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Determination of 35S-aminoacyl-transfer ribonucleic acid specific radioactivity in small tissue samples
ANALYTICAL
BIOCHEMISTRY
118,
Determination Acid Specific ALLEN
155-161 (1981)
of %-Aminoacyl-Transfer Ribonucleic Radioactivity in Small Tissue Samples
M. SAMAREL,
EDWARD A. OGUNRO, AND MICHAEL LPSCH
ALAN
G. FERGUSON,
Section of Cardiology. Department of Medicine, Northwestern University School of Medicine, 303 E. Chicago Avenue, Chicago, Illinois 6061 I Received March 16, 198 1 Rate determination of protein synthesis utilizing tracer amino acid incorporation requires accurate assessment of the specific radioactivity of the labeled precursor aminoacyl-tRNA pool. Previously published methods presumably useful for the measurement of any aminoacyltRNA were unsuccessful when applied to [%]methionine, due to the unique chemical properties of this amino acid. Herein we describe modifications of these methods necessary for the measurement of 35S-aminoacyl-tRNA specific radioactivity from small tissue samples incubated in the presence of [‘5S]methionine. The use of [35S]methionine of high specific radioactivity enables analysis of the methionyl-tRNA from less than 100 mg of tissue. Conditions for optimal recovery of 35S-labeled dansyl-amino acid derivatives are presented and possible applications of this method are discussed.
Since its introduction for use as a fluorescent amino ligand in the endgroup determination of peptides and proteins, the reaction of I-dimethylaminonaphthalene-5sulfonyl chloride (dansyl chloride’) with primary amines has been used in a wide variety of biochemical analyses (1). Experiments in this and other laboratories (2,3) based upon the original observations of Regier and Kafatos (4) have utilized radiolabeled dansyl chloride in the determination of the specific radioactivity of total intracellular amino acids derived from small tissue samples. These methods have recently been adapted for measuring the quantity and specific radioactivity of amino acids bound to tRNA (5,6). Whereas these methods were presumed to be of use in analyzing any intracellular amino acid, difficulties were encountered in measuring the specific radioactivity of methionyl-tRNA in rabbit
left ventricular slices incubated in the presence of [%]methionine. Utilizing previously described methods (2,5,6) only a small fraction of the labeled amino acid was recovered after tRNA deacylation and reaction with dansyl chloride. In addition, the dansyl-35Samino acid derivatives recovered migrated during ascending thin-layer chromatography (tic) as spots not previously recognized as characteristic of dansyl-methionine. As [ 35S]methionine has become an important tool in studies of in vitro protein synthesis (owing to its commercial availability at specific radioactivities in excess of 1000 Ci/ mmol), the purpose of this brief report is to point out the modifications necessary for adapting previous methods of determining aminoacyl-tRNA specific radioactivity for use with [35S]methionine. MATERIALS AND METHODS
Reagents. Methionine, methionine sulfoxide, methionine sulfone, dansyl chloride, dansyl-cysteic acid, and dansyl-methionine
’ Abbreviations used: dansyl chloride, l-dimethylaminonaphthalene-5-sulfonyl chloride; tic, thin-layer chromatography; SA, specific radioactivity. 155
0003-2697/8 l/l701 55-07$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
156
SAMAREL
were obtained from Sigma Chemical Company, St. Louis, Missouri. L-[3sS]Methionine ( - 1000 Ci/mmol) and [ G-3H]dansyl chloride (13.7 Ci/mmol) were obtained from Amersham Corporation, Arlington Heights, Illinois. Soluene 350, Rimaiume 30, and Instage1 were obtained from Packard Instruments, Downers Grove, Illinois. All other reagents were of the highest analytical grade commercially available and were obtained from Fisher Scientific, Itasca, Illinois or from Scientific Products, McGaw Park, Illinois. Preparation of dansyl-amino acid derivatives. Dansylation reactions were per-
formed in 10 X 75-mm siliconized borosilicate glass test tubes. Amino acid samples were dissolved in 10 to 100 ~1 of 200 mM sodium bicarbonate-carbonate buffer, pH 9.5, and added to an equal volume of 5 mM dansyl chloride in acetone. In experiments utilizing [ G-3H]dansyl chloride, the 3H-labeled compound was added to a solution of unlabeled dansyl chloride to a final concentration of 4.5 mM, 50 &i/ml with a specific radioactivity of Il. 1 mCi/mmol. The dansyl chloride concentration was confirmed by using the molar absorption coefficient of 3.67 X lo3 M-’ cm-’ at 369 nm (7). The reaction tubes were sealed with laboratory plastic and were incubated for 1 h at 37°C in the dark. The acetone was then removed under a stream of N2 and the mixtures were lyophilized to dryness. The dried residues either were redissolved in appropriate test buffers (100 ~1) and extracted twice with ethyl acetate (200 PI), or were dissolved directly in 50% (v/v) aqueous pyridine (20 ~1) for tic. Thin-layer chromatography of dansylamino acid derivatives. Two-dimensional
ascending tic was performed as previously described (4) using 7.5 X 7.5cm micropolyamide sheets and a three-solvent system. Using these solvents dansyl-cysteic acid comigrated with dansyl hydroxide, but these derivatives were readily separated using a fourth solvent ( 1 M NH~OH:ethanol, ( 1: 1)) in the direction of solvents II and III (8).
ET AL.
