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A convenient assay for mono-, di-, and oligophenylalanines
ANAWTICAL
28, 376-384
BIOCHEMISTRY
A
Convenient
(1969)
Assay
for
Mono-,
Di-,
and
Oligophenylalanines SIDNEY
PESTKA, EDWARD MI. SCOLNICK AND BARBARA H. HECK Institutes of Health Bethesda, Maryland 2001.4
National
Received September 12, 1968 In studies of protein synthesis, it was useful to have a procedure for separating mono-, di-, and higher oligophenylalanines that could be carried out rapidly and conveniently with many samples (1). Analyses of phenylalanine peptides have been carried out in several ways (14) but no rapid method for their determination has been reported. This communication describes a procedure which can conveniently and quickly separate mono-, di-, and the higher oligophenylalanines on small disposable columns. MATERIALS
AND
EXPERIMENTAL
PROCEDURE
Radioactive isotopes and materials. SH- and 14C-L-phenylalanine (1500 and 495 mc/mmole, respectively) were obtained from New England Nuclear and Nuclear Chicago, respectively. W-L-Phenylalanine and W-L-diphenylalanine were obtained from Mann and Miles-Yeda, respectively. W-L-Tetraphenylalanine, 12C-L-phenylalanine benzyl ester and 12C-L-phenylalanyl-L-phenylalanine benzyl ester were obtained from Cycle Chemical Co. 14C-L-Phenylalanine was diluted with 12C-L-phenylalanine to a specific activity of 2mc/ mmole for use in the synthesis of di- and triphenylalanines. Radioactive di- and triphenylalanines were prepared chemically by reacting the carbobenzoxy derivative of 14C-L-phenylalanine with 12C-L-phenylalanine benzyl ester and with L-phenylalanyl-L-phenylalanine benzyl ester to obtain the blocked derivatives of the dipeptide and tripeptide, respectively (5) . The carbobenzoxybenzyl esters were converted to the free peptides by hydrogenation with a palladium catalyst. The 14C-di- and -triphenylalanines were further purified by chromatography in n-butanol/concentrated ammonium hydroxide/water ( 100/3/B) (1) . 376
MONO-,
DI-,
AND
OLIGOPHENYLALANINE
ASSAY
377
Radioactive tetraphenylalanine was prepared by modification of the method of Marshall and Merrifield (6). W-L-Phenylalanine (367 mc/mmole, New England Nuclear) was diluted with Wphenylalanine to a specific activity of 0.1 mc/mmole. The N-tbutoxycarbonyl derivative of the phenylalanine was prepared by reacting the free amino acid with t-butoxycarbonyl azide. The 12C-L-triphenylalanine resin ester was prepared from unlabeled t-butoxycarbonyl-L-phenylalanine resin ester and N-t-butoxycarbonyl-L-phenylalanine obtained from Schwarz BioResearch (6) . The N-t-butoxycarbonyl-W-L-phenylalanine was coupled to the L-triphenylalanine resin ester with dicyclohexylcarbodiimide at equimolar amounts of the three reactants in methylene chloride W-L-tetraphenylalanine was for 16 hours at 24”. The resultant removed from resin by HBr hydrolysis in methylene chloride and trifluoroacetic acid (30/70 = v/v). Benzoylated dietl~ylaminoethylcellulose (BD-cellulose) was prepared and analyzed by the method of Gilliam et al. (7). The resultant BD-cellulose contained 2.5 moles of benzoyl residues per mole of anhydroglucose. Short columns of BD-cellulose were prepared in disposable Pasteur pipets plugged with glass wool. Columns were 2 em high by 0.5 em in diameter. Procedure for sepnl-ation of ,mono-, di-, and triphenylalanines. The columns of BD-cellulose were equilibrated routinely with solution I (0.05 M potassium acetate, pH 5.7) by passing about 5 ml of the potassium acetate solution through the column. However, neither the pH nor the salt concentration was critical, as will be discussed later. All procedures were carried out at room temperature. Samples were applied to the column in the same potassium acetate buffer in a volume of 1 ml unless otherwise noted. After application of the sample, the column was then eluted successively with an additional 2 ml solution I, followed by 3 ml solution II (formamide/ethanol/ water = 32/30/38 by volume), and last by 3 ml solz-dion III (formamide/ethanol/water = 65/30/5 by volume). The entire procedure took about 15 minutes. In every case, 9776 or more of the isotope placed on the column was recovered within the 3 successive elutions. For counting radioactive samples, the fractions were placed in 5 times their volume of a scintillation fluor consisting of 1 volume Triton X-100 (Packard Instrument Co.) and 2 volumes toluene containing 0.55 “/o (w/v) 2,5-diphenyloxazole (PPO) , 0.015 % (w/v) 1,4-bis-2- (5-phenyloxazolyl) benezene (POPOP) , and 0.0125% (w/v) 1,4-bis-2- (4-methyl-5-phenyloxazolyl) benezene (dimethyl POPOP) (8).
