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Solid-phase edman degradation: A new method for attaching peptides to insoluble resins using trifluoroacetic anhydride
ANALYTICAL
BIOCHEMISTRY
89, 136- 142 (1978)
Solid-Phase Edman Degradation: A New Method for Attaching Peptides to Insoluble Resins Using Trifluoroacetic Anhydride HOW-MING LEE’ ANDJAMES F. RIORDAN~ Department of Biological Chemistry, Harvard Medical School and Division of Medical Biology, Pefer Bent Brigham Hospital, Boston, Massachusetts 0211.5 Received February 8, 1978 A method has been developed for attaching peptides to solid supports through their carboxyl groups. The carboxyl groups are activated by mixed anhydride formation in the presence of trifluoroacetic acid and trifluoroacetic anhydride. The activated peptides are then attached to an insoluble resin and subjected to the usual procedures for solid-phase sequencing.
Solid-phase methods for peptide sequencing were first introduced by Laursen in 1971 (1) and since then this technique has been employed widely in many laboratories. A large number of technical improvements have been made and these have been summarized in a recent review (2). The success of solid-phase sequencing relies primarily on the mode of attachment of the peptide to the solid support. There are three basic means for attaching peptides that apply to all of the many types of resin available. These attachment routes employ various peptide functional groups: (i) Lysine-containing peptides can be attached via their e-amino groups by use of the bifunctional reagent, p-phenylenediisothiocyanate (3). (ii) Cyanogen bromide cleavage products can be attached through their C-terminal homoserine lactone residues (4). (iii) Carboxyl groups can be attached with various coupling reagents such as carbonyldiimidazole (5) or carbodiimides (6). The first two methods are acknowledged to have high attachment yields and reproducibility but not all peptides contain either lysine or homoserine. On the other hand, low yields, lengthy blocking procedures (7), and lack of reliability have dissuaded sequence analyzers from using the third method unless all other efforts have failed. For this reason we have developed a convenient method for activating carboxyl groups so that they can be attached readily to insoluble carriers. ’ Present New York 2 Author Peter Bent
address: Department of Microbiology, Mt. Sinai School of Medicine, New York, 10029. to whom correspondence should be addressed: Biophysics Research Laboratory, Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115.
0003-2697/78/0891-0136$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
136
SOLID-PHASE
AUTO
CYCLE
EDMAN
65
I
137
DEGRADATION
‘,
I
I
COUNT
raEl
PART.
VALVE 65
ADVANCE
Li
20
I 40
60
MINUTES
FIG. 1. Sequencing program used with Sequemat 12K sequencer. the bars are the minutes for which each step is in operation.
The
numbers
within
METHODS Materials. Oxidized insulin A and B chains and glucagon were obtained from Sigma, Angiotensin III inhibitor and substance P from Peninsula Labs, and trifluoroacetic anhydride was from Aldrich. Dimethylformamide (Fisher) was distilled from ninhydrin at 40 mmHg and stored over Linde type 3A molecular sieves (Fisher). All other sequencing chemicals were obtained from Pierce and used without further purification. TETA-resin3 (50 mg) was washed successively with 1 ml of DMF containing 50 ~1 of triethylamine and with 2 ml of DMF prior to use. For each washing, the resin was stirred in solvent for at least 3 min. Activation of carboxyl groups. The dry peptide sample (106 nmol of oxidized insulin A chain) is dissolved by stirring in 0.5 ml of F,CCOOH for 5 to 10 min. Trifluoroacetic anhydride (0.2 ml) is introduced and the Parafilm-sealed sample is allowed to stand at room temperature for 30 to 40 min. The solvent and unreacted anhydride are removed by evaporating over potassium hydroxide in a vacuum dessicator (less than 10 min is required to reach dryness). Trace amounts of residual reagent are removed by blowing a gentle stream of dry nitrogen over the sample. The dry peptidyl anhydride is immediately sealed and subjected to further treatment. Attachment to insoluble resin. The peptidyl-mixed anhydride is dissolved in 100 to 150 ~1 of DMF, and the solution is added to a test tube containing 50 mg of wet, prewashed TETA-resin (see Materials). The sample tube is rinsed successively with 100 ~1 of DMF containing 20 ~1 of HzO, 100 ~1 of DMF containing 50 ~1 of triethylamine, and 3 Abbreviations used: TETA-resin, triethylenetetramine polystyrene methylformanide; TFA, trifluoroacetic acid; PTH, phenylthiohydantoin; DCE, dichloroethane; and PITC, phenylisothiocyanate.
