o n of m e t h i o n i n e c a r b o x y m e t h y l s u l f o n i u msalt to h o m o s e r i n e a n d methylthioacetate permitted d e t e r m i n a t i o n of the a m o u n t s of radioactivity i n the 4 - c a r b o n a n d methylthio groups of methionine. T h e results o b t a i n e d b y d e g r a d a t i o n of authentic labeled samples of m e t h i o n i n e c a r b o x y m e t h y l s u l f o n i u m salt (Table 2) validate this method. For" all samples of degraded m e t h i o n i n e c a r b o x y m e t h y l s u l f o n i u m salt, radioactivity was recovered virtually quantitatively i n methylthioacetate a n d homoserine. M o r e t h a n 99% of the radioactivity derived from the methyl group of m e t h i o n i n e was recovered i n methylthioacetate ( E x p e r i m e n t 1), a n d more t h a n 99% of radioactivity from the 4 - c a r b o n group was recovered i n h o m o s e r i n e ( E x p e r i m e n t 2). T h e d i s t r i b u t i o n of 14C i n methylthioacetate a n d h o m o s e r i n e derived from authentic [U-14C]methionine was in excellent agreement with that expected (Experiment 3). E x p e r i m e n t 4 shows that the a d d i t i o n a l presence of a labeled sulfur group did n o t affect the validity of the assay of 14C i n the methyl a n d 4 - c a r b o n moieties of methionine. The d i s t r i b u t i o n of 14C i n the methyl a n d 4 - c a r b o n moieties of [14C]methionine isolated from the trichloroacetic acid-soluble fraction of Lemna is given i n E x p e r i m e n t 5. F o r samples c o n t a i n i n g b o t h 35S a n d 14C, radioactivity i n methylthioacetate is a m e a s u r e of that present in b o t h the sulfur a n d methyl moieties of m e t h i o n i n e . The
8
TABLE 3 WET COMBUSTION OF METHYLTHIOACETATE Compound [35S]Methylthioacetate
[rnethyl-14C]MethyltbAoaceta~e [35S.14C]Methylttuoacetate
Radioactivity in sulfate fraction (%) a 98.0 b (100) 0.25 c (0) 69.8 a (68.8) ~
Samp!es of authentic radioactive methylthtoacetate were derived from appropriately labeled methioninecarboxymethylsulfonium salt. a Percentage of radioactivity m methylthioacetate that was recovered in sulfate fractmn Expected values are shown in parentheses. b Mean of 4 determinations (SE, 0.8) ° Mean of 3 determinauons (SE. 0.1). d Mean of 3 determinations (SE, 0.6) e The expected value of 68.8% was calculated from the relative amounts of radmactivlty in the mixture of [35S]methionine and [U-14C]methionine tha~ was used for ultimate converszon t o [35S,14C]methylthioacetate as described in Experiment 4 of Table 2.
relative amounts of radioactivity in each of the latter moieties was determined by wet oxidation of methylthioacetate to inorganic sulfate and CO 2. Radioactivity in the sulfur moiety of methionine is given by that in [35S]sulfate, while subtraction of the latter value from the combined radioactivity in methylthioacetate yields 14C in the methyl moiety. Wet combustion of methylthioacetate requires quantitative recovery of the sulfur of methylthioacetate as sulfate, with negligible contamination of the latter by radioactivity from the methyl group. The results of Table 3 show these requirements to be satisfied. Further validation of the assay was provided by the excellent agreement between the observed and predicted values for combustion of a sample of methylthioacetate containing radioactivity in both sulfur and methyl groups. The importance of maintaining the incubation temperature at 250°C was demonstrated by the finding that incubations at 230°C for 12 or 25 h resulted in incomplete (80%) conversion of the sulfur of methyltfiioacetate to inorganic sulfate, and failed to convert 50-80% of the radioactivity of [rnethyl-14C]methylthioacetate to a volatile form.