After drying, the separated dansyl-amino acids (and reaction by-products) were visualized under short uv light. Performic acid oxidation of [35S]methionine. [ 35S]Methionine (10 mCi/ml) was
added to a solution of nonradioactive methionine in water to a final concentration of 100 FM and 10’ dpm/ml. This solution was diluted lo-fold with 3% (v/v) HZ02 in 88% (w/v) formic acid (performic acid) and incubated at 25°C for 4 h (9). The reaction mixture was then dried in a vacuum dessicator overnight and the residue was redissolved to the original volume with water. Thin-layer chromatography of methionine and methionine oxidation products. Methionine, methionine sulfoxide, and methionine
sulfone were separated by ascending tic in one dimension using precoated cellulose sheets (#13255, Eastman Chemical Co., Rochester, N. Y.) in a solvent system consisting of 1-butanol:pyridine:water ( 1: 1: 1). After chromatography the sheets were dried under a stream of cool air and sprayed with ninhydrin (0.3% w/v in I-butanol). Isolation, oxidation, and dansylation of amino acids acylated to tRNA. Slices of rab-
bit left ventricular myocardium (OS- to lmm thick, 40- to 60-mg wet weight) were incubated in Krebs bicarbonate buffer containing 5 mM glucose and varying concentrations of [ “S]methionine. Amino acids acylated to tRNA were isolated by the method of Airhart et al. (6). The dried amino acids were dissolved in 100 ~1 of performic acid, incubated for 4 h at 2S°C, and dried by vacuum dessication. The performic acid-oxidized amino acids were dissolved in 100 ~1 of sodium bicarbonate-carbonate buffer and added to an equal volume of [G‘H]dansyl chloride in acetone. After dansylation the acetone was removed under a stream of Nz and the mixtures were lyophilyzed to dryness. The dried residues were dissolved in 100 ~1 of 0.5 M sodium citrate buffer, pH 4.0, and extracted twice with 200 ~1 of ethyl acetate. The ethyl acetate layer was evaporated to dryness under a stream
AMINOACYL-tRNA
SPECIFIC RADIOACTIVITY
157
of air and the residues were dissolved in 20 ~1 of 50% aqueous pyridine for tic. Radioassay. The tic spots containing radioactivity were cut from the plastic foil plates and placed in scintillation vials con0’ taining 500 ~1 of Soluene 350. After 18 h at 25”C, 10 ml of Dimalume 30 was added and ii . the samples were counted in a Model 3375 Packard Tri-Carb liquid scintillation spectrometer. Aqueous and organic solvent samples were counted in 10 ml of Instagel. For double-labeled samples, tritium was counted at an efficiency of 10% and was completely excluded from the “S-channel. ?S was counted at an efficiency of 75% in the 35Schannel and 9% in the tritium channel. Efficiencies were calculated by the external -SOLVENT Istandards method (10) and corrected for quenching utilizing a programmable calcuFIG. 1. Ascending two-dimensional tic of dansylamino acid derivatives of methionine. (A) A solution of lator.
I :,
:, I d “0 I
RESULTS AND DISCUSSION
Products and Yield of Methionine Dansylation When [35S]methionine (derived from rabbit left verticular tRNA) was reacted with dansyl chloride and isolated as previously described for other amino acids (2,5,6), a surprisingly low yield (1% of the total 35Sradioactivity present prior to dansylation) was recovered. In addition, the dansyl-35Samino acid derivatives migrated during twodimensional tic as spots not characteristic of dansyl-methionine. In order to determine the cause of the low yield of the dansylation reaction for methionine and the nature of the additional derivatives of methionine separated after reaction with dansyl chloride, [ 35S]methionine was dansylated, lyophilyzed to dryness, redissolved directly in 50% pyridine, and separated by ascending tic (Fig. 1A). Each fluorescent spot was cut from the chromatogram and counted for radioactivity. Total recovery of applied radioactivity was in excess of 80%; however, only 7% of the counts recovered were present as dansylmethionine. Most of the recovered radioac-
L-methionine (100 PM, containing 10’ dpm [?S]methionine/ml) was added to an equal volume of dansyl chloride (5 mM in acetone) and incubated for 1 h at 37°C. The reaction mixture was dried, redissolved in 20 ~1 of 50% pyridine, and subjected to ascending tic. Each spot visualized under short bv light was cut from the chromatogram and counted for “S-radioactivity. The identity of each spot was determined by comparing the migration of dansyl-amino acid standards to the mobility of the reaction by-products dansyl amine, 5, and dansyl hydroxide, 6. Percentages of recovered radioactivity were for 1, dansyl-methionine sulfone, 39%; 2, dansyl-methionine, 7%; 3, dansyl-methionine sulfoxide, 13%; and 4, dansyl-methionine sulfoxide, 41%. (B) A similar solution of [“Slmethionine was oxidized with performic acid prior to reaction with dansyl chloride. All of the recovered radioactivity was present as dansylmethionine sulfone, 1. Solvent I, formic acid (88% w/ v):water (3:lOO); solvent II, benzene:glacial acetic acid (9O:lO); and solvent III, ethyl acetate:methanol:glacial acetic acid (20: 1:1).
tivity was present as two separable dansylamino acid derivatives. Reaction of dansyl chloride with methionine sulfoxide and methionine sulfone, followed by tic, demonstrated that these additional radioactive derivatives were oxidation products of [ 35SJmethionine. Interestingly, the dansylderivative labeled 3 in Fig. 1A (and accounting for 13% of the total recovered radioac-
158
SAMAREL
ET AL.
