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A method for determination of methionine containing radioactivity in the thiomethyl moiety
AN.ALTTI(‘.~L
BIOCHEMISTRY
A Method
BRIAN
61,
(1974)
for Determination of Methionine Containing Radioactivity in the Thiomethyl Moiety’ R. CLARK, AND
Department
243-247
of Chemistry, Received
HILARY ASHE, RICHARD ROBERTS A. SMITH University
January
of California, 10, 1974;
accepted
M. HALPERN,
Los Angeles, March
California
900%$
28, 1974
B method is described for the determination of methionine containing a radioactive label in the thiomethyl moiety. Cyanogen bromide in aqueous acetic acid is used to convert the labeled thiomethyl moiety to labeled methylthiocyanate which is then extracted into toluene and counted by liquid scintillation.
Cyanogen bromide is widely used as a reagent for cleavage of polypeptides at methionine residues. The cleavage occurs at amide bonds, the acyl groups of which arise from methionine. A proposed reaction mechanism in acid solution involves conversion of methionine residues to the cyanosulfonium bromide derivative which then decomposes to give methylthiocyanate as one of the products. Free methionine is also quantitatively cleaved to methylthiocyanate by the same general mechanism (1). This communication describes the use of cyanogen bromide to measure the amount of free [methyl-W] methionine synthesized from [I%?methyl] 5-met,hyltetrahydrofolate and homocysteine in a reaction catalyzed by the enzyme, 5-methyltetrahydrofolate-homocysteine transmethylase. The [methyl-W] methylthiocyanate formed is extracted into toluene and counted by liquid scintillation. The method is obviously applicable as well to the determination of free or bound methionine containing tritium or sulfur-35 in the thiomethyl moiety. METHODS
Cyanogen bromide was purchased from Matheson, Coleman, and Bell, Norton, Ohio. [ Methyl-‘*Cl methionine, 50 mCi/mmole, was obtained from ICN, Irvine, California. ‘This Dorothy partment Copyright All rights
work was supported by the Andrew Fried Foundation, and USPHS Grant publication number 3270. @ 1974 by Academic Press, of reproduction in any form
243 Inc. reserved.
Norman 5 TO1
Foundation, GM 00463-13.
the Julius Chemistry
and Dr-
244
CLARK
ET
AL,
[‘4C-methyl ]5methyltetrahydrofolate, 61 mCi/mmole, was purchased from Amersham/Searle Corp., Arlington Heights, Illinois, and diluted to 3 mCi/mmole with nonradiolabeled 5-methyltetrahydrofolate obtained from Sigma Chemical Co., St. Louis, Missouri. Homocysteine thiolactone was purchased from Aldrich Chemical Co., Milwaukee, Wisconsin, and converted to homocysteine just prior to use by treatment wit’h 0.3 N NaOH for 20 min and neutralization with 0.3 N KH,PO,. Cyanocobalamin was purchased from General Biochemicals Inc., Chagrin Falls, Ohio. Enzyme source. The 5-methyltetrahydrofolate-homocysteine transmethylase activity was contained in the supernatant from a 1-hr, 50,OOOg centrifugation of rat liver homogenized in 0.1 M phosphate buffer, pH 7.5, containing 1 mM reduced glutathione. Enzyme reaction conditions. The reaction conditions for the transmethylase-catalyzed synthesis of [methyl-14C]methionine from homocysteine and [14C-methyl]5-methyltetrahydrofolate were the same as those described by Kamely et al. (2). Each reaction mixture was run in duplicate. Quantitative measurement of [methyl-14C]methionine. The enzyme reaction was terminated by addition of isopropanol to a concentration of 75% (v/v). After 30 min at 4”C, the protein precipitate was removed by centrifugation and the supernatant (isopropanol extract) was recovered for determination of [methyl-14C]methionine by our cyanogen bromide method and for comparison, by a paper chromatographic method (3). Both assays were performed using aliquots from the same supernatant. Cyanogen bromide method. To minimize exposure to cyanogen bromide, all the following operations were carried out in a well-ventilated fume hood. 0.1 ml of supernatant (isopropanol extract) was transferred to a 12 ml screw-top centrifuge tube and 0.25 ml of freshly prepared 5% cyanogen bromide in 0.1 M acetic acid was added. The tubes were tightly capped with screw tops containing Teflon-coated pressure linings and heated for 5 min in a boiling water bath. The tubes were placed in a water-ice bath for 2 min and then allowed to come to room temperature; 1.0 ml toluene was added, the tubes were capped, shaken vigorously, and centrifuged; 0.5 ml of the toluene layer was transferred to a scintillation vial containing 10 ml of toluene-based scintillation fluid and counted in a Beckman LS-100 scintillation counter. Paper chromatographic method. 0.1 ml of the supernatant (isopropanol extract) was chromatographed on Whatman 3mm paper as described by Mudd et al. (3). The spots corresponding to methionine were cut out, placed in scintillation vials, and counted as described above.