378
PESTKA,
SCOLNICK,
AND
HECK
Analysis of phenylalanine peptides on amino acid analyzer. For analysis of tetraphenylalanine and the lower homologs, a Beckman model 12OC amino acid analyzer was used with an 8 X 0.9 cm diameter column of type PA-35 resin (Beckman). Elution was performed with 0.38 M sodium citrate, pH 5.28, containing 2.4% (v/v) of benzyl alcohol and 3.2 % (v/v) n-propanol. Mono-, di-, tri-, and tetraphenylalanines were well separated under these conditions and were eluted in that order. Nonradioactive peptides were checked for homogeneity with the use of the amino acid analyzer. Analysis of radioactive phenylalanine peptides. Each radioactive peptide was checked for homogeneity and correspondence to a nonradioactive standard in two chromatographic systems (1). In addition, W-di-, tri-, and- tetraphenylalanine were each digested to 12Cand W-phenylalanine with both leucine aminopeptidase and carboxypeptidase A under conditions reported previously (1). RESULTS
Separation of mono-, di-, nine, 14C-L-diphenylalanine, placed on a separate 2 cm cated under the procedure phenylalanines. The results which indicate that mono-,
and triphenylalanines. 3H-r.,-Phenylalaand W-L-triphenylalanine were each BD-cellulose column and eluted as indifor separation of mono-, di-, and triare presented in Figures lA, B, and C, di-, and triphenylalanines are recovered
FIG. 1. Elution of SH-t.-phenylalanine, 1%-L-diphenylalanine, and "C-Ltriphenylalanine on BD-cellulose columns. Elutions with solutions I, II, and III were carried out as described under “Experimental Procedure.” (A) 90,000 cpm 3H-L-phenylalanine was applied to a 2 cm BD-cellulose column in solution I in a total volume of 0.1 ml. (B) 2,400 cpm W-L-diphenylalanine was applied to the column in a volume of 1 ml of solution I. (C) 1,880 cpm I%-L-triphenylalanine was applied to a column in a volume of 1 ml of solution I.
MONO-,
Separation
DI-, AND OLIGOPHENYLALANINE
of Mono-,
TABLE 1 Di-, and Triphenylalanines To of total
Phenylalanine isotope
---___
radioactivity
379
ASSAY
on BD-Cellulose
Columns0
in fraction
I
II
III
IV
98.4 4.5 1.3
1.5 88.7 9.7
0.1 6.4 88.3
0.02 0.4 0.7
.-
3H-mono14C-& W-tria The results Fractions I, II, respectively; IV hydroxide. The
presented in this table quantitate the data presented in Figure 1. and III represent the 3 ml elutions with solutions I, II, and III, represents that fraction eluting with 2 ml ethanolic 0.5 N potassium procedure is described in the legend to Figure 1.
chiefly in the first, second, and third elutions, respectively. The data of Table 1 summarize these graphs quantitatively, indicating that 98% of the phenylalanine is recovered in the first 3 ml elution, whereas only 4.5% of di-, and 1.3% of triphenylalanine elutes in this fraction. With the second 3 ml elution (solution II), almost 89% of the diphenylalanine is eluted from the column, but only 1.5% of the mono- and 9.7% of the triphenylalanine. With the third 3 ml elution (solution III), 88% of the triphenylalanine is eluted while only 0.1 “/o of mono- and 6 % diphenylalanine appear in this fraction. Fraction IV is a control that elutes all the phenylalanine peptides from the column by hydrolyzing the benzoyl residues which retain them. EfSect of protein synthesis reaction mixtures on the separation of mono-, di-, and triphenylalanines. To assess the effect of reaction
mixtures containing the components necessary for cell-free protein synthesis on the separations, similar elutions were carried out using mono-, di-, and triphenylalanines, with the components of reaction mixtures for protein synthesis applied to the column simultaneously with the isotope. The elutions were otherwise similar to those described for Figure 1 and Table 1. These results are presented in Table 2 and indicate that the components such as ribosomes, polyuridylic acid, transfer ribonucleic acid, and some additional salts do not interfere with elution from these columns. The data of Tables 1 and 2 are comparable. Binding of di-, tri-, and tetraphenylalanines QS a function of formamide concentration. 14C-Di-, tri-, and tetraphenylalanines were
bound to BD-cellulose columns. The columns were then eluted with 3 ml of a formamide/ethanol/water solution. The elution of each peptide as a function of formamide concentration is presented in Figure 2. As can be seen, diphenylalanine elutes from the column
380
PESTKA,
SCOLNICK,
AND
HECK
TABLE ‘2 Effect of Protein Synthesis Reaction Mixtures on Separation Mono-, Di-, and Triphenylalaninesn To of total Phenylalanine isotope
3H-mono14C-di“C-tri-
radioactivity
of
in fraction
I
II
III
IV
98.7 4.4 1.7
1.2 87.5 7.5
0.1 7.6 90.0
0.00 0.5 0.8
0 The procedure was similar to that described in the legend to Table 1 with the exception that SH-phenylalanine, 14C-diphenylalanine, and W-triphenylalanine were applied to each column in a volume of 1 ml, which also contained 0.075 ml of a reaction mixture utilized for studies of protein synthesis; these conditions approximate those anticipated during studies of di- and tripeptide synthesis with cell-free extracts. This 0.075 ml reaction mixture contained 0.74 A 26(1unit of ribosomes, 0.3 A?sa unit of polyuridylic acid, 0.08 A280 unit of transfer ribonudeic acid, and 0.3 M potassium chloride. These mixtures were deacylated in 0.3 N potassium hydroxide at 37°C for 5 minutes and then neutralized with hydrochloric acid-this accounts for the presence of potassium chloride (1).