tivity) had the same mobility as dansyl-methionine during separation in the first dimension, and the same mobility as dansylmethionine sulfoxide during separation in the second dimension, This derivative was also present on chromatography of the purified dansyl-methionine standard commercially obtained. This spot was therefore produced by oxidation of dansyl-methionine to dansyl-methionine sulfoxide during drying of the chromatogram between the first and second solvent runs. Performing the dansylation reaction and chromatography under Nz atmosphere decreased, but did not eliminate, the oxidation of methionine. These results indicated that the yield of the dansylation products of methionine after tic was in excess of 80’70, but that the reaction and chromatography led to the loss of most of the dansyl-methionine by oxidation. Performic
Acid Oxidation
of Methionine
In an attempt to isolate all of the 35S-radioactivity to a single dansyl-amino acid derivative, [ 35S]methionine was exposed to performic acid prior to dansylation. Incubation of [35S]methionine at 25°C for 4 h in the presence of performic acid quantitatively oxidized methionine to methionine sulfone (Fig. 2). Dansylation of performic acid-oxidized [ “Slmethionine, followed by ascending tic, produced a single radioactive derivative migrating to the same position as the dansylation product of methionine sulfone (Fig. 1B). Thus performic acid oxidation prior to reaction with dansyl chloride eliminated all but one of the radioactive dansylamino acid derivatives of methionine. Extraction and Recovery of DansylMethionine Sulfone into Organic Solvents The presence of large amounts of salts and dansylation by-products (especially dansyl hydroxide) can lead to variations in the migration and streaking of the dansyl-amino acid derivatives during tic ( 11). Extraction
FIG. 2. Performic acid oxidation of methionine. A solution of L-methionine (100 al of a 1 mM solution containing IO6 dpm [%]methionine) was oxidized with performic acid, lyophilyzed to dryness, and redissolved to its original volume with water. One tenth of this sampel (10 11) was applied to a cellulose sheet (lane l), as were lo-p1 samples of 1 mM methionine (lane 2). methionine sulfoxide (lane 3) and methionine sulfone (lane 4). Ascending chromatography in one dimension was performed in a standard tic tank containing l-butanol:pyridine:water (1:l: 1:). After separation, the sheet was dried and sprayed with ninhydrin. The regions in lane 1 corresponding to the migration of methionine, methionine sulfoxide, and methionine sulfone were cut from the chromatogram and counted for “S-radioactivity. Recovery of applied counts was 91%. Percentages of recovered radioactivity were for methionine, 1%; for methionine sulfoxide, 4%; and for methionine sulfone, 95%.
of dansylation reaction mixtures into nonpolar organic solvents can be used to separate most of. the dansyl-amino acid derivatives from excess dansyl hydroxide and inorganic salts. However, care must be exercised in the choice of solvents used, as the solubility of some dansyl-amino acid deriv-
AMiNOACY~tRNA
pH AQUEOUS
LAYER
FIG. 3. Extraction of methionine sulfone and dansylmethionine sulfone into ethyl acetate. Methionine sulfone (10 ~1 of a 100 pM solution containing lo5 dpm of performic acid-oxidized [)?S]methionine) was reacted with an equal volume of dansyl chloride (5 mM in actone), lyophily~ to dryness, and diisolved in 100 ~1 of aqueous solutions of varying pH. The samples were then extracted twice with ethyl acetate (200 ~1) and the organic layers counted for 35S-radioactivity (0). Solutions of [?S]methionine sulfone not subjected to dansylation were treated similarly (0). Recovery of “Sradioactivity was expressed as the percentage of total counts extracted into the organic layer.
atives in organic solvents is limited (1). We found that extraction of dansyl-methionine sulfone into ethyl acetate was highly dependent upon the pH of the aqueous layer (Fig. 3). Extraction of dansyl-methionine sulfone with ethyl acetate was nearly complete if the pH of the aqueous phase was between 3 and 5. Acid extraction removed most of the salts, dansyl hydroxide, and unreacted methionine sulfone from the dansyl-methionine sulfone and minimized streaking during tic. As seen in Fig. 3, ethyl acetate extraction at pH 7.5, as described in our previous method (2), resulted in the loss of most of the dansyl-meth~onine sulfone and accounted in part for the low recovery of 35S-radioactivity noted during the isolation of [35Slmethionine bound to tRNA. Determination Radioactivity Pools
159
SPECIFIC RADIOACTIVITY
of the Specijic of “~-Ami~~acyl-tR~A
Amino acids (isolated by deacylation of rabbit left ventricular tRNA) were oxidized
with performic acid, reacted with [G3H]dansyl chloride (of known specific radioactivity), and extracted with ethyl acetate at pH 4 as described under Materials and Methods. The dansyl-amino acid derivatives were then subjected to ascending tic and counted for 3H- and 35S-radioactivity (Fig. 4). The efficiency of dansylation of oxidized 3sS-amino acids derived from tRNA was lo20%. The recovered 35S-radioactivity was localized only to dansyl-methionine sulfone and dansyl-cysteic acid (the sole oxidation product of cysteine). The specific radioactivities of the [35S]methionyland (“S]cysteinyl-tRNA pools were determined by obtaining the ratios of “S- to 3H-counts present in the corresponding tic spots and comparing the ratios obtained to a standard curve (Fig. 5). The standard curve was obtained by performic acid oxidation of methionine solutions of known specific radio-
-SOLVENT
I -
FOG. 4. Tw~dimensional tic of dansyl-amino acids derived from rabbit left ventricular tRNA. Slices of rabbit left ventricular myocardium (-500 mg of wet tissue) were incubated (37°C) in 4 ml of oxygenated Krebs bicarbonate buffer containing 5 mM glucose and 165 &i of [35S]methionine (1000 Ci/mmol, final concentration 41 nM). After incubation for 4 h, the tissue was rapidly homogenized in 2 ml of iced 250 mM sodium cacadylate buffer, pH 6.8, containing 1% (w/v) sodium dodecyl sulfate. Amino acids acylated to tRNA were isolated by the method of Airhart et al. (6), oxidized with performic acid, reacted with [G-‘Hldansyl chloride, and extracted with ethyl acetate at pH 4 as described in the-text. The symbols and solvents used are the same as in Fig. I. In this system dansyl-cysteic acid comigrated with dansyl hydroxide, but these derivatives were readily separated using a fourth solvent (1 M NH,OH:ethanol, 1:I) run in the direction of solvents II and III.