RADIOACTIVE 20
METHIONINE
/
2G.5
ASSAY
/ 0 . / /
I3 -
* o_ x IOI
EXPERIMENTAL
t
CONTROL
(minus
CNBd .
2.4
yMOLES
3.0
METHIONINE
FIG. 1. Cleavage of increasing amounts of [methyl-Wmethionine by excess cyanogen bromide under the conditions described in the text. (O&-Theoretical (radioactivity added as [methyl-‘4CImethionine) ; (@)-experimental (radioactivity recovered in the toluene layer after cyanogen bromide treatment); (A&-control (radioactivity recovered in toluene layer when cyanogen bromide was omitted from the reaction tubes.
RESULTS
Figure 1 shows t.he results of cyanogen bromide cleavage of commercial [methyl-W] methionine followed by extraction of the [methyl-‘%] methylthiocyanate product into toluene. The toluene extracts were analyzed for radioactivity with an efficiency of counting of 92%. -4 comparison of the experimental curve with the theoretical curve (total cpm added) reveals that more than 90% of the methionine is cleaved to methylthiocyanate. The lowest curve represents control tubes to which no cyanogen bromide was added, Table 1 compares the results obtained by our cyanogen bromide method with those obt.ained by the paper chromatographic method of Mudd et al, (3). The [methyl-Wlmethionine was generated enzymically from [‘*Cmethyl] B-methyl tetrahydrofolate and homocysteine as described in METHODS. From tubes containing denatured enzyme (and therefore containing no [methyl-W] methionine) , it was calculated that less than 1% of the total cpm [W-methyl]5-methyltetrahydrofolate was recovered in the toluene layer after cyanogen bromide treatment. As can be seen, the results of the two methods agree quite well. In addition, a tube con-
246
CLARK
Comparison Measurement
ET AL.
TABLE 1 of Cpanogen Bromide and Paper Chromatographie Assays for the of W-Methyltetrahydrofolatehomocysteine Met,hyltransferase Activity in Rat Liver Extracts”
(nmoles
methionine
Cyanogen Sample
plus
(1) Boiled enzyme plus Bi2 in reaction mixture (2) Active enzyme plus Bn in reaction mixture (3) Active enzyme minus B12 in reaction mixture
CNBr
Activity formed/mg
protein/hr)
bromide minus
Paper chromatography recovered with authentic carrier methionine
CNB
Net
0.011
-
0.18
* 0.090
0.16
k 0.008
0.21
f
4.2
f 0.210
0.37
+ 0.019
3.8
3.4
k 0.170
2.9
f 0.145
0.26
+ 0.013
2.6
2.5
i- 0.125
0 The reaction mixtures contained 20 Fmoles sodium phosphate buffer pH 7.0, 50 nmoles S-adenosylmethionine, 50 nmoles DLhomocysteine, 120 nmoles DL [methyli4C!]5-methyltetrahydrofolate (sp act 650 cpm/nmole), 25 rmoles mercaptoethanol, 50 nmoles cyanocobalamin, and 0.1 ml rat liver supernatant in a final volume of 0.2 ml. Any deletions or additions are indicated in the tables. The mixtures were incubated and assayed as described in the text (METHODS). Samples 1 and 2 were incubated anaerobically under nitrogen whereas 3 was incubated aerobically under air.