at formamide concentrations that elute very little tri- or tetraphenylalanine. Tri- and tetraphenylalanines, however, are not separable to any significant extent on these miniature columns under these conditions. All the bound oligopeptides could be quantitatively removed from the BD-cellulose columns by 3 ml 0.5 M NaOH or KOH in ethanol. Therefore, by using ethanolic 0.5 M NaOH or KOH after elution with solution II, the oligophenylalanines of chain length greater than three could be eluted in one fraction. Effect of salt, acid, and base on the binding of mono-, di-, tri-, and tetraphenylalanines to the column. The presence of 1 M sodium chloride, 1 N potassium hydroxide, or 2 N hydrochloric acid in the sample applied to the column did not interfere with the binding of di-, tri-, or tetraphenylalanine to the BD-cellulose columns. In addition, these solutions did not interfere with the elution of 3Hphenylalanine from these columns. Although a high ionic strength, strong base or strong acid did not significantly interfere with the column separations, extremes of pH should be avoided to minimize breakdown of the BD-cellulose. Column capacity. Each 2 cm high column of BD-cellulose could quantitatively retain at least 1 pmole of tri- or tetraphenylalanine. The capacity for diphenylalanine was approximately 0.1 pmole before substantial additional diphenylalanine appeared in the first 3 ml elution.
MONO-,
DI-,
AND
OLIGOPHENYLALANINE
ASSAY
381
FIG. 2. Elution of W-L-di-, tri-, and tetraphenylalanines on BD-cellulose columns. ‘*C-L-diphenylalanine (1,230 cpm) , l*C-L-triphenylalanine (940 and W-L-tetraphenylalanine (2,640 cpm) were applied to separate cm), columns of BD-cellulose as described under “Experimental Procedure.” Each point represents elution of bound peptide from a separate column in 3 ml of a solution containing formamide, ethanol, and water (v/v/v). Ethanol concentration was constant at 30%. Formamide concentration is given on the abscissa.
Some properties of phenylalanine peptides. Since phenylalanine peptides are formed frequently in poly U-dependent ribosomal systems synthesizing proteins (l-4)) it was of interest to examine some characteristics of the peptides. The ability of trichloroacetic acid to precipitate the various peptides as a function of concentration of the compound is given in Table 3. *C-Phenylalanine was retained by the filter to the extent of 0.3 % . This probably represents nonspecific adsorption to the nitrocellulose filter. ‘“C-D& and triphenylalanines were retained by the filter to the extent of 2 and 5%, respectively, regardless of the total concentration up to 0.5 pmole/ ml trichloroacetic acid. “C-Tetraphenylalanine was precipitated by trichloroacetic acid, the extent of precipitation being dependent on concentration. At 0.5 pmole/ml, 67% of the tetraphenylalanine was retained by the filter. Therefore, by adding unlabeled tetraphenylalanine as carrier, radioactive tetraphenylalanine could be precipitated by trichloroacetic acid.
382
PESTKA,
Trichloroacetic
SCOLNICK,
HECK
TABLE 3 of Mono-, Di-, Tri-, and Tetraphenylalaninesa
Acid Precipitability
y. of total
Phenylalanine
‘%-mono14C-diW-triW-tetra-
AND
fimole/ml
0.3 2 4 19
counts
on Millipore
0.25 ~mole/ml
0.3 2 5 65
filter 0.5 /.lmole/ml
0.3 2 5 67
a Trichloroacetic acid precipitability of each fraction was measured by adding 2 ml 10 7o trichloroacetic acid to each tube containing mono-, di-, tri-, or tetraphenylalanine in a volume of 0.10 ml. Each tube was kept in an ice bath for 5 minutes and the contents then filtered through a Millipore filter. The tubes and filter were washed with three 5 ml portions of cold 57, trichloroaeetic acid. The filters were dried and counted in a scintillation spectrometer as previously reported (1). Tubes designated contained 20,000 cpm W-mono-, 670 cpm I%-di-, 1060 cpm W-tri-, or 2000 cpm W-tetraphenylalanines. Nonradioactive mono-, di-, tri-, or tetraphenylalanine was added to tubes to bring the concentration to 0.25 or 0.5 pmole amino acid or peptide per milliliter of trichloroacetic acid. The column designated
At concentrations below 0.01 Pmole/ml the solubility of each peptide in ethyl acetate was examined at various hydrogen ion concentrations of the aqueous phases (Table 4). Mono-, di-, tri-, and tetraphenylalanines were dissolved in ethyl acetate in order of increasing solubility, respectively. Almost all the tetraphenylalanine was soluble in the ethyl acetate phase. The solubility of the peptides was greater at pH 1 and pH 6 than at pH 13. DISCUSSION
The results indicate that a useful separation of mono-, di-, and triphenylalanines can be obtained rapidly with the use of miniature BD-cellulose columns. Tetraphenylalanine is eluted from the column under conditions that elute triphenylalanine. Ethanolic 0.5 N sodium or potassium hydroxide can be used to elute the higher oligopeptides; their release probably occurs via hydrolysis of the benzoyl groups in ethanolic potassium hydroxide. In studies of protein biosynthesis using polyuridylic acid as a synthetic template, it is thus relatively easy to obtain a separation of the products into three classes by appropriate elutions from the small BD-cellulose columns: monophenylalanine, using solution I or almost any solution not substantially interfering with aromatic interactions; diphenylalanine, using solution II; and the oligophenylalanines, using 0.5 N ethanolic sodium or potassium hydroxide.