160
SAMAREL 24
IO
[3%]
20
METHIONINE
30 SPECIFIC (DPM/pMOL)
40
I
I
50
60
RADIOACTIVITY
FIG. 5. Typical results of a standard curve for the dansyl chloride assay of methionine. Varying amounts of [%]methionine were added to a solution of nonradioactive methionine (100 pM) to yield standards of [“Slmethionine of known specific radioactivity. These standards were oxidized with performic acid, reacted with [ G-‘Hldansyl chloride ( 11.1 mCi/mmol), and extracted with ethyl acetate at pH 4 prior to tic. The ratio of “S-radioactivity to ‘H-radioactivity in the dansylmethionine sulfone spots (cut from each chromatogram) was determined and plotted as a function of [ “Slmethionine specific radioactivity. Similar results were obtained when methionine solutions of varying concentrations (1 mM to 1 M) were added to a constant amount of [ %]methionine.
activity, followed by dansylation with [G3H]dansyl chloride, and then by extraction and tic of the dansyl-methionine sulfone produced. The linearity and the precision of the double-label radioassay was demonstrated by the results obtained for these [‘%Imethionine standards. As stated previously (2,5,6) this technique circumvents the need to obtain a quantitative reaction between the amino acid and the dansyl chloride and does not require the complete recovery of dansylamino acid derivatives. Owing to the sensitivity of fluorescent detection, the excellent separation of dansyl-amino acid derivatives by tic, the high specific radioactivities of commercially available [ 35S]-
ET AL.
methionine and [G-3H]dansyl chloride, and the improvements in the methods mentioned above, quantities of dansyl-methionine sulfone and dansyl-cysteic acid as small as 2 pmol were readily detected and assayed. To determine the rate of protein synthesis of an isolated tissue or organ, it is essential to know the specific radioactivity of the pool of amino acids that serves as a precursor of peptide bond formation ( 12). Previous measurements of protein synthesis based upon measurements of total intracellular amino acid specific radioactivity have been demonstrated to be inaccurate because of compartmentation of amino acids within the intracellular pool (5,13,14). The measurement of aminoacyl-tRNA specific radioactivity, however, assesses the intracellular pool directly serving as the precursor of newly synthesized proteins. Experiments using 14Caminoacyl-tRNA specific radioactivity (as determined by conventional column chromatographic methods of amino acid analysis) have been limited to the use of large quantities of tissue pooled from numerous experimental animals due to the low concentration (5-20 nmol/g) of aminoacyltRNA within the cell (15) and the low specific radioactivity of commercially available 14C-labeled amino acids. The use of [35S]methionine of high specific radioactivity and the application of ultramicromethods for isolating amino acids derived from tRNA (6) allows for the accurate assessment of precursor pool specific radioactivity derived from less than 100 mg of tissue. The modified methods described in this report may be applied to the measurement of methionylbased protein synthetic rates in a number of experimental model systems. Metabolic conversion of methionine to cysteine during cell culture or organ perfusion can be assessed by simultaneous specific radioactivity measurements of tRNA-derived dansylation derivatives of methionine sulfone and cysteic acid using the same tic plate. The rate of synthesis of a single protein may be determined if (a) the specific radioactivities of
AMINOACYL-tRNA
161
SPECIFIC RADIOACTIVITY
methionyi-tRNA and cysteinyl-tRNA remain relatively constant during the period of incubation or perfusion, (b) the accumulation of ?%radioactivity into the protein is linear with time, (c) the amino acid composition of the protein is known, and (d) the protein can be quantitatively isolated in purified form. If these conditions are met, the synthetic rate of the specific protein can be calculated using the formula synthetic rate (pmol g tissue-’ h-‘) 35S-incorporation (dpm g tissue-’ h-‘) = Wbewx~~ (dpm/pmol)) GG) + (SAcys-trw (dpmlpmol)) W where K, and K2 are the number of picomoles of methionine and cysteine per picomoles of specific protein, respectively. We suggest that this procedure may be applicable to the study of a wide variety of individual proteins synthesized in nanogram quantities in isolated organ and tissue culture systems. ACKNOWLEDGMENTS These studies were supported in part by USPHS grant NHLBI-19648 and a grant from the Oppenheimer Family Foundation. A.M.S. was a recipient of an Owen L. Coon Foundation Fellowship at Northwestern University during the time these studies were performed.
REFERENCES 1. Seiler, N. (1970) in Methods of Biochemical Analysis (Click, D., ed.), Vol. 18, pp. 259-337, Interscience, New York. 2. Peterson, M. B., Ferguson, A. G., and Lesch, M. (1973)
J. MoL
Cell
Cardiol.
5, 547-552.
3. Milstone, L., and Piatigorsky, J. (1975)
Bid.
Devdop.
43, 91-100.
4. Regier, J., and Kafatos, F. (197 1) J. Biol. Chem. i46,6480-6488.
5. McKee, E. E., Cheung, J. Y., Rannefs, D. E., and Morgan, H. E. ( 1978) J. B&l. Chem. 253,10301040. 6.
Airhart, J., Kelley, J., Brayden, J. E., Low, R. B., and Stirewalt, W. S. (1979) Anal. Biochem. 96, 4.5-55.
7. Brie& G., and Neuhoff, V. (1972) copse-~e~fer’s Z. Physiol. Chem. 353,540-553. 8. Hartley, B. S. (1970) Biochem. J. X19,805-822. 9. Toennies, G., and Homiller, R. P. (1942) .I. Amer. Chem.
Sot.
64, 3054-3055.
10. Kobayashi, Y., and Maudsley, D. V. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D., ed.), pp. 76-85, Grune & Stratton, New York. 1I, Needleman, S. (1970) Protein Sequence Determination, p. 45, Springer-Verlag, New York/Berlin. 12. Rannels, D. E., McKee, E. E., and Morgan, H. E. (1977) in Biochemical Actions of Hormones (Litwak, G., ed.), Vol. 4, pp. 135-195, Academic Press, New York. 13. Airhart, J., Vidrich, A., and Khairaliah, E. A. (1974)
Khairallah,
Biochem.
I. 140,539-548.
E. A., and Mortimore, G. E. (1976) J. Biol. Chem. 251, 1375-1384. 15. Liu, D. K., McKee, E. E., and Fritz, P. J. (1976) Bid. Neonafe 28, 27-35.