Discussion
The procedure for purification of [14C]methionine exploits the large changes in R f values associated with the chemical modification of methionine and certain of its derivatives. [14C]Methionine was purified by successive paper c h r o m a t o g r a p h y first in the form of methionine (Fig. 1A), then as the sulfoxide (Fig. 1B) and finally as the carboxymethylsulfonium salt (Fig. 1C). 14C in methionine was determined from that in methioninecarboxymethylsulfonium salt, which was shown to be radiopure b y its co-migration with the authentic c o m p o u n d (Fig. 1C), and b y quantitative conversion
of 14C in this derivative to methylthioacetate and homoserine (Table 2, Fig. 1D). The effectiveness of the determination is demonstrated by the isolation of [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from Lemna containing a 2000-fold excess of 14C is undefined compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. The procedure could also be adapted to determination of [3H]methionine tabeled in the methyl and 2-carbons, since 3H on these carbons is stable throughout the purification procedures. Assay of [3H]methionine labeled on the remaining carbon atoms should also be feasible, since 3H in these positions would also be expected to be s t a r e during purification. In addition, we have used the method for the less demanding assay of protein [14C,35S]methionine in Lemna grown in the presence of [a4C,35Slmethionine [11]. Since methionine is essentially the only amino acid labeled under theseconditions, the procedures described here may be abbreviated by converting the methionine fraction eluted from the chromatogram of Fig. 1A directly to methioninecarboxymethylsulfonium salt without passage through methionine sulfoxide. An added advantage of the assay is that it is easily adapted to measurement of radioactivity in the 4-carbon, methyl, and sulfur moieties of methionine. The relative amounts of 14C in the 4-carbon moiety of both non-protein (Table 2) and protein [12] methionine isolated from Lemna grown for at least two generations in the presence of [U-14C]sucrose were less than the value of 80% expected for uniform distribution. Although the reason for this finding is not clear, it is suggested that (unlabeled) CO 2 may contribute preferentially to the 4-carbon moiety of methionine under the mixotrophic growth conditions employed. Exchange of CO 2 with the carboxyl groups of malate and oxaloacetate could be catalyzed by known plant enzymes [13], and these dicarboxylic acids are readily converted to aspartate, from which the 4-carbon moiety of methionine is derived [14]. Attempts to determine also the chemical amounts (and hence specific radioactivities) of [I4C]methionine by use of iodo[3H]acetate of known specific radioactivity * were prechided by the facile lability of 3H on the (carboxy)methylene carbon of methioninecarboxymethylsulfoniumsalt. Our studies suggest that this lability of 3 H results from the combined presence of adjacent sulfonium and carboxyl groups, since no labilization was observed of 3 H 0 n the methylene carbon of carboxymethylcysteine, nor on the methyl group of methioninecarboxymethylsulfonium salt. It is proposed that labilization of 3H in methioninecarb.oxymethylsulfoniumsalt proceeds by dissociation of ~H to yield a sulfur ylid CH3-S(R)-CH-COOH , where R = CH2-CH2-CH(NH2)COOH. This ylid would be expected to be stabilized through delocalization of carbanion electrons through the carboxyl group, and into the vacant 3d orbital of sulfur [15].
* In theory, 3H incorporated into methiouinecarboxymethylsulfomum salt from iodo[3H]acetate could be distinguished from that derived from [2-3H]methiomne by degradation of methaoninecarboxymethylsulfonium salt. Thus 3H derived from [2-3H]methiouine would appear only in homoserine, while that from iodoacetate would appear ouly m methylthioacetate.
10
Simplified description of the method and its advantages Simple and inexpensive procedures are described for isolation and deterrmnation of []4C]methionine in the presence of a 2000-fold excess of 14C in undefined compounds. Purification of [14C]methionme to radiopurity was accomphshed in approximately 40% yield by successive paper chromatography first in the form of methionine, then as the sulfoxide, and finally as the carboxymethylsulfonium salt. [3 H]Methionine added during extraction of the tissues provided a convenient marker during chromatography, and permitted correction for losses during purification. Determination of [14Ctmethionine was calculated from the ratio of 14C/3}-[ in methioninecarboxsanethylsutfonium salt of proven radiopurity. The method is unique in providing an assay of [14C]methionine in the non-protein fraction of tissues (especially plants) extensively labeled with 14C, and containing large excesses of 14C in a variety of compounds, many undefined. The method is applicable also to assay- of radioactive methionine under less demanding con&tions, and offers an attractive alternative to the use of more expensive and sophisticated equipment and ancillary skilled operators. An added advantage is that a determination of radioactivity in all three moieties of methionine is readily obtained by degradatmn of the purified derivative, methioninecarboxymethylsulfonium salt. Radioactivity in the 4-carbon and methyl groups of [14C]methionine was de*.ermined from the respective amounts of 14C in homoserine and methykhioacetate formed by this degradation~ In samples of methianine containing both 14C and SSs, methylthioacetate was oxidized to CO2, water and inorganic sulfate. Radioactivity in the latter is eqnivalent to 3SS in the sulfur group of methionine. The difference between this value and the total radioactivity in methylthioacetate is equivalent to 14C in the methyl group of methionine. Alternatively, ff time permits, the amount of 14C in the methyl moiety may be determ~aed directly after 35S is allowed to decay to a negli~ble amount; 35S is then calculated from the difference between the total radioactivity origmally present (before decay of 35S) in methylthioacetate, and that present in 14C. If the methyl group of methykhioacetate is known to be labeled only with 3H, [he separate amounts of 3H and 35S can, of course, be determined directly with a dual channel liquid scintillation spectrometer. Attempts to extend the method to determination of the specific radioactivity of methionine by use of ~odo[3H]acetate for synthesis of methioninecarboxymethylsulfonium salt were precluded by a facile tabilization of 3H on the (carboxy)methylene group of the latter compound.
Acknowledgements We wish to thank Professor F.C.C. Hoskin, Illinois Institute of Technology, Chicago, for his advice on methods for wet oxidation, and Dr. Anne H. Datko for growth of plants and helpful discussions during this work.