MONO-,
Solubility
DI-, AND OLIGOPHENYLALANINE TABLE 4 Di-, Tri-, and Tetraphenylalanines
of Mono-,
PH 1 % cpm ethyl acetate
W-mono14C-diW-triW-tetra-
1.5 21 60 96
in
in Ethyl
ethyl acetate
430 25 4.4 0.26
1.3 17 64 9’7
Acetatea pH
PH 6
y. epm in KD
383
ASSAY
13
% cpm in KD
520 32 3.7 0.19
ethyl acetate
1.4 6.6 13 60
KD
490 94 46 4.5
a Tubes contained the amounts of W-mono-, W-di-, I%-tri-, or W-tetraphenylalanine indicated in the legend to Table 3. No carrier amino acid or peptides were present. Each radioactive compound was placed in 0.3 ml of each of the following aqueous solutions: 0.1 N HCl (pH l), 0.5 M KAc, pH 5.7 (pH 6), and 0.1 N NaOH (pH 13). Each tube was then extracted with 2 ml ethyl acetate saturated with the same solution. A 1.5 ml portion of the ethyl acetate fraction was counted in 10 ml of the Triton-toluene scintillation fluor. The columns “7’0 cpm in ethyl acetate” refer to the percentage of radioactive peptide extracted into the et.hyl acetate fraction. KD is the distribution ratio of the radioactive peptide partitioned between the two phases and is equal to the ratio of the concentrations of the peptide in the aqueous and ethyl acetate phases, respectively. The results in the table are the average of duplicate determinations.
For optimal results, it was found that solutions containing formamide should be freshly made. Also, it might prove necessary for the separations to use somewhat different concentrations of formamide with batches of BD-cellulose containing significantly greater or lesser amounts of benzoyl substitutions. Although other means may be used to interfere with aromatic interactions on the BD-cellulose columns, such as higher temperatures or the addition of competing aromatic compounds, the use of formamide has proved convenient and reliable. If desired, the separations can be improved further by increasing the size of the columns. Furthermore, the BD-cellulose column might prove useful for the rapid separation of other peptides containing aromatic residues. SUMMARY
A convenient and rapid separation of mono-, di-, and triphenylalanines on miniature columns of benzoylated diethylaminoethylcellulose is described. Phenylalanine passes directly through the column which retains the di- and tripeptides. The di- and triphenylalanines are then successively eluted from the column with solutions of formamide, ethanol, and water. Tetraphenylalanine elutes essen-
384
PESTKA,
SCOLNICK,
AND
HECK
tially with triphenylalanine under the conditions examined. For studies of protein biosynthesis using a synthetic polyuridylic acid template, three classes of peptides can be readily separated: mono-, di-, and oligophenylalanines (the latter containing peptides of chain length three and greater). ACKNOWLEDGMENT We would like to thank Dr. W. M. Lovenberg for the use of his amino acid analyzer, E. C. Bruckwick for analyzing our peptides on it, and Drs. M. Wilchek, A. Zeiger, and A. Marglin for advice in synthesizing the radioactive peptides. REFERENCES 1. 2. 3. 4.
PESTKA, S., J. Biol. Chem, 243,281O (1968). ARLINGHAUS, R., SHAEFFER, J., AND SCHWEET, R., Proc. Natl. U.S., 51, 1291 (1964). GORWN, J., AND LIPMANN, F., J. Mol. Biol. 23, 23 (1967). BRETTHAUER, R. K., AND GOLICHOWSKI, A. M., Biochim. Biophys.
Acad. Sci.
Acta 155, 549 (1967). (translated by E. Gross), 5. SCHRODER, E., AND LOBICE, K., “The Peptides” Vol. I, pp. 26, 108. Academic Press, New York, 1965. 6. MARSHALL, G. R., AND MERRIFIELD, R. B., Biochemistry 4, 2394 (1965). 7. GILLIAM, I., MILLWARD, S., BLEW, D., VON TIGERSTROM, M., WIMMER, E., AND TENER, G. M., Biochemistry 6,3043 (1967). 8. PATTERSON, M. S., AND GREENE, R. C., Anal. Chem. 37, 854 (1965).