14.
BIOCHEMISTRY
118,
Determination Acid Specific ALLEN
155-161 (1981)
of %-Aminoacyl-Transfer Ribonucleic Radioactivity in Small Tissue Samples
M. SAMAREL,
EDWARD A. OGUNRO, AND MICHAEL LPSCH
ALAN
G. FERGUSON,
Section of Cardiology. Department of Medicine, Northwestern University School of Medicine, 303 E. Chicago Avenue, Chicago, Illinois 6061 I Received March 16, 198 1 Rate determination of protein synthesis utilizing tracer amino acid incorporation requires accurate assessment of the specific radioactivity of the labeled precursor aminoacyl-tRNA pool. Previously published methods presumably useful for the measurement of any aminoacyltRNA were unsuccessful when applied to [%]methionine, due to the unique chemical properties of this amino acid. Herein we describe modifications of these methods necessary for the measurement of 35S-aminoacyl-tRNA specific radioactivity from small tissue samples incubated in the presence of [‘5S]methionine. The use of [35S]methionine of high specific radioactivity enables analysis of the methionyl-tRNA from less than 100 mg of tissue. Conditions for optimal recovery of 35S-labeled dansyl-amino acid derivatives are presented and possible applications of this method are discussed.
Since its introduction for use as a fluorescent amino ligand in the endgroup determination of peptides and proteins, the reaction of I-dimethylaminonaphthalene-5sulfonyl chloride (dansyl chloride’) with primary amines has been used in a wide variety of biochemical analyses (1). Experiments in this and other laboratories (2,3) based upon the original observations of Regier and Kafatos (4) have utilized radiolabeled dansyl chloride in the determination of the specific radioactivity of total intracellular amino acids derived from small tissue samples. These methods have recently been adapted for measuring the quantity and specific radioactivity of amino acids bound to tRNA (5,6). Whereas these methods were presumed to be of use in analyzing any intracellular amino acid, difficulties were encountered in measuring the specific radioactivity of methionyl-tRNA in rabbit
left ventricular slices incubated in the presence of [%]methionine. Utilizing previously described methods (2,5,6) only a small fraction of the labeled amino acid was recovered after tRNA deacylation and reaction with dansyl chloride. In addition, the dansyl-35Samino acid derivatives recovered migrated during ascending thin-layer chromatography (tic) as spots not previously recognized as characteristic of dansyl-methionine. As [ 35S]methionine has become an important tool in studies of in vitro protein synthesis (owing to its commercial availability at specific radioactivities in excess of 1000 Ci/ mmol), the purpose of this brief report is to point out the modifications necessary for adapting previous methods of determining aminoacyl-tRNA specific radioactivity for use with [35S]methionine. MATERIALS AND METHODS
Reagents. Methionine, methionine sulfoxide, methionine sulfone, dansyl chloride, dansyl-cysteic acid, and dansyl-methionine
’ Abbreviations used: dansyl chloride, l-dimethylaminonaphthalene-5-sulfonyl chloride; tic, thin-layer chromatography; SA, specific radioactivity. 155
0003-2697/8 l/l701 55-07$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
156
SAMAREL
were obtained from Sigma Chemical Company, St. Louis, Missouri. L-[3sS]Methionine ( - 1000 Ci/mmol) and [ G-3H]dansyl chloride (13.7 Ci/mmol) were obtained from Amersham Corporation, Arlington Heights, Illinois. Soluene 350, Rimaiume 30, and Instage1 were obtained from Packard Instruments, Downers Grove, Illinois. All other reagents were of the highest analytical grade commercially available and were obtained from Fisher Scientific, Itasca, Illinois or from Scientific Products, McGaw Park, Illinois. Preparation of dansyl-amino acid derivatives. Dansylation reactions were per-
formed in 10 X 75-mm siliconized borosilicate glass test tubes. Amino acid samples were dissolved in 10 to 100 ~1 of 200 mM sodium bicarbonate-carbonate buffer, pH 9.5, and added to an equal volume of 5 mM dansyl chloride in acetone. In experiments utilizing [ G-3H]dansyl chloride, the 3H-labeled compound was added to a solution of unlabeled dansyl chloride to a final concentration of 4.5 mM, 50 &i/ml with a specific radioactivity of Il. 1 mCi/mmol. The dansyl chloride concentration was confirmed by using the molar absorption coefficient of 3.67 X lo3 M-’ cm-’ at 369 nm (7). The reaction tubes were sealed with laboratory plastic and were incubated for 1 h at 37°C in the dark. The acetone was then removed under a stream of N2 and the mixtures were lyophilized to dryness. The dried residues either were redissolved in appropriate test buffers (100 ~1) and extracted twice with ethyl acetate (200 PI), or were dissolved directly in 50% (v/v) aqueous pyridine (20 ~1) for tic. Thin-layer chromatography of dansylamino acid derivatives. Two-dimensional
ascending tic was performed as previously described (4) using 7.5 X 7.5cm micropolyamide sheets and a three-solvent system. Using these solvents dansyl-cysteic acid comigrated with dansyl hydroxide, but these derivatives were readily separated using a fourth solvent ( 1 M NH~OH:ethanol, ( 1: 1)) in the direction of solvents II and III (8).
ET AL.