References 1 Becket, R.R. (1967) Methods Enzymol. 11, t08-121 2 Pfeifer, R.F. and Hill, D.W. (1983) Adv Chromatogr. 22, 37-69 3 Seigler, D.S. (1981) in The Biochemistry of Plants (Corm, E.E., ed.), Vol. 7, pp. 139-176, Academic Press, New York 4 Rosenthal, G.A. (1982) Plant Nonprotein Amino and Imino Acids, pp. 2 and 57-242, Academic Press, New York 5 Giovanelli, J., Mudd, S.H. and Datko, A.H. (1974) Plant Physiol. 54, 725-736 6 Datko, A.H., Mudd, S.H. and Giovanelli, J. (1980) Plant Physiol. 65, 906-912 7 Giovanelli, J., Mudd, S.H. and Datko, A.H. (1978) J. Biol. Chem. 253, 5665-5677 8 Lipton, S.H. and Bodwell, C.E. (1976) J. Agric. Food Chem. 24, 26-31 9 Gundlach, H.G., Moore, S. and Stein, W.H. (1959) J. Biol. Chem. 234, 1761-1764
11 10 11 12 13 14
Toennies, G. and Bakay, B. (1953) Anal. Chem. 25, 160-165 Giovanelli, J., Mud& S.H. and Datko, A.H. (1981) Biochem. Biophys. Res. Commun. 100, 831-839 Giovanelli, J., Mudd, S.H. and Datko, A.H. (1984) Plant Physiol., 77, 450-455 Davies, D.D. (1979) Annu. Rev. Plant Physiol. 30. 131-158 Bryan, J.K. (1980) in The Biochemistry of Plants (Miflin, B.J., ed.), VoL 5, pp. 403-452, Academic Press, New York 15 Trost, B.M. and Melvin. L.S., Jr. (1975) Sulfur Ylides, pp. 23-25 and 29-30, Acadermc Press, New York
1
Elsevier BBM 00466
Radioactive methionine" determination, and distribution of radioactivity in the sulfur, methyl and 4-carbon moieties John Giovanelli and S. Harvey Mudd Laboratory of General and Comparative Biochemtstry, National Institute of Mental Health, Bethesda, MD 20205, U.S.A.
(Received 10 July 1984) (Accepted 2 January 1985)
Summary A simple and inexpensive method is described for isoIation and determinauon of [14C]methionine in the non-protein fraction of tissues extenstvely labeled with 14C, The effectiveness of the method was demonstrated by isolation of non-protein [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from the plant Lemna which had been grown for a number of generations on [U-14C]sucrose and contained a 2000-fold excess of 14C in undefined non-protein compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. This method also offers an attractive alternative to the use of more expensive and sophisticated equipment for assay of radioactive methionine under less demanding conditions. An advantage is that the isolated methiomnecarboxymethylsulfonmm salt is readily degraded to permit separate determination of radioactivity in the 4-carbon, methyl and sulfur moieties of methionine. During this work, a facale labilization of 3H attached to the (carboxy)methylene carbon of methiomnecarboxymethylsulfoniumsalt was observed. This labilizauon is ascribed to formation of a sulfur ylid. Key words: methiomne; methionine sulfoxide; methiomnecarboxymethylsulfonium salt; paper chromatography; Lemna; sulfur ylid.
Introduction Determination of [14C]methionine in protein of tissues extensively labeled with ~4C is relatively straightforward, since other 14C compounds in protein are limited to known amino acids. A variety of methods for such determinations, including Address for correspondence: John Giovanelli, Bmlding 32, Room 101, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A.
automated amino acid analysis [1] and high-performance liquid chromatography [2], has been described. By contrast, comparable assay of non-protein [14C]methionine requires its resolution from large excesses of 14C in complex mixtures containing undefined compounds. Such an assay is especially difficult in plants since, in addition to the complement of carbon compounds common to all organisms, they also contain many unusual secondary products in the non-protein fraction [3,41. For example, over 200 non-protein amino acids, some of which comprise the major portion of soluble nitrogen, have been found in the plant kingdom [41. As illustrated by Becker [1] and the results of this work, currently available methods should be applied to the assay of non-protein [t4C]methionine only with caution, since they do not provide proof of the radiopurity of the amino acid. Here we describe the isolation and determination of non-protein [14C]methionine of established radiopurity from a plant extensively labeled with ~4C by long-term growth on [U-14Ctsucrose. The procedure is based on successive paper chromatographic purification first of methionine, then of the sulfoxide and carboxymethylsulfonium salt derivatives. Degradation of the latter derivative further provides, for the first time, a method for determination of radioactivity in each of the three moieties of methionine.
Materials and Methods
Chemicals Unlabeled methioninecarboxymethylsulfoniumsalt and methylthioacetic acid were prepared and isolated by Dowex 50-H + chromatography as described below for the radioactive compounds. Other radioactive compounds were obtained from Amersham, New England Nuclear, or ICN.
Chromatography Chromatography with Dowex 50-H + was performed as described [5]. Paper chromatograms on Whatman No. 1 paper were developed with the following solvents: solvent 1, 1-butanol/propionic acid/31.4 mM aqueous mercaptoethanol (250:124: 175, v/v); solvent 2, 2-propano1/88% formic acid/50 mM aqueous 2-mercaptoethanol (7 : 1 : 2, v/v).
Growth of plants Lemna paucicostata Hegelm. 6746 was grown for 2.1 generations mixotrophically with 20 /xM inorganic sulfate [61 and [U-14C]sucrose of specific activity 0.761 nCi/nmol. Plants grew with a normal doubling time of 36 h.