28, 376-384
BIOCHEMISTRY
A
Convenient
(1969)
Assay
for
Mono-,
Di-,
and
Oligophenylalanines SIDNEY
PESTKA, EDWARD MI. SCOLNICK AND BARBARA H. HECK Institutes of Health Bethesda, Maryland 2001.4
National
Received September 12, 1968 In studies of protein synthesis, it was useful to have a procedure for separating mono-, di-, and higher oligophenylalanines that could be carried out rapidly and conveniently with many samples (1). Analyses of phenylalanine peptides have been carried out in several ways (14) but no rapid method for their determination has been reported. This communication describes a procedure which can conveniently and quickly separate mono-, di-, and the higher oligophenylalanines on small disposable columns. MATERIALS
AND
EXPERIMENTAL
PROCEDURE
Radioactive isotopes and materials. SH- and 14C-L-phenylalanine (1500 and 495 mc/mmole, respectively) were obtained from New England Nuclear and Nuclear Chicago, respectively. W-L-Phenylalanine and W-L-diphenylalanine were obtained from Mann and Miles-Yeda, respectively. W-L-Tetraphenylalanine, 12C-L-phenylalanine benzyl ester and 12C-L-phenylalanyl-L-phenylalanine benzyl ester were obtained from Cycle Chemical Co. 14C-L-Phenylalanine was diluted with 12C-L-phenylalanine to a specific activity of 2mc/ mmole for use in the synthesis of di- and triphenylalanines. Radioactive di- and triphenylalanines were prepared chemically by reacting the carbobenzoxy derivative of 14C-L-phenylalanine with 12C-L-phenylalanine benzyl ester and with L-phenylalanyl-L-phenylalanine benzyl ester to obtain the blocked derivatives of the dipeptide and tripeptide, respectively (5) . The carbobenzoxybenzyl esters were converted to the free peptides by hydrogenation with a palladium catalyst. The 14C-di- and -triphenylalanines were further purified by chromatography in n-butanol/concentrated ammonium hydroxide/water ( 100/3/B) (1) . 376
MONO-,
DI-,
AND
OLIGOPHENYLALANINE
ASSAY
377
Radioactive tetraphenylalanine was prepared by modification of the method of Marshall and Merrifield (6). W-L-Phenylalanine (367 mc/mmole, New England Nuclear) was diluted with Wphenylalanine to a specific activity of 0.1 mc/mmole. The N-tbutoxycarbonyl derivative of the phenylalanine was prepared by reacting the free amino acid with t-butoxycarbonyl azide. The 12C-L-triphenylalanine resin ester was prepared from unlabeled t-butoxycarbonyl-L-phenylalanine resin ester and N-t-butoxycarbonyl-L-phenylalanine obtained from Schwarz BioResearch (6) . The N-t-butoxycarbonyl-W-L-phenylalanine was coupled to the L-triphenylalanine resin ester with dicyclohexylcarbodiimide at equimolar amounts of the three reactants in methylene chloride W-L-tetraphenylalanine was for 16 hours at 24”. The resultant removed from resin by HBr hydrolysis in methylene chloride and trifluoroacetic acid (30/70 = v/v). Benzoylated dietl~ylaminoethylcellulose (BD-cellulose) was prepared and analyzed by the method of Gilliam et al. (7). The resultant BD-cellulose contained 2.5 moles of benzoyl residues per mole of anhydroglucose. Short columns of BD-cellulose were prepared in disposable Pasteur pipets plugged with glass wool. Columns were 2 em high by 0.5 em in diameter. Procedure for sepnl-ation of ,mono-, di-, and triphenylalanines. The columns of BD-cellulose were equilibrated routinely with solution I (0.05 M potassium acetate, pH 5.7) by passing about 5 ml of the potassium acetate solution through the column. However, neither the pH nor the salt concentration was critical, as will be discussed later. All procedures were carried out at room temperature. Samples were applied to the column in the same potassium acetate buffer in a volume of 1 ml unless otherwise noted. After application of the sample, the column was then eluted successively with an additional 2 ml solution I, followed by 3 ml solution II (formamide/ethanol/ water = 32/30/38 by volume), and last by 3 ml solz-dion III (formamide/ethanol/water = 65/30/5 by volume). The entire procedure took about 15 minutes. In every case, 9776 or more of the isotope placed on the column was recovered within the 3 successive elutions. For counting radioactive samples, the fractions were placed in 5 times their volume of a scintillation fluor consisting of 1 volume Triton X-100 (Packard Instrument Co.) and 2 volumes toluene containing 0.55 “/o (w/v) 2,5-diphenyloxazole (PPO) , 0.015 % (w/v) 1,4-bis-2- (5-phenyloxazolyl) benezene (POPOP) , and 0.0125% (w/v) 1,4-bis-2- (4-methyl-5-phenyloxazolyl) benezene (dimethyl POPOP) (8).