After drying, the separated dansyl-amino acids (and reaction by-products) were visualized under short uv light. Performic acid oxidation of [35S]methionine. [ 35S]Methionine (10 mCi/ml) was
added to a solution of nonradioactive methionine in water to a final concentration of 100 FM and 10’ dpm/ml. This solution was diluted lo-fold with 3% (v/v) HZ02 in 88% (w/v) formic acid (performic acid) and incubated at 25°C for 4 h (9). The reaction mixture was then dried in a vacuum dessicator overnight and the residue was redissolved to the original volume with water. Thin-layer chromatography of methionine and methionine oxidation products. Methionine, methionine sulfoxide, and methionine
sulfone were separated by ascending tic in one dimension using precoated cellulose sheets (#13255, Eastman Chemical Co., Rochester, N. Y.) in a solvent system consisting of 1-butanol:pyridine:water ( 1: 1: 1). After chromatography the sheets were dried under a stream of cool air and sprayed with ninhydrin (0.3% w/v in I-butanol). Isolation, oxidation, and dansylation of amino acids acylated to tRNA. Slices of rab-
bit left ventricular myocardium (OS- to lmm thick, 40- to 60-mg wet weight) were incubated in Krebs bicarbonate buffer containing 5 mM glucose and varying concentrations of [ “S]methionine. Amino acids acylated to tRNA were isolated by the method of Airhart et al. (6). The dried amino acids were dissolved in 100 ~1 of performic acid, incubated for 4 h at 2S°C, and dried by vacuum dessication. The performic acid-oxidized amino acids were dissolved in 100 ~1 of sodium bicarbonate-carbonate buffer and added to an equal volume of [G‘H]dansyl chloride in acetone. After dansylation the acetone was removed under a stream of Nz and the mixtures were lyophilyzed to dryness. The dried residues were dissolved in 100 ~1 of 0.5 M sodium citrate buffer, pH 4.0, and extracted twice with 200 ~1 of ethyl acetate. The ethyl acetate layer was evaporated to dryness under a stream
AMINOACYL-tRNA
SPECIFIC RADIOACTIVITY
157
of air and the residues were dissolved in 20 ~1 of 50% aqueous pyridine for tic. Radioassay. The tic spots containing radioactivity were cut from the plastic foil plates and placed in scintillation vials con0’ taining 500 ~1 of Soluene 350. After 18 h at 25”C, 10 ml of Dimalume 30 was added and ii . the samples were counted in a Model 3375 Packard Tri-Carb liquid scintillation spectrometer. Aqueous and organic solvent samples were counted in 10 ml of Instagel. For double-labeled samples, tritium was counted at an efficiency of 10% and was completely excluded from the “S-channel. ?S was counted at an efficiency of 75% in the 35Schannel and 9% in the tritium channel. Efficiencies were calculated by the external -SOLVENT Istandards method (10) and corrected for quenching utilizing a programmable calcuFIG. 1. Ascending two-dimensional tic of dansylamino acid derivatives of methionine. (A) A solution of lator.
I :,
:, I d “0 I
RESULTS AND DISCUSSION
Products and Yield of Methionine Dansylation When [35S]methionine (derived from rabbit left verticular tRNA) was reacted with dansyl chloride and isolated as previously described for other amino acids (2,5,6), a surprisingly low yield (1% of the total 35Sradioactivity present prior to dansylation) was recovered. In addition, the dansyl-35Samino acid derivatives migrated during twodimensional tic as spots not characteristic of dansyl-methionine. In order to determine the cause of the low yield of the dansylation reaction for methionine and the nature of the additional derivatives of methionine separated after reaction with dansyl chloride, [ 35S]methionine was dansylated, lyophilyzed to dryness, redissolved directly in 50% pyridine, and separated by ascending tic (Fig. 1A). Each fluorescent spot was cut from the chromatogram and counted for radioactivity. Total recovery of applied radioactivity was in excess of 80%; however, only 7% of the counts recovered were present as dansylmethionine. Most of the recovered radioac-
L-methionine (100 PM, containing 10’ dpm [?S]methionine/ml) was added to an equal volume of dansyl chloride (5 mM in acetone) and incubated for 1 h at 37°C. The reaction mixture was dried, redissolved in 20 ~1 of 50% pyridine, and subjected to ascending tic. Each spot visualized under short bv light was cut from the chromatogram and counted for “S-radioactivity. The identity of each spot was determined by comparing the migration of dansyl-amino acid standards to the mobility of the reaction by-products dansyl amine, 5, and dansyl hydroxide, 6. Percentages of recovered radioactivity were for 1, dansyl-methionine sulfone, 39%; 2, dansyl-methionine, 7%; 3, dansyl-methionine sulfoxide, 13%; and 4, dansyl-methionine sulfoxide, 41%. (B) A similar solution of [“Slmethionine was oxidized with performic acid prior to reaction with dansyl chloride. All of the recovered radioactivity was present as dansylmethionine sulfone, 1. Solvent I, formic acid (88% w/ v):water (3:lOO); solvent II, benzene:glacial acetic acid (9O:lO); and solvent III, ethyl acetate:methanol:glacial acetic acid (20: 1:1).
tivity was present as two separable dansylamino acid derivatives. Reaction of dansyl chloride with methionine sulfoxide and methionine sulfone, followed by tic, demonstrated that these additional radioactive derivatives were oxidation products of [ 35SJmethionine. Interestingly, the dansylderivative labeled 3 in Fig. 1A (and accounting for 13% of the total recovered radioac-
158
SAMAREL
ET AL.