Harvesting and extraction of plants Plants (219 colonies) were harvested, washed to remove external radioactivity, frozen in liquid nitrogen, and homogenized in 10% trichloroacetic acid containing 50 nmol (181 000 dpm) of authentic [2-3H]methionine. The homogenate was separated into trichloroacetic acid-soluble and -insoluble fractions by centrifugation. Radioac-
tivity in the latter fraction was determined on an aliquot dissolved in 0.4 ml of Protosol (New England Nuclear).
Determination of [14C]methionine After removal of trichloroacetic acid by extraction with ether, the trichloroacetic acid-soluble fraction was adjusted to p H 5.0 with N H g O H , and boiled for 10 min to convert relatively unstable S-adenosylmethionine to 5'-methylthioadenosine and homoserine [7]. The boiled solution was chromatographed on Dowex 50-H +, and the N H 4 O H eluate evaporated to dryness. Any methionine sulfoxide was reduced to methionine by incubation of the eluted material at 100°C for 1 h in 0.2 ml of 0.7 M 2-mercaptoethanol in 10 mM potassium phosphate, p H 7.5. The incubation products were chromatographed with solvent 1. The peak localized with [3H]methionine was eluted, and chromatographed on Dowex 50-H +. The N H 4 O H eluate was evaporated to dryness, and the ehited material oxidized in 0.2 ml of 50% (v/v) dimethylsulfoxide in 3 N HC1 [8]. After 50-fold dilution with water, the reaction mixture was chromatographed on Dowex 50-H +. [14C]Methionine sulfoxide was recovered in the N H a O H ehiate and was chromatographed with solvent 1. The peak localized by [3H]methionine sulfoxide was eluted, reduced with 2-mercaptoethanol as described above, and evaporated to dryness. The residue was immediately dissolved in 1 ml of 0.215 M sodium iodoacetate in 20 mM formic acid (pH 5) and incubated at 40°C for 20 h to convert methionine to methioninecarboxymethylsulfonium salt [9]. The incubation mixture was diluted with 10 ml of water, and chromatographed on Dowex 50-H + to yield an N H 4 O H eluate. The latter was chromatographed with solvent 2 and the peak localized by [3H]methioninecarboxymethylsulfonium salt eluted for determination of the ratio of a4C/3H. This ratio was used to calculate the amount of [14C]methionine originally present in the plants.
Degradation of methioninecarboxymethylsulfonium salt Radioactive methioninecarboxymethylsulfonium salt was converted in 98% yield to homoserine (via the lactone) and methylthioacetate [9] by incubation for 80 min in 0.5 ml of 0.1 M Tris-HC1, p H 8.0, containing 100 nmol carrier methioninecarboxymethylsulfonium salt. The mixture was applied to a column of Dowex 50-H +, and radioactivity in methylthioacetate recovered in the effluent and water wash. These combined fractions were neutralized with Tris base, and radioactivity determined on an aliquot. Homoserine was eluted from the column with 5 ml of 3 N N H 4 O H and its radioactivity determined.
Wet combustion of methylthioacetate Conversion of methylthioacetate to inorganic sulfate (plus CO 2 and water) was based on the method of Toennies and Bakay [10]. The sample, together with Tris base (100 /~mol), Na2SO 4 (10 /~mol) and methylthioacetic acid (1 /~mol), was transferred to a vacuum hydrolysis tube (Kontes Glass, catalog No. K-896860-4015) equipped with an adjustable teflon seal. The solution was evaporated to dryness, and 0.4 ml of fuming H N O 3 and 0.05 ml of concentrated HC1 added to the residue. The contents of the tube were immediately frozen in a solid CO2-ethanol mixture, and
the tube evacuated. The bottom of the tube was immersed in a bath of silicone oil (HTF-100 Ucon fluid, Blue M Electric Co., Blue Island, Illinois), the surface of which was covered with a sheet (3 mm thick) of compressed asbestos ('Transite'). The major portion of the hydrolysis tube was allowed to protrude through a hole drilled in the cover. The cover helped to maintain a constant bath temperature, and was essential in preventing heat deformation of the seal and ensuing explosive release of the sample. After incubation at 250°C for 12 h, the contents of the tube were frozen, the seal removed, and 50 /xl of 1 M N a N O 3 added to the frozen contents. The contents were thawed, transferred quantitatively with water washes to a counting vial, and evaporated to dryness on a steam bath. To ensure complete removal of any 14CO2 formed by oxidation of the methyl moiety, 1 ml of 0.1 M N a H C O 3 was added to the residue, followed by 0.1 ml of 5 M HNO3, and the solution again evaporated before determination of radioactivity.
Results
Validation of [3H]methionine as internal marker Addition to the sample of a known amount of radioactivity in [3H]methionine provides a convenient marker during the chromatography of methionine and its derivatives, and allows corrections for losses during the purification of these compounds. Use of [3H]methionine for these purposes requires that no labilization of 3H occurs during the overall procedure. No significant change in 14C/3H was detected when mixtures of authentic [3H]- and [14C]methionine were carried through the procedures for isolation of methioninecarboxymethylsulfonium salt. These mixtures contained [methyl-3H]methionine or [2-3 H]methionine, each in the presence of either [methyl-14C] - or [1-14Clmethionine. This finding establishes the stability of 3H in [methyl-3H] - and [2-3Hlmethionine, and validates the use of these 3H compounds in the assay.