resin; DMF, diCyA, cysteic acid;
138
LEE AND RIORDAN TABLE SOLID
-PHASE
EDMAN
DEGRADATION
1 OF OXIDIZED
INSULIN
A CHAIN”
Counts per minute minus background Ala y-Me-Glub
Origin
Gly
3
0 0 0
0 0 0
0 0 0
0 0 0
4 5 6 I 8
255 888 1064 1194 143
0 14 9 0 59
CYSO, 0 22 421 475 143
Glu 3 127 42 0 0
12 164 9 3 -464 Gln 0 214 74 14 0
Cycle No. Solvent I 1 2
Solvent II 4 5 6 7 8
Met
Val
Ile
Leu
Pro
0
0
0
0
0 15 13 16 0
0 1678 92 19 0 0 0
1958 -iii 0 0 0 0 0
179 22 0 0 0 0 0
135 0 0 0 0 0 0
GUY 0 30 12 0 4
Ala 0 0 28 21 404 -
Amino acid
(GUY)
Be Val
Ala
(GM Gln CYSO, CYSOS Ala
(1Forty percent of the PTH sample obtained at each cycle was chromatographed on a silica gel sheet in solvent I (CHCl,:C,H,OH/98:2). Another 40% was chromatographed in solvent II (CHCl,:CH,OW9:1). The FTHs were visualized under uv light. For quantitation, areas corresponding to FTH spots were cut out and counted. The N-terminal residue was recovered as a nonradioactive methylthiohydantoin since methylisothiocyanate was used to block the excess amino groups of resin. b Methyl ester of PTH-Glu migrates near PT’H-Ala.
finally 100 ~1 of DMF containing 20 ~1 of H20. The resin mixture plus the washings are stirred at 45°C for 90 min or longer. Excess amino groups on the resin are blocked by adding 100 ~1 of sequencing buffer (1) [l M N-methylmorpholinium trifluoroacetate at pH 8.1 and pyridine (2:3)] and 100 ~1 of CH,NCS:CH&N (1: 1) to the reaction mixture. Stirring is continued for an additional 75 min. After being washed with methanol, the resin is dried under vacuum. Degradation of peptides and analysis of phenylthiohydantoins. The peptidyl resin sample is mixed with glass beads and submitted to the FIG. 2. Thin-layer chromatography of PTH-amino acids from solid-phase Edman degradation of insulin A chain coupled to TETA-resin using trifluoroacetic anhydride. Samples I, II, and III are standards containing the PTH-amino acids indicated by the single letter code. Samples 1 to 8 are from cycles 1 to 8 of the Edman degradation. The upper chromatogram was run in CHCl,:C,H,OH (98:2), and the lower one was run in CHCl,:CH,OH (9:l). The extraneous spots that appear in the lower chromatogram in the region around serine arise from the TETA-resin. They are not eliminated by extending the methanol wash time. Also they are not radioactive and, hence, are not included in the quantitative analyses.
