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14C-labeling of synthetic peptides
Journal of Immunological Methods, 110 (1988) 271-273
271
Elsevier JIM04781 Technical note
14C-labeling of synthetic peptides A l b e r t o Chersi, M a r i a Luisa T r i n c a a n d M a t i l d e C a m e r a Istituto Regina Elenafor Cancer Research, Rome, Italy
(Received 19 November1987, accepted 18 January 1988)
Two methods are described for the labeling of synthetic peptides using iodo[a4C]acetic acid. The first procedure may be employed when the synthetic fragment contains a cysteine with a free sulfhydryl group. Alternatively, a commercial amino-protected cysteine may be carboxymethylated using radioactive iodoacetic acid. This derivative can be added to the growing peptide chain in the manual or automatic solid-phase synthesis of the fragment. Key words: Syntheticpeptide; 14C-labeling; Carboxymethylation
In the last 4 years, synthetic peptides have been widely used in immunology for the production and detection of antibodies. In the latter case, immune sera that are supposed to contain antibody subsets to linear antigenic determinants, are tested against synthetic peptides that correspond to known amino acid sequences of the immunogen (Chersi et al., 1987; Ulrich et al., 1987). For this assay, the synthetic peptides are generally radioiodinated at a tyrosine residue by the chloramine-T method (Greenwood et al., 1963), or, alternatively, at a lysine residue (Bolton and Hunter, 1973; Church et al., 1983). Although iodination of peptides (and proteins) is generally easy to accomplish, the technique suffers the disadvantage that the nuclide decays rapidly (half life: 60 days). Moreover, the introduction into the molecule of
Correspondence to: A. Chersi, Istituto Regina Elena for Cancer Research, Laboratory of Biochemistry, Viale Regina Elena 291, 00161 Rome, Italy. Abbreviations: DCM, dichloromethane; DCC, dicyclohexylcarbodimide; TFA, trifluoroacetic acid; MBS; m-maleimidobenzoyl-N-hydroxysuccinimide ester; EtOH, ethanol; tBoc, tert-butyloxycarbonyl; OBzl, O-benzyl.
the heavy iodine atom may sometimes result in a partial loss of the biological activity of the labeled compound. According to our experience, the labeling of peptides by iodine can be advantageously replaced, in many cases, by aac-labeling using iodo[14C] acetic acid, provided that the peptide contains a cysteine. Carboxymethylation of proteins by this reagent has been widely employed over the last 20 years; cysteines are converted into stable, labeled derivatives, and carboxy-methyl cysteine-containing peptides can be easily identified. Carboxymethylation of a cysteine-containing peptide is easy and safe. Alternatively, one can prepare N-protected [carboxy-14C]methylcysteine, and introduce this labeled amino acid directly during synthesis of a polypeptide, to obtain a labeled fragment. Both of the above methods will be described here. The first, which is more obvious, can be associated with a device for separating reacted, labeled peptide from the unreacted material, in order to obtain higher specific activity. The second needs a manual or automatic synthesizer, and is suitable for labeling all peptides produced over a long period of time.