Lability of 3H on (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt A facile labilization of 3H on the (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt was observed during the synthesis of this compound from iodo[3H]acetate and [35S]methionine. 35S was incorporated in good yield into methioninecarboxymethylsulfonium salt, but no incorporation of 3H was detected. Authenticity of the iodo[3H]acetate was established by the stoichiometric incorporation of 3H into carboxymethylcysteine following incubation of iodo[3H]acetate with [U -14 Clcysteine.
Purification and determination of [~ 4C]methionine Since cellular carbon of Lemna growing under the standard mixotrophic conditions is derived predominantly from sucrose [6], extensive labeling with 14C was observed. Thus the plants incorporated 5.223 × 107 dpm 14C into the trichloroacetic acid-insoluble fraction and 9.018 × 106 dpm into the trichloroacetic-soluble fraction. Table 1 summarizes the main steps in purification of [14C]methionine in the latter
fraction, together with the degree of purification and recovery of 3H and 14C obtained in each of these steps. Fig. 1A-C illustrates the paper chromatographic purification of [14C]methionine and its sulfoxide and carboxymethylsulfonium salt derivatives. Paper chromatography of fraction 2 of Table 1 revealed a peak of 14C localized by [3H]methionine that comprised about 6% of 14C in this fraction (Fig. 1A). Although at first sight the approximate correspondence of 14C and 3H in this peak might suggest that the ratio of these radioactivities would provide an estimate of [14C]methionine, the results presented below show that [14C]methionine comprised only 2% of 14C in this peak. Radioactivity localized by [3H]methionine was eluted from the chromatogram of Fig. 1A and recovered in the NH4OH eluate after Dowex 50-H + chromatography (fraction 4 of Table 1). During this step a 3.6-fold purification occurred, the reason for which was not clarified. The NH4OH eluate, after oxidation with dimethylsulfoxide, was rechromatographed in the same solvent as used for the chromatogram of Fig. 1A. Use of the same solvent system ensures that only 14C material sensitive to oxidation undergoes a change in migration rate. Fig. 1B shews that oxidation resulted in essentially complete conversion of [3H]methionine to a compound with an appreciably lower Rf corresponding to that of methionine sulfoxide; only 11% of the 14C was converted to material migrating with methionine sulfoxide. The ratio of 14C/3H in the area corresponding to methionine sulfoxide indicated that an overall 1465-fold purification of [14C]methionine had been achieved (Table 1), but the 14C shoulder on the leading edge showed that 14C impurity was still present. Radioactivity in the methionine sulfoxide peak was eluted and, after reduction, incubated with iodoacetate. 14C and 3H now co-migrated during paper chromatography as a major peak with a mobility corresponding to that of methioninecarboxy-
TABLE 1 PURIFICATION OF [14C]METHIONINE
14C
Fraction
3H
Number Description
dpm (X10 -3)
%
181
(100) 9018
1 2 3 4 5 6
Trichloroacetic acid-soluble fraction NH4OH eluate from Dowex 50-H + Methiomne peak (eluate) NH4OH eluate from Dowex 50-H + Methionine sulfoxide peak (eluate) Methioninecarboxymethylsulfonium salt peak (eluate)
164 132
91 73
145
80
125
69 38
68.7
dpm (X10 -3)
2682 146 44.7
14C/3H %
Purification (-fold) a
(100) 30 1.6
49.8
(1.0)
16.3 1.11
3.0 45
0.50
0.308
162
4.27
0.047
0.034
1465
1.67
0.019
0.024
2075
a Calculated by dividing 49.8 (14C/3H in fraction 1) by 14C/3H in fraction.
A.
B.
[~CD
150,000 [I
I
100/
~
~4000
I
I
2000~
:
~
,
j L;
5o
~20~
~____~1~ ~ - o
!
"
1~0 i
,
4000
,
3000
~
20~
s ~ ~ ~ o
IO®~
o
4~
~
72000
~ 0
10
20
30
,~
Ioo
(2~)
@
J1000
50 )
0
40
~
0
~ 10
20
30
40
DISTANCE FROM ORIGIN(cm)
Fig. 1. Paper c h r o m a t o g r a p h i c purification of n o n - p r o t e i n [14C]metb~omne f r o m Lemna g r o w n u p o n
[U-14C]sucrose. The positions of authentic methmmne (1), methionine sulfoxide (2), methiomnecarboxymethylsulfomum salt (3), and homoserine (4) chromatographed in parallel were visualized with nimhydnn. Brackets indicate those sections of chromatograms that were eluted for further study. (A) Fraction 2 (see Tabte 1) chromatographed with solvent 1. (B) Peak localized by [3H]methiomne (indicated by bracket) from A after oxidation with dimethylsulfoxade, and chromatography as in A. (C) Peak locahzed by [3H]methiomne sulfoxlde (indicated by bracket) from B after reduction and incubation with iodoacetate Chromatography was with solvent 2. (D) Peak localized by [3H]methioninecarboxymethylsulfomum salt (indicated by bracket) from C after incubation at pH 8.0. The NH4OH eluate obtamed by Dowex 50-H+ chromatography of the incubation products was mixed with authentic homoserine and chromatographed with solvent 2. The shaded area shows the &stnbution of homoserine detected with
ninhydrin.