11234IL5678m: 139
140
LEE AND RIORDAN
usual chemical treatments for solid-phase Edman degradation (1) using [3H]phenylisothiocyanate. The reaction time of sequencing reagents is shown in Fig. 1. The thiazolinone is isomerized at 80” for 10 min in 20% TFA to a phenylthiohydantoin (1) which is identified by thin-layer chromatography (see Table 1, footnote). Ultraviolet-absorbing spots are cut out and analyzed for radioactivity (Table 1). Automatic solid-phase Edman degradation was performed using a Sequemat Model 12 sequencer. The sequencing program shown in Fig. 1 exposes the peptidyl resin to phenylisothiocyanate for 25 min and to TFA for 20 min at 45°C. RESULTS AND DISCUSSION
The method described provides an alternative means for attaching peptides to insoluble resins for solid-phase Edman degradation. It has been tested by carrying out a sequence analysis of oxidized bovine insulin A chain. As shown in Fig. 2 and Table 1, the sequence NH,-Gly-Ile-ValXXX-Gln-CyA-CyA-Ala can be identified through eight cycles without ambiguity. These results are consistent with the known amino terminal sequence of the A chain of bovine insulin. The absence of radioactivity released at cycle 4 indicates that this residue must be Glu or Asp (actually it is Glu) and that the corresponding PTH derivative remains attached to the solid support. The appearance of PTH derivatives following cycle 4 (cycles 5 to 8) demonstrates that the degradation cycle does not stop at the point of peptide attachment although the appearance of PTH-CyA (it remains at the origin in both TLC solvents) at cycles 6 and 7 (Table 1) indicates that cysteic acid residues are not attached to the solid support. Presumably the sulfonic acid group does not form a mixed anhydride on treatment with trifluoroacetic anhydride under the conditions employed. The attachment yield of peptide to resin via formation of the trifluoroacetyl mixed anhydride was determined by attaching several peptides separately to the TETA-resin according to the procedure described above (Table 2). After washing the peptidyl resin with TFA, water, and then TABLE ATTACHMENT
YIELDS
OF PEPTIDES
2
TO TETA-RESIN
USING
TRIFLUOROACETK
ANHYDRIDE
Peptide
Carboxyl side chains
C-Terminus
Yield
Insulin A chain Insulin B chain Glucagon Angiotensin III inhibitor Substance P
1 2 3
Asn Ala Thr
98% 93% 78%
0 0
Ile Amide
55% 1%
SOLID-PHASE H,N-CHR,-CO
------NH-CH-CO-
+H,N-CHR,-CO
----NH-CH-CO-
EDMAN
141
DEGRADATION NH-CHR,-COOH
NH-CHR,-COOH COOH 1 I,,
+H,N-CHR,-
CO -NH-CH-CO-
+H,N-CHR,-
CO -NH-CH-CO-
ANHYDRIDE NH-CHR,-CO-0-CO-CF,
NH-CHR,-CO i co
1’
I
-RESIN
EDMAN
e---d. DEGRADATION
SCHEME 1. Peptide attachment to TETA-resin and subsequent Edman degradation. Dissolution of the peptide in trifluoroacetic acid before introduction of the anhydride prevents acylation of the a-amino group.
methanol to remove any noncovalently bound peptide, hydrolysis was carried out with norleucine added as an internal standard. The amount of peptide attached in each case was determined based on its known amino acid composition. High yields were obtained with insulin A and B chains and glucagon, similar to those reported by Horn and Laursen (4) for attaching cyanogen bromide peptides to TETA-resin under essentially identical conditions. In that case homoserine was cyclized to the lactone in the presence of trifluoroacetic acid. Attachment yields could likely be improved by reducing the volume of DMF employed to dissolve the activated peptide prior to mixing with the resin. In the instances studied, this did not appear to be a problem, but for more general practice this should be kept in mind. Angiotensin III inhibitor, Arg-Val-Tyr-Ile-His-Pro-Ile, which contains no carboxyl side chain and has isoleucine at the C-terminus, gave a relatively low yield. This low yield may be due to the presence of a bulky side chain (Ile) near the carboxyl group which could hinder either mixed anhydride formation or nucleophilic replacement. Substance P, Arg-ProLys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH,, lacks a carboxyl group and hence coupling would not be expected. The trifluoroacetic acid/anhydride pair serves several advantages: The two reagents are miscible, trifluoroacetic acid is a very good solvent for peptides, and in strong acid amino groups are protonated and hence are not acylated. Since trifluoroacetate is the better leaving group, the predominant point of attack by the amino resin will be at the carbonyl carbon of the glutamyl or aspartyl w-carboxyl group rather than at that of the trifluoroacetyl moiety, as shown in Scheme 1 (step 3).