Comparison
of Cyanogen Bromide of [Methyl-W]Methionine
TABLE 2 and Paper Chromatographic Added to Incubation
Assays Mixtures
for Recovery
To Recovery Cyanogen
bromide
Sample
plus
Boiled Enzyme - Jh 5-Me&F’ +Ci* methionine (150,000 cpm) The reaction conditions a N6-methyltetrahydrofolic
CNBr
91
minus
CNBr
1
Net
Paper chromatography recovered with authentic carrier methionine
90
were the same as those described acid (5-methyltetrahydrofolate).
64
in Table
1.
RADIOACTIVE
METHIONINE
ASSAY
247
boiled enzyme, and commercial [methylJ”C] taining homocysteine, methionine was included to verify the recovery of authentic label depicted in Fig. 1 (see Table 2). DISCUSSION
The reaction of cyanogen bromide with free methionine to form methylthiocyanate apparently has not been exploited as a means of measuring methionine levels in biological samples. This, in part, may be due to lack of a sensitive method for measurement of methylthiocyanate. The results show that methionine can be quantitatively measured by our cyanogen bromide method when it contains radioactive label in the thiomethyl moiety and that the method may be used to monitor enzymatic formation of methionine. The sensitivity of the assay using [methyl-l%] methionine with a specific activity of 50 mCi/mmole is 0.5 nmoles. The advantages of our cyanogen bromide method over other specific methods used to measure thiomethyl-labeled methionine are its simplicity, rapidity, reproducibility, and avoidance of the problems inherent in the ion-exchange method of Weissbach (4). REFERENCES 1. GROSS, E., AND WITKOP, B. (1962) J. Viol. Chem. 237, 1856. 2. KAMELY, D.. LITTLEFIELD, J. W., AND ERBE, R. W. (1973) Proc. Nut. Acad. Sci. 70, 2585. 3. MUDD. H. S.. LEVY, H. L., AND MORROW III, G. (1970) Bkchem. Med. 4, 193. 4. WEISSBACH, H., PETERKOFSKY, A., REDFIELD, B. G., .~ND DICKERMAN, H. (1963) J. Biol. Chem. 238, 3318.
BIOCHEMISTRY
A Method
BRIAN
61,
(1974)
for Determination of Methionine Containing Radioactivity in the Thiomethyl Moiety’ R. CLARK, AND
Department
243-247
of Chemistry, Received
HILARY ASHE, RICHARD ROBERTS A. SMITH University
January
of California, 10, 1974;
accepted
M. HALPERN,
Los Angeles, March
California
900%$
28, 1974
B method is described for the determination of methionine containing a radioactive label in the thiomethyl moiety. Cyanogen bromide in aqueous acetic acid is used to convert the labeled thiomethyl moiety to labeled methylthiocyanate which is then extracted into toluene and counted by liquid scintillation.
Cyanogen bromide is widely used as a reagent for cleavage of polypeptides at methionine residues. The cleavage occurs at amide bonds, the acyl groups of which arise from methionine. A proposed reaction mechanism in acid solution involves conversion of methionine residues to the cyanosulfonium bromide derivative which then decomposes to give methylthiocyanate as one of the products. Free methionine is also quantitatively cleaved to methylthiocyanate by the same general mechanism (1). This communication describes the use of cyanogen bromide to measure the amount of free [methyl-W] methionine synthesized from [I%?methyl] 5-met,hyltetrahydrofolate and homocysteine in a reaction catalyzed by the enzyme, 5-methyltetrahydrofolate-homocysteine transmethylase. The [methyl-W] methylthiocyanate formed is extracted into toluene and counted by liquid scintillation. The method is obviously applicable as well to the determination of free or bound methionine containing tritium or sulfur-35 in the thiomethyl moiety. METHODS
Cyanogen bromide was purchased from Matheson, Coleman, and Bell, Norton, Ohio. [ Methyl-‘*Cl methionine, 50 mCi/mmole, was obtained from ICN, Irvine, California. ‘This Dorothy partment Copyright All rights
work was supported by the Andrew Fried Foundation, and USPHS Grant publication number 3270. @ 1974 by Academic Press, of reproduction in any form
243 Inc. reserved.
Norman 5 TO1
Foundation, GM 00463-13.