m e t h y l s u l f o n i u m salt (Fig. 1C). R a d i o a c t i v i t y localized b y a u t h e n t i c [3H]methioninecarboxymethylsulfonium salt (indicated by bracket in Fig. 1C) was eluted, and the ratio of 1 4 C / 3 H in this eluate (fraction 6 of Table 1) used to calculate [14 C]methionine originally present in the tissue. The radiopurity of 14C was confirmed by the finding that incubation of the eluate at p H 8.0 resulted in quantitative conversion of 14C to homoserine and methylthioacetate (see Table 2). Paper c h r o m a t o g r a p h y of the radioactive products of this incubation retained b y D o w e x 50-H + is illustrated in Fig. 1D. One major peak of 1 4 C w a s observed that migrated appreciably faster than the parent c o m p o u n d of Fig. 1C. This change in migration rate results specifically from incubation at p H 8.0, since identical solvent systems were used for the chromatograms of Fig. 1C, D. The 14C p r o d u c t co-migrated with authentic [3 H]homoserine produced during the degradation, and with authentic carrier homoserine visualized with ninhydrin. One minor peak of radioactivity was also detected, and was ascribed to a trace of undegraded methioninecarboxymethylsulfonium salt. Radioactive methylthioacetic acid, the other expected p r o d u c t of the
TABLE 2 DETERMINATION OF 14C IN METHYL AND 4-CARBON GROUPS OF METHIONINE Experiment No.
Ortgin of methioninecarboxymethylsulfonium salt
Relative 14C in: Methylthioacetate (%) a
Homoserine (%) a
1
[methyl-14C]methionine
2 3 4 5
[2-14 C]methionine [U-14C]methionine [35S, U-~4C]methionine non-protein [14C]methionine
99.2 (100) 0.8 (0) 20.5 b (20.0) 21.0 cd (20.0) 36.8 e
0.8 (0) 99.2 (100) 79.5 b ( 8 0 . 0 ) 79.0 ° (80.0) 63.2 e
The same procedures (including paper chromatography steps) as outlined in Fig. 1 were followed for conversion of authentic radioactive methionine to radioactwe methioninecarboxymethylsulfoniumsalt. a Values calculated from the amounts of 14C in each fraction relative to the total recovery of 14C, which was essentially quantitative. Expected values are shown in parentheses. No corrections were made for the small proportions (2% or less) in undegraded methioninecarboxymethylsulfoniumsalt, smce these correctmns would have decreased the relative 14C in homoserine by less than 1 percentage unit. b Mean of 3 values (SE, 0.5). ° Mean of 11 values (SE, 0.4). d The amount of 14C in methylthioacetate was calculated by subtracting the amount of 3Ss, determined by wet combustion of methylthioacetate, from the total radioactivity in methylthioacetate. In this experiment there was an approximately 2-fold excess of 3Ss over 14C in methylthioacetate. e Mean value (SE, 2.0) obtained for fraction 6 (Table 1) and an additional sample isolated from Lemna growing under identical conditions. degradation, was characterized b y its r e t e n t i o n b y Dowex 1-formate at p H 5.5, b u t n o t b y Dowex 50-H +.
Determination of relative radioactivity in three moieties of methionine D e g r a d a t i o n of m e t h i o n i n e c a r b o x y m e t h y l s u l f o n i u msalt to h o m o s e r i n e a n d methylthioacetate permitted d e t e r m i n a t i o n of the a m o u n t s of radioactivity i n the 4 - c a r b o n a n d methylthio groups of methionine. T h e results o b t a i n e d b y d e g r a d a t i o n of authentic labeled samples of m e t h i o n i n e c a r b o x y m e t h y l s u l f o n i u m salt (Table 2) validate this method. For" all samples of degraded m e t h i o n i n e c a r b o x y m e t h y l s u l f o n i u m salt, radioactivity was recovered virtually quantitatively i n methylthioacetate a n d homoserine. M o r e t h a n 99% of the radioactivity derived from the methyl group of m e t h i o n i n e was recovered i n methylthioacetate ( E x p e r i m e n t 1), a n d more t h a n 99% of radioactivity from the 4 - c a r b o n group was recovered i n h o m o s e r i n e ( E x p e r i m e n t 2). T h e d i s t r i b u t i o n of 14C i n methylthioacetate a n d h o m o s e r i n e derived from authentic [U-14C]methionine was in excellent agreement with that expected (Experiment 3). E x p e r i m e n t 4 shows that the a d d i t i o n a l presence of a labeled sulfur group did n o t affect the validity of the assay of 14C i n the methyl a n d 4 - c a r b o n moieties of methionine. The d i s t r i b u t i o n of 14C i n the methyl a n d 4 - c a r b o n moieties of [14C]methionine isolated from the trichloroacetic acid-soluble fraction of Lemna is given i n E x p e r i m e n t 5. F o r samples c o n t a i n i n g b o t h 35S a n d 14C, radioactivity i n methylthioacetate is a m e a s u r e of that present in b o t h the sulfur a n d methyl moieties of m e t h i o n i n e . The
8
TABLE 3 WET COMBUSTION OF METHYLTHIOACETATE Compound [35S]Methylthioacetate
[rnethyl-14C]MethyltbAoaceta~e [35S.14C]Methylttuoacetate
Radioactivity in sulfate fraction (%) a 98.0 b (100) 0.25 c (0) 69.8 a (68.8) ~
Samp!es of authentic radioactive methylthtoacetate were derived from appropriately labeled methioninecarboxymethylsulfonium salt. a Percentage of radioactivity m methylthioacetate that was recovered in sulfate fractmn Expected values are shown in parentheses. b Mean of 4 determinations (SE, 0.8) ° Mean of 3 determinauons (SE. 0.1). d Mean of 3 determinations (SE, 0.6) e The expected value of 68.8% was calculated from the relative amounts of radmactivlty in the mixture of [35S]methionine and [U-14C]methionine tha~ was used for ultimate converszon t o [35S,14C]methylthioacetate as described in Experiment 4 of Table 2.