142
LEE AND RIORDAN
The low yield and distribution of radioactivity at cycle 5 (Table 1) seem to imply that partial deamination of glutamine may have occurred. It is not clear, however, whether and to what extent deamination occurred during the attachment manipulation. If deamination occurred during anhydride treatment, the resultant Glu would become attached to the resin and would be lost from cycle 5. On the other hand, if it happened during the trifluoroacetic acid cycle of the Edman degradation, both F’TH-Glu and PTH-Gln would be produced. Since some Glu is detected at this step, the latter possibility seems to be favored. In other words, if Asp or Glu is detected at a particular step it most likely means that the actual sequence position is occupied by the corresponding amide. One potential difficulty that was not addressed in this study has to do with peptides that contain aspartic acid residues. Activation of the pcarboxyl group could result in the formation of a cyclic imide by displacement of trifluoroacetic acid. If this were to occur, then it would not be possible to continue the Edman degradation past this position. Sequencing studies with such peptides would have to be carried out in order to ascertain the significance of this possibility.* ACKNOWLEDGMENT We would like to thank Prof. R. A. Laursen for his advice and for use of the Sequemat Model 12 sequencer. We thank Prof. B. L. Vallee for his continued support and encouragement. This work was supported by Grant-in-Aid GM 15003 from The National Institute of General Medical Sciences of the Department of Health, Education and Welfare.
REFERENCES 1. Laursen, R. A. (1971)Eur. J. Biochem. 20, 89. 2. Laursen, R. A. (ed.) (1975) Solid Phase Methods in Protein Sequence Analysis, Pierce Chemical Co., Rockford, Ill. 3. Laursen, R. A., Horn, M. J., and Bonner, A. G. (1972) FEB.5 Letr. 21, 67. 4. Horn, M. J., and Laursen, R. A. (1973) FEBS Left. 36, 285. 5. Laursen, R. A. (1966) J. Amer. Chem. Sot. 88, 5344. 6. Previero, A., Derancourt, J., Coletti-Previero, M-A., and Laursen, R. A. (1973) FESS Let?. 33, 135. 7. Mross, G. A., and Doolittle, R. F. (1971) Fed. Proc. 30, 1241. 4 This possibility was pointed out by one of the referees.
BIOCHEMISTRY
89, 136- 142 (1978)
Solid-Phase Edman Degradation: A New Method for Attaching Peptides to Insoluble Resins Using Trifluoroacetic Anhydride HOW-MING LEE’ ANDJAMES F. RIORDAN~ Department of Biological Chemistry, Harvard Medical School and Division of Medical Biology, Pefer Bent Brigham Hospital, Boston, Massachusetts 0211.5 Received February 8, 1978 A method has been developed for attaching peptides to solid supports through their carboxyl groups. The carboxyl groups are activated by mixed anhydride formation in the presence of trifluoroacetic acid and trifluoroacetic anhydride. The activated peptides are then attached to an insoluble resin and subjected to the usual procedures for solid-phase sequencing.
Solid-phase methods for peptide sequencing were first introduced by Laursen in 1971 (1) and since then this technique has been employed widely in many laboratories. A large number of technical improvements have been made and these have been summarized in a recent review (2). The success of solid-phase sequencing relies primarily on the mode of attachment of the peptide to the solid support. There are three basic means for attaching peptides that apply to all of the many types of resin available. These attachment routes employ various peptide functional groups: (i) Lysine-containing peptides can be attached via their e-amino groups by use of the bifunctional reagent, p-phenylenediisothiocyanate (3). (ii) Cyanogen bromide cleavage products can be attached through their C-terminal homoserine lactone residues (4). (iii) Carboxyl groups can be attached with various coupling reagents such as carbonyldiimidazole (5) or carbodiimides (6). The first two methods are acknowledged to have high attachment yields and reproducibility but not all peptides contain either lysine or homoserine. On the other hand, low yields, lengthy blocking procedures (7), and lack of reliability have dissuaded sequence analyzers from using the third method unless all other efforts have failed. For this reason we have developed a convenient method for activating carboxyl groups so that they can be attached readily to insoluble carriers. ’ Present New York 2 Author Peter Bent
address: Department of Microbiology, Mt. Sinai School of Medicine, New York, 10029. to whom correspondence should be addressed: Biophysics Research Laboratory, Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115.