the Julius Chemistry
and Dr-
244
CLARK
ET
AL,
[‘4C-methyl ]5methyltetrahydrofolate, 61 mCi/mmole, was purchased from Amersham/Searle Corp., Arlington Heights, Illinois, and diluted to 3 mCi/mmole with nonradiolabeled 5-methyltetrahydrofolate obtained from Sigma Chemical Co., St. Louis, Missouri. Homocysteine thiolactone was purchased from Aldrich Chemical Co., Milwaukee, Wisconsin, and converted to homocysteine just prior to use by treatment wit’h 0.3 N NaOH for 20 min and neutralization with 0.3 N KH,PO,. Cyanocobalamin was purchased from General Biochemicals Inc., Chagrin Falls, Ohio. Enzyme source. The 5-methyltetrahydrofolate-homocysteine transmethylase activity was contained in the supernatant from a 1-hr, 50,OOOg centrifugation of rat liver homogenized in 0.1 M phosphate buffer, pH 7.5, containing 1 mM reduced glutathione. Enzyme reaction conditions. The reaction conditions for the transmethylase-catalyzed synthesis of [methyl-14C]methionine from homocysteine and [14C-methyl]5-methyltetrahydrofolate were the same as those described by Kamely et al. (2). Each reaction mixture was run in duplicate. Quantitative measurement of [methyl-14C]methionine. The enzyme reaction was terminated by addition of isopropanol to a concentration of 75% (v/v). After 30 min at 4”C, the protein precipitate was removed by centrifugation and the supernatant (isopropanol extract) was recovered for determination of [methyl-14C]methionine by our cyanogen bromide method and for comparison, by a paper chromatographic method (3). Both assays were performed using aliquots from the same supernatant. Cyanogen bromide method. To minimize exposure to cyanogen bromide, all the following operations were carried out in a well-ventilated fume hood. 0.1 ml of supernatant (isopropanol extract) was transferred to a 12 ml screw-top centrifuge tube and 0.25 ml of freshly prepared 5% cyanogen bromide in 0.1 M acetic acid was added. The tubes were tightly capped with screw tops containing Teflon-coated pressure linings and heated for 5 min in a boiling water bath. The tubes were placed in a water-ice bath for 2 min and then allowed to come to room temperature; 1.0 ml toluene was added, the tubes were capped, shaken vigorously, and centrifuged; 0.5 ml of the toluene layer was transferred to a scintillation vial containing 10 ml of toluene-based scintillation fluid and counted in a Beckman LS-100 scintillation counter. Paper chromatographic method. 0.1 ml of the supernatant (isopropanol extract) was chromatographed on Whatman 3mm paper as described by Mudd et al. (3). The spots corresponding to methionine were cut out, placed in scintillation vials, and counted as described above.
RADIOACTIVE 20
METHIONINE
/
2G.5
ASSAY
/ 0 . / /
I3 -
* o_ x IOI
EXPERIMENTAL
t
CONTROL
(minus
CNBd .
2.4
yMOLES
3.0
METHIONINE
FIG. 1. Cleavage of increasing amounts of [methyl-Wmethionine by excess cyanogen bromide under the conditions described in the text. (O&-Theoretical (radioactivity added as [methyl-‘4CImethionine) ; (@)-experimental (radioactivity recovered in the toluene layer after cyanogen bromide treatment); (A&-control (radioactivity recovered in toluene layer when cyanogen bromide was omitted from the reaction tubes.
RESULTS
Figure 1 shows t.he results of cyanogen bromide cleavage of commercial [methyl-W] methionine followed by extraction of the [methyl-‘%] methylthiocyanate product into toluene. The toluene extracts were analyzed for radioactivity with an efficiency of counting of 92%. -4 comparison of the experimental curve with the theoretical curve (total cpm added) reveals that more than 90% of the methionine is cleaved to methylthiocyanate. The lowest curve represents control tubes to which no cyanogen bromide was added, Table 1 compares the results obtained by our cyanogen bromide method with those obt.ained by the paper chromatographic method of Mudd et al, (3). The [methyl-Wlmethionine was generated enzymically from [‘*Cmethyl] B-methyl tetrahydrofolate and homocysteine as described in METHODS. From tubes containing denatured enzyme (and therefore containing no [methyl-W] methionine) , it was calculated that less than 1% of the total cpm [W-methyl]5-methyltetrahydrofolate was recovered in the toluene layer after cyanogen bromide treatment. As can be seen, the results of the two methods agree quite well. In addition, a tube con-
246
CLARK
Comparison Measurement
ET AL.