relative amounts of radioactivity in each of the latter moieties was determined by wet oxidation of methylthioacetate to inorganic sulfate and CO 2. Radioactivity in the sulfur moiety of methionine is given by that in [35S]sulfate, while subtraction of the latter value from the combined radioactivity in methylthioacetate yields 14C in the methyl moiety. Wet combustion of methylthioacetate requires quantitative recovery of the sulfur of methylthioacetate as sulfate, with negligible contamination of the latter by radioactivity from the methyl group. The results of Table 3 show these requirements to be satisfied. Further validation of the assay was provided by the excellent agreement between the observed and predicted values for combustion of a sample of methylthioacetate containing radioactivity in both sulfur and methyl groups. The importance of maintaining the incubation temperature at 250°C was demonstrated by the finding that incubations at 230°C for 12 or 25 h resulted in incomplete (80%) conversion of the sulfur of methyltfiioacetate to inorganic sulfate, and failed to convert 50-80% of the radioactivity of [rnethyl-14C]methylthioacetate to a volatile form.
Discussion
The procedure for purification of [14C]methionine exploits the large changes in R f values associated with the chemical modification of methionine and certain of its derivatives. [14C]Methionine was purified by successive paper c h r o m a t o g r a p h y first in the form of methionine (Fig. 1A), then as the sulfoxide (Fig. 1B) and finally as the carboxymethylsulfonium salt (Fig. 1C). 14C in methionine was determined from that in methioninecarboxymethylsulfonium salt, which was shown to be radiopure b y its co-migration with the authentic c o m p o u n d (Fig. 1C), and b y quantitative conversion
of 14C in this derivative to methylthioacetate and homoserine (Table 2, Fig. 1D). The effectiveness of the determination is demonstrated by the isolation of [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from Lemna containing a 2000-fold excess of 14C is undefined compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. The procedure could also be adapted to determination of [3H]methionine tabeled in the methyl and 2-carbons, since 3H on these carbons is stable throughout the purification procedures. Assay of [3H]methionine labeled on the remaining carbon atoms should also be feasible, since 3H in these positions would also be expected to be s t a r e during purification. In addition, we have used the method for the less demanding assay of protein [14C,35S]methionine in Lemna grown in the presence of [a4C,35Slmethionine [11]. Since methionine is essentially the only amino acid labeled under theseconditions, the procedures described here may be abbreviated by converting the methionine fraction eluted from the chromatogram of Fig. 1A directly to methioninecarboxymethylsulfonium salt without passage through methionine sulfoxide. An added advantage of the assay is that it is easily adapted to measurement of radioactivity in the 4-carbon, methyl, and sulfur moieties of methionine. The relative amounts of 14C in the 4-carbon moiety of both non-protein (Table 2) and protein [12] methionine isolated from Lemna grown for at least two generations in the presence of [U-14C]sucrose were less than the value of 80% expected for uniform distribution. Although the reason for this finding is not clear, it is suggested that (unlabeled) CO 2 may contribute preferentially to the 4-carbon moiety of methionine under the mixotrophic growth conditions employed. Exchange of CO 2 with the carboxyl groups of malate and oxaloacetate could be catalyzed by known plant enzymes [13], and these dicarboxylic acids are readily converted to aspartate, from which the 4-carbon moiety of methionine is derived [14]. Attempts to determine also the chemical amounts (and hence specific radioactivities) of [I4C]methionine by use of iodo[3H]acetate of known specific radioactivity * were prechided by the facile lability of 3H on the (carboxy)methylene carbon of methioninecarboxymethylsulfoniumsalt. Our studies suggest that this lability of 3 H results from the combined presence of adjacent sulfonium and carboxyl groups, since no labilization was observed of 3 H 0 n the methylene carbon of carboxymethylcysteine, nor on the methyl group of methioninecarboxymethylsulfonium salt. It is proposed that labilization of 3H in methioninecarb.oxymethylsulfoniumsalt proceeds by dissociation of ~H to yield a sulfur ylid CH3-S(R)-CH-COOH , where R = CH2-CH2-CH(NH2)COOH. This ylid would be expected to be stabilized through delocalization of carbanion electrons through the carboxyl group, and into the vacant 3d orbital of sulfur [15].