0003-2697/78/0891-0136$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
136
SOLID-PHASE
AUTO
CYCLE
EDMAN
65
I
137
DEGRADATION
‘,
I
I
COUNT
raEl
PART.
VALVE 65
ADVANCE
Li
20
I 40
60
MINUTES
FIG. 1. Sequencing program used with Sequemat 12K sequencer. the bars are the minutes for which each step is in operation.
The
numbers
within
METHODS Materials. Oxidized insulin A and B chains and glucagon were obtained from Sigma, Angiotensin III inhibitor and substance P from Peninsula Labs, and trifluoroacetic anhydride was from Aldrich. Dimethylformamide (Fisher) was distilled from ninhydrin at 40 mmHg and stored over Linde type 3A molecular sieves (Fisher). All other sequencing chemicals were obtained from Pierce and used without further purification. TETA-resin3 (50 mg) was washed successively with 1 ml of DMF containing 50 ~1 of triethylamine and with 2 ml of DMF prior to use. For each washing, the resin was stirred in solvent for at least 3 min. Activation of carboxyl groups. The dry peptide sample (106 nmol of oxidized insulin A chain) is dissolved by stirring in 0.5 ml of F,CCOOH for 5 to 10 min. Trifluoroacetic anhydride (0.2 ml) is introduced and the Parafilm-sealed sample is allowed to stand at room temperature for 30 to 40 min. The solvent and unreacted anhydride are removed by evaporating over potassium hydroxide in a vacuum dessicator (less than 10 min is required to reach dryness). Trace amounts of residual reagent are removed by blowing a gentle stream of dry nitrogen over the sample. The dry peptidyl anhydride is immediately sealed and subjected to further treatment. Attachment to insoluble resin. The peptidyl-mixed anhydride is dissolved in 100 to 150 ~1 of DMF, and the solution is added to a test tube containing 50 mg of wet, prewashed TETA-resin (see Materials). The sample tube is rinsed successively with 100 ~1 of DMF containing 20 ~1 of HzO, 100 ~1 of DMF containing 50 ~1 of triethylamine, and 3 Abbreviations used: TETA-resin, triethylenetetramine polystyrene methylformanide; TFA, trifluoroacetic acid; PTH, phenylthiohydantoin; DCE, dichloroethane; and PITC, phenylisothiocyanate.
resin; DMF, diCyA, cysteic acid;
138
LEE AND RIORDAN TABLE SOLID
-PHASE
EDMAN
DEGRADATION
1 OF OXIDIZED
INSULIN
A CHAIN”
Counts per minute minus background Ala y-Me-Glub
Origin
Gly
3
0 0 0
0 0 0
0 0 0
0 0 0
4 5 6 I 8
255 888 1064 1194 143
0 14 9 0 59
CYSO, 0 22 421 475 143
Glu 3 127 42 0 0
12 164 9 3 -464 Gln 0 214 74 14 0
Cycle No. Solvent I 1 2
Solvent II 4 5 6 7 8
Met
Val
Ile
Leu
Pro
0
0
0
0
0 15 13 16 0
0 1678 92 19 0 0 0
1958 -iii 0 0 0 0 0
179 22 0 0 0 0 0
135 0 0 0 0 0 0
GUY 0 30 12 0 4
Ala 0 0 28 21 404 -
Amino acid
(GUY)
Be Val
Ala
(GM Gln CYSO, CYSOS Ala
(1Forty percent of the PTH sample obtained at each cycle was chromatographed on a silica gel sheet in solvent I (CHCl,:C,H,OH/98:2). Another 40% was chromatographed in solvent II (CHCl,:CH,OW9:1). The FTHs were visualized under uv light. For quantitation, areas corresponding to FTH spots were cut out and counted. The N-terminal residue was recovered as a nonradioactive methylthiohydantoin since methylisothiocyanate was used to block the excess amino groups of resin. b Methyl ester of PTH-Glu migrates near PT’H-Ala.