378
PESTKA,
SCOLNICK,
AND
HECK
Analysis of phenylalanine peptides on amino acid analyzer. For analysis of tetraphenylalanine and the lower homologs, a Beckman model 12OC amino acid analyzer was used with an 8 X 0.9 cm diameter column of type PA-35 resin (Beckman). Elution was performed with 0.38 M sodium citrate, pH 5.28, containing 2.4% (v/v) of benzyl alcohol and 3.2 % (v/v) n-propanol. Mono-, di-, tri-, and tetraphenylalanines were well separated under these conditions and were eluted in that order. Nonradioactive peptides were checked for homogeneity with the use of the amino acid analyzer. Analysis of radioactive phenylalanine peptides. Each radioactive peptide was checked for homogeneity and correspondence to a nonradioactive standard in two chromatographic systems (1). In addition, W-di-, tri-, and- tetraphenylalanine were each digested to 12Cand W-phenylalanine with both leucine aminopeptidase and carboxypeptidase A under conditions reported previously (1). RESULTS
Separation of mono-, di-, nine, 14C-L-diphenylalanine, placed on a separate 2 cm cated under the procedure phenylalanines. The results which indicate that mono-,
and triphenylalanines. 3H-r.,-Phenylalaand W-L-triphenylalanine were each BD-cellulose column and eluted as indifor separation of mono-, di-, and triare presented in Figures lA, B, and C, di-, and triphenylalanines are recovered
FIG. 1. Elution of SH-t.-phenylalanine, 1%-L-diphenylalanine, and "C-Ltriphenylalanine on BD-cellulose columns. Elutions with solutions I, II, and III were carried out as described under “Experimental Procedure.” (A) 90,000 cpm 3H-L-phenylalanine was applied to a 2 cm BD-cellulose column in solution I in a total volume of 0.1 ml. (B) 2,400 cpm W-L-diphenylalanine was applied to the column in a volume of 1 ml of solution I. (C) 1,880 cpm I%-L-triphenylalanine was applied to a column in a volume of 1 ml of solution I.
MONO-,
Separation
DI-, AND OLIGOPHENYLALANINE
of Mono-,
TABLE 1 Di-, and Triphenylalanines To of total
Phenylalanine isotope
---___
radioactivity
379
ASSAY
on BD-Cellulose
Columns0
in fraction
I
II
III
IV
98.4 4.5 1.3
1.5 88.7 9.7
0.1 6.4 88.3
0.02 0.4 0.7
.-
3H-mono14C-& W-tria The results Fractions I, II, respectively; IV hydroxide. The
presented in this table quantitate the data presented in Figure 1. and III represent the 3 ml elutions with solutions I, II, and III, represents that fraction eluting with 2 ml ethanolic 0.5 N potassium procedure is described in the legend to Figure 1.
chiefly in the first, second, and third elutions, respectively. The data of Table 1 summarize these graphs quantitatively, indicating that 98% of the phenylalanine is recovered in the first 3 ml elution, whereas only 4.5% of di-, and 1.3% of triphenylalanine elutes in this fraction. With the second 3 ml elution (solution II), almost 89% of the diphenylalanine is eluted from the column, but only 1.5% of the mono- and 9.7% of the triphenylalanine. With the third 3 ml elution (solution III), 88% of the triphenylalanine is eluted while only 0.1 “/o of mono- and 6 % diphenylalanine appear in this fraction. Fraction IV is a control that elutes all the phenylalanine peptides from the column by hydrolyzing the benzoyl residues which retain them. EfSect of protein synthesis reaction mixtures on the separation of mono-, di-, and triphenylalanines. To assess the effect of reaction
mixtures containing the components necessary for cell-free protein synthesis on the separations, similar elutions were carried out using mono-, di-, and triphenylalanines, with the components of reaction mixtures for protein synthesis applied to the column simultaneously with the isotope. The elutions were otherwise similar to those described for Figure 1 and Table 1. These results are presented in Table 2 and indicate that the components such as ribosomes, polyuridylic acid, transfer ribonucleic acid, and some additional salts do not interfere with elution from these columns. The data of Tables 1 and 2 are comparable. Binding of di-, tri-, and tetraphenylalanines QS a function of formamide concentration. 14C-Di-, tri-, and tetraphenylalanines were
bound to BD-cellulose columns. The columns were then eluted with 3 ml of a formamide/ethanol/water solution. The elution of each peptide as a function of formamide concentration is presented in Figure 2. As can be seen, diphenylalanine elutes from the column
380
PESTKA,
SCOLNICK,
AND
HECK
TABLE ‘2 Effect of Protein Synthesis Reaction Mixtures on Separation Mono-, Di-, and Triphenylalaninesn To of total Phenylalanine isotope
3H-mono14C-di“C-tri-
radioactivity
of
in fraction
I
II
III
IV
98.7 4.4 1.7
1.2 87.5 7.5
0.1 7.6 90.0
0.00 0.5 0.8
0 The procedure was similar to that described in the legend to Table 1 with the exception that SH-phenylalanine, 14C-diphenylalanine, and W-triphenylalanine were applied to each column in a volume of 1 ml, which also contained 0.075 ml of a reaction mixture utilized for studies of protein synthesis; these conditions approximate those anticipated during studies of di- and tripeptide synthesis with cell-free extracts. This 0.075 ml reaction mixture contained 0.74 A 26(1unit of ribosomes, 0.3 A?sa unit of polyuridylic acid, 0.08 A280 unit of transfer ribonudeic acid, and 0.3 M potassium chloride. These mixtures were deacylated in 0.3 N potassium hydroxide at 37°C for 5 minutes and then neutralized with hydrochloric acid-this accounts for the presence of potassium chloride (1).