* In theory, 3H incorporated into methiouinecarboxymethylsulfomum salt from iodo[3H]acetate could be distinguished from that derived from [2-3H]methiomne by degradation of methaoninecarboxymethylsulfonium salt. Thus 3H derived from [2-3H]methiouine would appear only in homoserine, while that from iodoacetate would appear ouly m methylthioacetate.
10
Simplified description of the method and its advantages Simple and inexpensive procedures are described for isolation and deterrmnation of []4C]methionine in the presence of a 2000-fold excess of 14C in undefined compounds. Purification of [14C]methionme to radiopurity was accomphshed in approximately 40% yield by successive paper chromatography first in the form of methionine, then as the sulfoxide, and finally as the carboxymethylsulfonium salt. [3 H]Methionine added during extraction of the tissues provided a convenient marker during chromatography, and permitted correction for losses during purification. Determination of [14Ctmethionine was calculated from the ratio of 14C/3}-[ in methioninecarboxsanethylsutfonium salt of proven radiopurity. The method is unique in providing an assay of [14C]methionine in the non-protein fraction of tissues (especially plants) extensively labeled with 14C, and containing large excesses of 14C in a variety of compounds, many undefined. The method is applicable also to assay- of radioactive methionine under less demanding con&tions, and offers an attractive alternative to the use of more expensive and sophisticated equipment and ancillary skilled operators. An added advantage is that a determination of radioactivity in all three moieties of methionine is readily obtained by degradatmn of the purified derivative, methioninecarboxymethylsulfonium salt. Radioactivity in the 4-carbon and methyl groups of [14C]methionine was de*.ermined from the respective amounts of 14C in homoserine and methykhioacetate formed by this degradation~ In samples of methianine containing both 14C and SSs, methylthioacetate was oxidized to CO2, water and inorganic sulfate. Radioactivity in the latter is eqnivalent to 3SS in the sulfur group of methionine. The difference between this value and the total radioactivity in methylthioacetate is equivalent to 14C in the methyl group of methionine. Alternatively, ff time permits, the amount of 14C in the methyl moiety may be determ~aed directly after 35S is allowed to decay to a negli~ble amount; 35S is then calculated from the difference between the total radioactivity origmally present (before decay of 35S) in methylthioacetate, and that present in 14C. If the methyl group of methykhioacetate is known to be labeled only with 3H, [he separate amounts of 3H and 35S can, of course, be determined directly with a dual channel liquid scintillation spectrometer. Attempts to extend the method to determination of the specific radioactivity of methionine by use of ~odo[3H]acetate for synthesis of methioninecarboxymethylsulfonium salt were precluded by a facile tabilization of 3H on the (carboxy)methylene group of the latter compound.
Acknowledgements We wish to thank Professor F.C.C. Hoskin, Illinois Institute of Technology, Chicago, for his advice on methods for wet oxidation, and Dr. Anne H. Datko for growth of plants and helpful discussions during this work.
References 1 Becket, R.R. (1967) Methods Enzymol. 11, t08-121 2 Pfeifer, R.F. and Hill, D.W. (1983) Adv Chromatogr. 22, 37-69 3 Seigler, D.S. (1981) in The Biochemistry of Plants (Corm, E.E., ed.), Vol. 7, pp. 139-176, Academic Press, New York 4 Rosenthal, G.A. (1982) Plant Nonprotein Amino and Imino Acids, pp. 2 and 57-242, Academic Press, New York 5 Giovanelli, J., Mudd, S.H. and Datko, A.H. (1974) Plant Physiol. 54, 725-736 6 Datko, A.H., Mudd, S.H. and Giovanelli, J. (1980) Plant Physiol. 65, 906-912 7 Giovanelli, J., Mudd, S.H. and Datko, A.H. (1978) J. Biol. Chem. 253, 5665-5677 8 Lipton, S.H. and Bodwell, C.E. (1976) J. Agric. Food Chem. 24, 26-31 9 Gundlach, H.G., Moore, S. and Stein, W.H. (1959) J. Biol. Chem. 234, 1761-1764
11 10 11 12 13 14
Toennies, G. and Bakay, B. (1953) Anal. Chem. 25, 160-165 Giovanelli, J., Mud& S.H. and Datko, A.H. (1981) Biochem. Biophys. Res. Commun. 100, 831-839 Giovanelli, J., Mudd, S.H. and Datko, A.H. (1984) Plant Physiol., 77, 450-455 Davies, D.D. (1979) Annu. Rev. Plant Physiol. 30. 131-158 Bryan, J.K. (1980) in The Biochemistry of Plants (Miflin, B.J., ed.), VoL 5, pp. 403-452, Academic Press, New York 15 Trost, B.M. and Melvin. L.S., Jr. (1975) Sulfur Ylides, pp. 23-25 and 29-30, Acadermc Press, New York