finally 100 ~1 of DMF containing 20 ~1 of H20. The resin mixture plus the washings are stirred at 45°C for 90 min or longer. Excess amino groups on the resin are blocked by adding 100 ~1 of sequencing buffer (1) [l M N-methylmorpholinium trifluoroacetate at pH 8.1 and pyridine (2:3)] and 100 ~1 of CH,NCS:CH&N (1: 1) to the reaction mixture. Stirring is continued for an additional 75 min. After being washed with methanol, the resin is dried under vacuum. Degradation of peptides and analysis of phenylthiohydantoins. The peptidyl resin sample is mixed with glass beads and submitted to the FIG. 2. Thin-layer chromatography of PTH-amino acids from solid-phase Edman degradation of insulin A chain coupled to TETA-resin using trifluoroacetic anhydride. Samples I, II, and III are standards containing the PTH-amino acids indicated by the single letter code. Samples 1 to 8 are from cycles 1 to 8 of the Edman degradation. The upper chromatogram was run in CHCl,:C,H,OH (98:2), and the lower one was run in CHCl,:CH,OH (9:l). The extraneous spots that appear in the lower chromatogram in the region around serine arise from the TETA-resin. They are not eliminated by extending the methanol wash time. Also they are not radioactive and, hence, are not included in the quantitative analyses.
11234IL5678m: 139
140
LEE AND RIORDAN
usual chemical treatments for solid-phase Edman degradation (1) using [3H]phenylisothiocyanate. The reaction time of sequencing reagents is shown in Fig. 1. The thiazolinone is isomerized at 80” for 10 min in 20% TFA to a phenylthiohydantoin (1) which is identified by thin-layer chromatography (see Table 1, footnote). Ultraviolet-absorbing spots are cut out and analyzed for radioactivity (Table 1). Automatic solid-phase Edman degradation was performed using a Sequemat Model 12 sequencer. The sequencing program shown in Fig. 1 exposes the peptidyl resin to phenylisothiocyanate for 25 min and to TFA for 20 min at 45°C. RESULTS AND DISCUSSION
The method described provides an alternative means for attaching peptides to insoluble resins for solid-phase Edman degradation. It has been tested by carrying out a sequence analysis of oxidized bovine insulin A chain. As shown in Fig. 2 and Table 1, the sequence NH,-Gly-Ile-ValXXX-Gln-CyA-CyA-Ala can be identified through eight cycles without ambiguity. These results are consistent with the known amino terminal sequence of the A chain of bovine insulin. The absence of radioactivity released at cycle 4 indicates that this residue must be Glu or Asp (actually it is Glu) and that the corresponding PTH derivative remains attached to the solid support. The appearance of PTH derivatives following cycle 4 (cycles 5 to 8) demonstrates that the degradation cycle does not stop at the point of peptide attachment although the appearance of PTH-CyA (it remains at the origin in both TLC solvents) at cycles 6 and 7 (Table 1) indicates that cysteic acid residues are not attached to the solid support. Presumably the sulfonic acid group does not form a mixed anhydride on treatment with trifluoroacetic anhydride under the conditions employed. The attachment yield of peptide to resin via formation of the trifluoroacetyl mixed anhydride was determined by attaching several peptides separately to the TETA-resin according to the procedure described above (Table 2). After washing the peptidyl resin with TFA, water, and then TABLE ATTACHMENT
YIELDS
OF PEPTIDES
2
TO TETA-RESIN
USING
TRIFLUOROACETK
ANHYDRIDE
Peptide
Carboxyl side chains
C-Terminus
Yield
Insulin A chain Insulin B chain Glucagon Angiotensin III inhibitor Substance P
1 2 3
Asn Ala Thr
98% 93% 78%
0 0
Ile Amide
55% 1%
SOLID-PHASE H,N-CHR,-CO
------NH-CH-CO-
+H,N-CHR,-CO
----NH-CH-CO-
EDMAN
141
DEGRADATION NH-CHR,-COOH
NH-CHR,-COOH COOH 1 I,,
+H,N-CHR,-
CO -NH-CH-CO-
+H,N-CHR,-
CO -NH-CH-CO-
ANHYDRIDE NH-CHR,-CO-0-CO-CF,
NH-CHR,-CO i co
1’
I
-RESIN
EDMAN
e---d. DEGRADATION
SCHEME 1. Peptide attachment to TETA-resin and subsequent Edman degradation. Dissolution of the peptide in trifluoroacetic acid before introduction of the anhydride prevents acylation of the a-amino group.