at formamide concentrations that elute very little tri- or tetraphenylalanine. Tri- and tetraphenylalanines, however, are not separable to any significant extent on these miniature columns under these conditions. All the bound oligopeptides could be quantitatively removed from the BD-cellulose columns by 3 ml 0.5 M NaOH or KOH in ethanol. Therefore, by using ethanolic 0.5 M NaOH or KOH after elution with solution II, the oligophenylalanines of chain length greater than three could be eluted in one fraction. Effect of salt, acid, and base on the binding of mono-, di-, tri-, and tetraphenylalanines to the column. The presence of 1 M sodium chloride, 1 N potassium hydroxide, or 2 N hydrochloric acid in the sample applied to the column did not interfere with the binding of di-, tri-, or tetraphenylalanine to the BD-cellulose columns. In addition, these solutions did not interfere with the elution of 3Hphenylalanine from these columns. Although a high ionic strength, strong base or strong acid did not significantly interfere with the column separations, extremes of pH should be avoided to minimize breakdown of the BD-cellulose. Column capacity. Each 2 cm high column of BD-cellulose could quantitatively retain at least 1 pmole of tri- or tetraphenylalanine. The capacity for diphenylalanine was approximately 0.1 pmole before substantial additional diphenylalanine appeared in the first 3 ml elution.
MONO-,
DI-,
AND
OLIGOPHENYLALANINE
ASSAY
381
FIG. 2. Elution of W-L-di-, tri-, and tetraphenylalanines on BD-cellulose columns. ‘*C-L-diphenylalanine (1,230 cpm) , l*C-L-triphenylalanine (940 and W-L-tetraphenylalanine (2,640 cpm) were applied to separate cm), columns of BD-cellulose as described under “Experimental Procedure.” Each point represents elution of bound peptide from a separate column in 3 ml of a solution containing formamide, ethanol, and water (v/v/v). Ethanol concentration was constant at 30%. Formamide concentration is given on the abscissa.
Some properties of phenylalanine peptides. Since phenylalanine peptides are formed frequently in poly U-dependent ribosomal systems synthesizing proteins (l-4)) it was of interest to examine some characteristics of the peptides. The ability of trichloroacetic acid to precipitate the various peptides as a function of concentration of the compound is given in Table 3. *C-Phenylalanine was retained by the filter to the extent of 0.3 % . This probably represents nonspecific adsorption to the nitrocellulose filter. ‘“C-D& and triphenylalanines were retained by the filter to the extent of 2 and 5%, respectively, regardless of the total concentration up to 0.5 pmole/ ml trichloroacetic acid. “C-Tetraphenylalanine was precipitated by trichloroacetic acid, the extent of precipitation being dependent on concentration. At 0.5 pmole/ml, 67% of the tetraphenylalanine was retained by the filter. Therefore, by adding unlabeled tetraphenylalanine as carrier, radioactive tetraphenylalanine could be precipitated by trichloroacetic acid.
382
PESTKA,
Trichloroacetic
SCOLNICK,
HECK
TABLE 3 of Mono-, Di-, Tri-, and Tetraphenylalaninesa
Acid Precipitability
y. of total
Phenylalanine
‘%-mono14C-diW-triW-tetra-
AND
fimole/ml
0.3 2 4 19
counts
on Millipore
0.25 ~mole/ml
0.3 2 5 65
filter 0.5 /.lmole/ml
0.3 2 5 67
a Trichloroacetic acid precipitability of each fraction was measured by adding 2 ml 10 7o trichloroacetic acid to each tube containing mono-, di-, tri-, or tetraphenylalanine in a volume of 0.10 ml. Each tube was kept in an ice bath for 5 minutes and the contents then filtered through a Millipore filter. The tubes and filter were washed with three 5 ml portions of cold 57, trichloroaeetic acid. The filters were dried and counted in a scintillation spectrometer as previously reported (1). Tubes designated contained 20,000 cpm W-mono-, 670 cpm I%-di-, 1060 cpm W-tri-, or 2000 cpm W-tetraphenylalanines. Nonradioactive mono-, di-, tri-, or tetraphenylalanine was added to tubes to bring the concentration to 0.25 or 0.5 pmole amino acid or peptide per milliliter of trichloroacetic acid. The column designated
At concentrations below 0.01 Pmole/ml the solubility of each peptide in ethyl acetate was examined at various hydrogen ion concentrations of the aqueous phases (Table 4). Mono-, di-, tri-, and tetraphenylalanines were dissolved in ethyl acetate in order of increasing solubility, respectively. Almost all the tetraphenylalanine was soluble in the ethyl acetate phase. The solubility of the peptides was greater at pH 1 and pH 6 than at pH 13. DISCUSSION
The results indicate that a useful separation of mono-, di-, and triphenylalanines can be obtained rapidly with the use of miniature BD-cellulose columns. Tetraphenylalanine is eluted from the column under conditions that elute triphenylalanine. Ethanolic 0.5 N sodium or potassium hydroxide can be used to elute the higher oligopeptides; their release probably occurs via hydrolysis of the benzoyl groups in ethanolic potassium hydroxide. In studies of protein biosynthesis using polyuridylic acid as a synthetic template, it is thus relatively easy to obtain a separation of the products into three classes by appropriate elutions from the small BD-cellulose columns: monophenylalanine, using solution I or almost any solution not substantially interfering with aromatic interactions; diphenylalanine, using solution II; and the oligophenylalanines, using 0.5 N ethanolic sodium or potassium hydroxide.