tivity) had the same mobility as dansyl-methionine during separation in the first dimension, and the same mobility as dansylmethionine sulfoxide during separation in the second dimension, This derivative was also present on chromatography of the purified dansyl-methionine standard commercially obtained. This spot was therefore produced by oxidation of dansyl-methionine to dansyl-methionine sulfoxide during drying of the chromatogram between the first and second solvent runs. Performing the dansylation reaction and chromatography under Nz atmosphere decreased, but did not eliminate, the oxidation of methionine. These results indicated that the yield of the dansylation products of methionine after tic was in excess of 80’70, but that the reaction and chromatography led to the loss of most of the dansyl-methionine by oxidation. Performic
Acid Oxidation
of Methionine
In an attempt to isolate all of the 35S-radioactivity to a single dansyl-amino acid derivative, [ 35S]methionine was exposed to performic acid prior to dansylation. Incubation of [35S]methionine at 25°C for 4 h in the presence of performic acid quantitatively oxidized methionine to methionine sulfone (Fig. 2). Dansylation of performic acid-oxidized [ “Slmethionine, followed by ascending tic, produced a single radioactive derivative migrating to the same position as the dansylation product of methionine sulfone (Fig. 1B). Thus performic acid oxidation prior to reaction with dansyl chloride eliminated all but one of the radioactive dansylamino acid derivatives of methionine. Extraction and Recovery of DansylMethionine Sulfone into Organic Solvents The presence of large amounts of salts and dansylation by-products (especially dansyl hydroxide) can lead to variations in the migration and streaking of the dansyl-amino acid derivatives during tic ( 11). Extraction
FIG. 2. Performic acid oxidation of methionine. A solution of L-methionine (100 al of a 1 mM solution containing IO6 dpm [%]methionine) was oxidized with performic acid, lyophilyzed to dryness, and redissolved to its original volume with water. One tenth of this sampel (10 11) was applied to a cellulose sheet (lane l), as were lo-p1 samples of 1 mM methionine (lane 2). methionine sulfoxide (lane 3) and methionine sulfone (lane 4). Ascending chromatography in one dimension was performed in a standard tic tank containing l-butanol:pyridine:water (1:l: 1:). After separation, the sheet was dried and sprayed with ninhydrin. The regions in lane 1 corresponding to the migration of methionine, methionine sulfoxide, and methionine sulfone were cut from the chromatogram and counted for “S-radioactivity. Recovery of applied counts was 91%. Percentages of recovered radioactivity were for methionine, 1%; for methionine sulfoxide, 4%; and for methionine sulfone, 95%.
of dansylation reaction mixtures into nonpolar organic solvents can be used to separate most of. the dansyl-amino acid derivatives from excess dansyl hydroxide and inorganic salts. However, care must be exercised in the choice of solvents used, as the solubility of some dansyl-amino acid deriv-
AMiNOACY~tRNA
pH AQUEOUS
LAYER
FIG. 3. Extraction of methionine sulfone and dansylmethionine sulfone into ethyl acetate. Methionine sulfone (10 ~1 of a 100 pM solution containing lo5 dpm of performic acid-oxidized [)?S]methionine) was reacted with an equal volume of dansyl chloride (5 mM in actone), lyophily~ to dryness, and diisolved in 100 ~1 of aqueous solutions of varying pH. The samples were then extracted twice with ethyl acetate (200 ~1) and the organic layers counted for 35S-radioactivity (0). Solutions of [?S]methionine sulfone not subjected to dansylation were treated similarly (0). Recovery of “Sradioactivity was expressed as the percentage of total counts extracted into the organic layer.
atives in organic solvents is limited (1). We found that extraction of dansyl-methionine sulfone into ethyl acetate was highly dependent upon the pH of the aqueous layer (Fig. 3). Extraction of dansyl-methionine sulfone with ethyl acetate was nearly complete if the pH of the aqueous phase was between 3 and 5. Acid extraction removed most of the salts, dansyl hydroxide, and unreacted methionine sulfone from the dansyl-methionine sulfone and minimized streaking during tic. As seen in Fig. 3, ethyl acetate extraction at pH 7.5, as described in our previous method (2), resulted in the loss of most of the dansyl-meth~onine sulfone and accounted in part for the low recovery of 35S-radioactivity noted during the isolation of [35Slmethionine bound to tRNA. Determination Radioactivity Pools
159
SPECIFIC RADIOACTIVITY
of the Specijic of “~-Ami~~acyl-tR~A
Amino acids (isolated by deacylation of rabbit left ventricular tRNA) were oxidized
with performic acid, reacted with [G3H]dansyl chloride (of known specific radioactivity), and extracted with ethyl acetate at pH 4 as described under Materials and Methods. The dansyl-amino acid derivatives were then subjected to ascending tic and counted for 3H- and 35S-radioactivity (Fig. 4). The efficiency of dansylation of oxidized 3sS-amino acids derived from tRNA was lo20%. The recovered 35S-radioactivity was localized only to dansyl-methionine sulfone and dansyl-cysteic acid (the sole oxidation product of cysteine). The specific radioactivities of the [35S]methionyland (“S]cysteinyl-tRNA pools were determined by obtaining the ratios of “S- to 3H-counts present in the corresponding tic spots and comparing the ratios obtained to a standard curve (Fig. 5). The standard curve was obtained by performic acid oxidation of methionine solutions of known specific radio-
-SOLVENT
I -
FOG. 4. Tw~dimensional tic of dansyl-amino acids derived from rabbit left ventricular tRNA. Slices of rabbit left ventricular myocardium (-500 mg of wet tissue) were incubated (37°C) in 4 ml of oxygenated Krebs bicarbonate buffer containing 5 mM glucose and 165 &i of [35S]methionine (1000 Ci/mmol, final concentration 41 nM). After incubation for 4 h, the tissue was rapidly homogenized in 2 ml of iced 250 mM sodium cacadylate buffer, pH 6.8, containing 1% (w/v) sodium dodecyl sulfate. Amino acids acylated to tRNA were isolated by the method of Airhart et al. (6), oxidized with performic acid, reacted with [G-‘Hldansyl chloride, and extracted with ethyl acetate at pH 4 as described in the-text. The symbols and solvents used are the same as in Fig. I. In this system dansyl-cysteic acid comigrated with dansyl hydroxide, but these derivatives were readily separated using a fourth solvent (1 M NH,OH:ethanol, 1:I) run in the direction of solvents II and III.