TABLE 1 of Cpanogen Bromide and Paper Chromatographie Assays for the of W-Methyltetrahydrofolatehomocysteine Met,hyltransferase Activity in Rat Liver Extracts”
(nmoles
methionine
Cyanogen Sample
plus
(1) Boiled enzyme plus Bi2 in reaction mixture (2) Active enzyme plus Bn in reaction mixture (3) Active enzyme minus B12 in reaction mixture
CNBr
Activity formed/mg
protein/hr)
bromide minus
Paper chromatography recovered with authentic carrier methionine
CNB
Net
0.011
-
0.18
* 0.090
0.16
k 0.008
0.21
f
4.2
f 0.210
0.37
+ 0.019
3.8
3.4
k 0.170
2.9
f 0.145
0.26
+ 0.013
2.6
2.5
i- 0.125
0 The reaction mixtures contained 20 Fmoles sodium phosphate buffer pH 7.0, 50 nmoles S-adenosylmethionine, 50 nmoles DLhomocysteine, 120 nmoles DL [methyli4C!]5-methyltetrahydrofolate (sp act 650 cpm/nmole), 25 rmoles mercaptoethanol, 50 nmoles cyanocobalamin, and 0.1 ml rat liver supernatant in a final volume of 0.2 ml. Any deletions or additions are indicated in the tables. The mixtures were incubated and assayed as described in the text (METHODS). Samples 1 and 2 were incubated anaerobically under nitrogen whereas 3 was incubated aerobically under air.
Comparison
of Cyanogen Bromide of [Methyl-W]Methionine
TABLE 2 and Paper Chromatographic Added to Incubation
Assays Mixtures
for Recovery
To Recovery Cyanogen
bromide
Sample
plus
Boiled Enzyme - Jh 5-Me&F’ +Ci* methionine (150,000 cpm) The reaction conditions a N6-methyltetrahydrofolic
CNBr
91
minus
CNBr
1
Net
Paper chromatography recovered with authentic carrier methionine
90
were the same as those described acid (5-methyltetrahydrofolate).
64
in Table
1.
RADIOACTIVE
METHIONINE
ASSAY
247
boiled enzyme, and commercial [methylJ”C] taining homocysteine, methionine was included to verify the recovery of authentic label depicted in Fig. 1 (see Table 2). DISCUSSION
The reaction of cyanogen bromide with free methionine to form methylthiocyanate apparently has not been exploited as a means of measuring methionine levels in biological samples. This, in part, may be due to lack of a sensitive method for measurement of methylthiocyanate. The results show that methionine can be quantitatively measured by our cyanogen bromide method when it contains radioactive label in the thiomethyl moiety and that the method may be used to monitor enzymatic formation of methionine. The sensitivity of the assay using [methyl-l%] methionine with a specific activity of 50 mCi/mmole is 0.5 nmoles. The advantages of our cyanogen bromide method over other specific methods used to measure thiomethyl-labeled methionine are its simplicity, rapidity, reproducibility, and avoidance of the problems inherent in the ion-exchange method of Weissbach (4). REFERENCES 1. GROSS, E., AND WITKOP, B. (1962) J. Viol. Chem. 237, 1856. 2. KAMELY, D.. LITTLEFIELD, J. W., AND ERBE, R. W. (1973) Proc. Nut. Acad. Sci. 70, 2585. 3. MUDD. H. S.. LEVY, H. L., AND MORROW III, G. (1970) Bkchem. Med. 4, 193. 4. WEISSBACH, H., PETERKOFSKY, A., REDFIELD, B. G., .~ND DICKERMAN, H. (1963) J. Biol. Chem. 238, 3318.