MONO-,
Solubility
DI-, AND OLIGOPHENYLALANINE TABLE 4 Di-, Tri-, and Tetraphenylalanines
of Mono-,
PH 1 % cpm ethyl acetate
W-mono14C-diW-triW-tetra-
1.5 21 60 96
in
in Ethyl
ethyl acetate
430 25 4.4 0.26
1.3 17 64 9’7
Acetatea pH
PH 6
y. epm in KD
383
ASSAY
13
% cpm in KD
520 32 3.7 0.19
ethyl acetate
1.4 6.6 13 60
KD
490 94 46 4.5
a Tubes contained the amounts of W-mono-, W-di-, I%-tri-, or W-tetraphenylalanine indicated in the legend to Table 3. No carrier amino acid or peptides were present. Each radioactive compound was placed in 0.3 ml of each of the following aqueous solutions: 0.1 N HCl (pH l), 0.5 M KAc, pH 5.7 (pH 6), and 0.1 N NaOH (pH 13). Each tube was then extracted with 2 ml ethyl acetate saturated with the same solution. A 1.5 ml portion of the ethyl acetate fraction was counted in 10 ml of the Triton-toluene scintillation fluor. The columns “7’0 cpm in ethyl acetate” refer to the percentage of radioactive peptide extracted into the et.hyl acetate fraction. KD is the distribution ratio of the radioactive peptide partitioned between the two phases and is equal to the ratio of the concentrations of the peptide in the aqueous and ethyl acetate phases, respectively. The results in the table are the average of duplicate determinations.
For optimal results, it was found that solutions containing formamide should be freshly made. Also, it might prove necessary for the separations to use somewhat different concentrations of formamide with batches of BD-cellulose containing significantly greater or lesser amounts of benzoyl substitutions. Although other means may be used to interfere with aromatic interactions on the BD-cellulose columns, such as higher temperatures or the addition of competing aromatic compounds, the use of formamide has proved convenient and reliable. If desired, the separations can be improved further by increasing the size of the columns. Furthermore, the BD-cellulose column might prove useful for the rapid separation of other peptides containing aromatic residues. SUMMARY
A convenient and rapid separation of mono-, di-, and triphenylalanines on miniature columns of benzoylated diethylaminoethylcellulose is described. Phenylalanine passes directly through the column which retains the di- and tripeptides. The di- and triphenylalanines are then successively eluted from the column with solutions of formamide, ethanol, and water. Tetraphenylalanine elutes essen-
384
PESTKA,
SCOLNICK,
AND
HECK
tially with triphenylalanine under the conditions examined. For studies of protein biosynthesis using a synthetic polyuridylic acid template, three classes of peptides can be readily separated: mono-, di-, and oligophenylalanines (the latter containing peptides of chain length three and greater). ACKNOWLEDGMENT We would like to thank Dr. W. M. Lovenberg for the use of his amino acid analyzer, E. C. Bruckwick for analyzing our peptides on it, and Drs. M. Wilchek, A. Zeiger, and A. Marglin for advice in synthesizing the radioactive peptides. REFERENCES 1. 2. 3. 4.
PESTKA, S., J. Biol. Chem, 243,281O (1968). ARLINGHAUS, R., SHAEFFER, J., AND SCHWEET, R., Proc. Natl. U.S., 51, 1291 (1964). GORWN, J., AND LIPMANN, F., J. Mol. Biol. 23, 23 (1967). BRETTHAUER, R. K., AND GOLICHOWSKI, A. M., Biochim. Biophys.
Acad. Sci.
Acta 155, 549 (1967). (translated by E. Gross), 5. SCHRODER, E., AND LOBICE, K., “The Peptides” Vol. I, pp. 26, 108. Academic Press, New York, 1965. 6. MARSHALL, G. R., AND MERRIFIELD, R. B., Biochemistry 4, 2394 (1965). 7. GILLIAM, I., MILLWARD, S., BLEW, D., VON TIGERSTROM, M., WIMMER, E., AND TENER, G. M., Biochemistry 6,3043 (1967). 8. PATTERSON, M. S., AND GREENE, R. C., Anal. Chem. 37, 854 (1965).