methanol to remove any noncovalently bound peptide, hydrolysis was carried out with norleucine added as an internal standard. The amount of peptide attached in each case was determined based on its known amino acid composition. High yields were obtained with insulin A and B chains and glucagon, similar to those reported by Horn and Laursen (4) for attaching cyanogen bromide peptides to TETA-resin under essentially identical conditions. In that case homoserine was cyclized to the lactone in the presence of trifluoroacetic acid. Attachment yields could likely be improved by reducing the volume of DMF employed to dissolve the activated peptide prior to mixing with the resin. In the instances studied, this did not appear to be a problem, but for more general practice this should be kept in mind. Angiotensin III inhibitor, Arg-Val-Tyr-Ile-His-Pro-Ile, which contains no carboxyl side chain and has isoleucine at the C-terminus, gave a relatively low yield. This low yield may be due to the presence of a bulky side chain (Ile) near the carboxyl group which could hinder either mixed anhydride formation or nucleophilic replacement. Substance P, Arg-ProLys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH,, lacks a carboxyl group and hence coupling would not be expected. The trifluoroacetic acid/anhydride pair serves several advantages: The two reagents are miscible, trifluoroacetic acid is a very good solvent for peptides, and in strong acid amino groups are protonated and hence are not acylated. Since trifluoroacetate is the better leaving group, the predominant point of attack by the amino resin will be at the carbonyl carbon of the glutamyl or aspartyl w-carboxyl group rather than at that of the trifluoroacetyl moiety, as shown in Scheme 1 (step 3).
142
LEE AND RIORDAN
The low yield and distribution of radioactivity at cycle 5 (Table 1) seem to imply that partial deamination of glutamine may have occurred. It is not clear, however, whether and to what extent deamination occurred during the attachment manipulation. If deamination occurred during anhydride treatment, the resultant Glu would become attached to the resin and would be lost from cycle 5. On the other hand, if it happened during the trifluoroacetic acid cycle of the Edman degradation, both F’TH-Glu and PTH-Gln would be produced. Since some Glu is detected at this step, the latter possibility seems to be favored. In other words, if Asp or Glu is detected at a particular step it most likely means that the actual sequence position is occupied by the corresponding amide. One potential difficulty that was not addressed in this study has to do with peptides that contain aspartic acid residues. Activation of the pcarboxyl group could result in the formation of a cyclic imide by displacement of trifluoroacetic acid. If this were to occur, then it would not be possible to continue the Edman degradation past this position. Sequencing studies with such peptides would have to be carried out in order to ascertain the significance of this possibility.* ACKNOWLEDGMENT We would like to thank Prof. R. A. Laursen for his advice and for use of the Sequemat Model 12 sequencer. We thank Prof. B. L. Vallee for his continued support and encouragement. This work was supported by Grant-in-Aid GM 15003 from The National Institute of General Medical Sciences of the Department of Health, Education and Welfare.
REFERENCES 1. Laursen, R. A. (1971)Eur. J. Biochem. 20, 89. 2. Laursen, R. A. (ed.) (1975) Solid Phase Methods in Protein Sequence Analysis, Pierce Chemical Co., Rockford, Ill. 3. Laursen, R. A., Horn, M. J., and Bonner, A. G. (1972) FEB.5 Letr. 21, 67. 4. Horn, M. J., and Laursen, R. A. (1973) FEBS Left. 36, 285. 5. Laursen, R. A. (1966) J. Amer. Chem. Sot. 88, 5344. 6. Previero, A., Derancourt, J., Coletti-Previero, M-A., and Laursen, R. A. (1973) FESS Let?. 33, 135. 7. Mross, G. A., and Doolittle, R. F. (1971) Fed. Proc. 30, 1241. 4 This possibility was pointed out by one of the referees.