160
SAMAREL 24
IO
[3%]
20
METHIONINE
30 SPECIFIC (DPM/pMOL)
40
I
I
50
60
RADIOACTIVITY
FIG. 5. Typical results of a standard curve for the dansyl chloride assay of methionine. Varying amounts of [%]methionine were added to a solution of nonradioactive methionine (100 pM) to yield standards of [“Slmethionine of known specific radioactivity. These standards were oxidized with performic acid, reacted with [ G-‘Hldansyl chloride ( 11.1 mCi/mmol), and extracted with ethyl acetate at pH 4 prior to tic. The ratio of “S-radioactivity to ‘H-radioactivity in the dansylmethionine sulfone spots (cut from each chromatogram) was determined and plotted as a function of [ “Slmethionine specific radioactivity. Similar results were obtained when methionine solutions of varying concentrations (1 mM to 1 M) were added to a constant amount of [ %]methionine.
activity, followed by dansylation with [G3H]dansyl chloride, and then by extraction and tic of the dansyl-methionine sulfone produced. The linearity and the precision of the double-label radioassay was demonstrated by the results obtained for these [‘%Imethionine standards. As stated previously (2,5,6) this technique circumvents the need to obtain a quantitative reaction between the amino acid and the dansyl chloride and does not require the complete recovery of dansylamino acid derivatives. Owing to the sensitivity of fluorescent detection, the excellent separation of dansyl-amino acid derivatives by tic, the high specific radioactivities of commercially available [ 35S]-
ET AL.
methionine and [G-3H]dansyl chloride, and the improvements in the methods mentioned above, quantities of dansyl-methionine sulfone and dansyl-cysteic acid as small as 2 pmol were readily detected and assayed. To determine the rate of protein synthesis of an isolated tissue or organ, it is essential to know the specific radioactivity of the pool of amino acids that serves as a precursor of peptide bond formation ( 12). Previous measurements of protein synthesis based upon measurements of total intracellular amino acid specific radioactivity have been demonstrated to be inaccurate because of compartmentation of amino acids within the intracellular pool (5,13,14). The measurement of aminoacyl-tRNA specific radioactivity, however, assesses the intracellular pool directly serving as the precursor of newly synthesized proteins. Experiments using 14Caminoacyl-tRNA specific radioactivity (as determined by conventional column chromatographic methods of amino acid analysis) have been limited to the use of large quantities of tissue pooled from numerous experimental animals due to the low concentration (5-20 nmol/g) of aminoacyltRNA within the cell (15) and the low specific radioactivity of commercially available 14C-labeled amino acids. The use of [35S]methionine of high specific radioactivity and the application of ultramicromethods for isolating amino acids derived from tRNA (6) allows for the accurate assessment of precursor pool specific radioactivity derived from less than 100 mg of tissue. The modified methods described in this report may be applied to the measurement of methionylbased protein synthetic rates in a number of experimental model systems. Metabolic conversion of methionine to cysteine during cell culture or organ perfusion can be assessed by simultaneous specific radioactivity measurements of tRNA-derived dansylation derivatives of methionine sulfone and cysteic acid using the same tic plate. The rate of synthesis of a single protein may be determined if (a) the specific radioactivities of
AMINOACYL-tRNA
161
SPECIFIC RADIOACTIVITY
methionyi-tRNA and cysteinyl-tRNA remain relatively constant during the period of incubation or perfusion, (b) the accumulation of ?%radioactivity into the protein is linear with time, (c) the amino acid composition of the protein is known, and (d) the protein can be quantitatively isolated in purified form. If these conditions are met, the synthetic rate of the specific protein can be calculated using the formula synthetic rate (pmol g tissue-’ h-‘) 35S-incorporation (dpm g tissue-’ h-‘) = Wbewx~~ (dpm/pmol)) GG) + (SAcys-trw (dpmlpmol)) W where K, and K2 are the number of picomoles of methionine and cysteine per picomoles of specific protein, respectively. We suggest that this procedure may be applicable to the study of a wide variety of individual proteins synthesized in nanogram quantities in isolated organ and tissue culture systems. ACKNOWLEDGMENTS These studies were supported in part by USPHS grant NHLBI-19648 and a grant from the Oppenheimer Family Foundation. A.M.S. was a recipient of an Owen L. Coon Foundation Fellowship at Northwestern University during the time these studies were performed.
REFERENCES 1. Seiler, N. (1970) in Methods of Biochemical Analysis (Click, D., ed.), Vol. 18, pp. 259-337, Interscience, New York. 2. Peterson, M. B., Ferguson, A. G., and Lesch, M. (1973)
J. MoL
Cell
Cardiol.
5, 547-552.
3. Milstone, L., and Piatigorsky, J. (1975)
Bid.
Devdop.
43, 91-100.
4. Regier, J., and Kafatos, F. (197 1) J. Biol. Chem. i46,6480-6488.
5. McKee, E. E., Cheung, J. Y., Rannefs, D. E., and Morgan, H. E. ( 1978) J. B&l. Chem. 253,10301040. 6.
Airhart, J., Kelley, J., Brayden, J. E., Low, R. B., and Stirewalt, W. S. (1979) Anal. Biochem. 96, 4.5-55.
7. Brie& G., and Neuhoff, V. (1972) copse-~e~fer’s Z. Physiol. Chem. 353,540-553. 8. Hartley, B. S. (1970) Biochem. J. X19,805-822. 9. Toennies, G., and Homiller, R. P. (1942) .I. Amer. Chem.
Sot.
64, 3054-3055.
10. Kobayashi, Y., and Maudsley, D. V. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D., ed.), pp. 76-85, Grune & Stratton, New York. 1I, Needleman, S. (1970) Protein Sequence Determination, p. 45, Springer-Verlag, New York/Berlin. 12. Rannels, D. E., McKee, E. E., and Morgan, H. E. (1977) in Biochemical Actions of Hormones (Litwak, G., ed.), Vol. 4, pp. 135-195, Academic Press, New York. 13. Airhart, J., Vidrich, A., and Khairaliah, E. A. (1974)
Khairallah,
Biochem.
I. 140,539-548.
E. A., and Mortimore, G. E. (1976) J. Biol. Chem. 251, 1375-1384. 15. Liu, D. K., McKee, E. E., and Fritz, P. J. (1976) Bid. Neonafe 28, 27-35.
14.