0022-1759/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
272 TABLE I A M I N O ACID SEQUENCE OF SYNTHETIC PEPTIDES SELECTED F O R 14C-LABELING The peptides correspond to sequences 61-73 and 133-143 of human histocompability antigens HLA-DR 2 and HLA-DQ fl chains. (M) Y N S Q K D I L E E A R A C (V) R N D Q E E T T G V V - P A M RESIN
(1) Peptide M (Table I) was kindly provided by Dr. R.A. Houghten, Scripps Clinic, La Jolla, CA. The peptide included a C-terminal cysteine at position 1. The determination of free - S H groups in the peptide by Ellman's reagent (Ellman, 1969) revealed that approximately 93% of those groups were available for reaction. For the labeling of the peptide, 300 /~1 of a 1 m g / m l solution of peptide in 0.05 M Tris-HC1 buffer, pH 8.2 (0.20 ttmol) were reacted for 2 h at room temperature with 5 #Ci (0.07 /~mol) of iodo[2-14C]acetic acid, in 200/~1 of the same buffer. The mixture of reacted and unreacted peptide was then recovered by chromatography on a 0.8 × 40 cm column of Sephadex G-25 superfine in 0.05 M acetic acid. The yield was approximately 270 ~g, as revealed by the absorbance of the peak at 206 nm; the total cpm were 6 × 106, the specific activity was 0.018/~Ci//~g. In order to obtain a higher specific activity, the sample was added to 500 /~1 of a 10% suspension of a gel prepared by reacting Sepharose-AH (1 g in 5 ml PBS) with 1 mg MBS (Liu et al., 1979) in 100 #1 dimethylformamide. The gel bound covalently the unreacted peptide with free - S H groups and this was then discarded by centrifugation. The peptide in the supernatant was 110/~g, cpm 5.8 × 10 6 (0.042/~Ci//zg). (2) As an alternative approach, one may introduce a radiolabeled carboxymethylcysteine directly at the N-terminal end of a peptide during synthesis. ~4C-labeled, N-protected amino acids have occasionally been successfully introduced into the growing chain of synthetic peptides (Kagnoff et al., 1984); however those derivatives are not generally available and are very expensive. We have developed therefore a procedure suitable for the preparation of N - B o c - [ c a r b o x y - 1 4 C ] m e t h y l c y s teine and its coupling to the N-terminal end of a synthetic fragment during synthesis. For this ex-
periment, we dissolved 0.66 mg (3/~mol) of commercial N-Boc-cysteine (Inalco) in 0.4 ml of 0.05 M N-ethylmorpholin-HC1 buffer, p H 8.2. 3 ttmol of iodo[2-14C]acetic acid (180 /~Ci) were then added in 0.2 ml of the same buffer; the reaction was allowed to proceed for 2 h. The sample was then taken to dryness in three aliquots. The specific activity of this protected amino acid was approximately 56/~Ci//tmol. The ll-residue peptide to be labeled (Table I) was prepared with the aid of a Vega Coupler, model 1000, starting with 0.3 g of Boc-Val-PAM resin (0.1 mmol of amino acid), and in conjunction with OBzl protecting groups for Asp, Glu, Thr. The guanidino group of arginine was left unprotected, since this was the last residue to be coupled. Details of the synthesis will be reported in detail elsewhere (Chersi et al., 1988). When the last residue (Arg) was coupled, the Boc-protecting group was cleaved by 40% T F A in DCM, the resin was neutralized and washed and then removed from the reservoir. Only 2% of the resin particles were then used for labeling: 6 mg (approximately 2 /~mol of peptide) were suspended in I ml DCM, and an aliquot of the previously prepared a4C-labeled N-Boc-S-carboxymethylcysteine (1 /~mol, 280 #g, 56 /~Ci) were added together with a six-fold excess of DCC in 1 ml DCM. The reaction was allowed to proceed in a 10 ml glass-stoppered flask, with continuous shaking, for about 2 h. Excess reagents were discarded by centrifugation in E t O H - D C M 2 : 1. Then the dry resin was treated with a 20-fold excess of trifluoromethansulfonic acid at 1 0 ° C for 30 rain (Yajima et al., 1975). After 1 in 3 dilution with water, and filtration through a Gelman Acrodisc, the free peptide was recovered by gel filtration on a 0.8 × 50 cm column of Sephadex G-25 superfine equilibrated in 0.05 M ammonia. The recovery of peptide was 1.4 ktmol (70%). The amino acid composition was within _+7% of the theoretical value and the cm-Cys content was 0.44 residues. Total cpm were 52 × 10 6 (or 30 /iCi//~mol, 0.028 /zCi//~g). Since the amino groups available for the reaction were in a 2 : 1 excess over the alkylating agent, the utilization of the radioactive compound was probably complete. No efforts were made to separate labeled from unlabeled peptide (approx. 1 : 1) since the specific
273
activity obtained was considered suitable for most purposes. Finally, if one uses for labeling N-protected, 14C-labeled carboxamidomethylcysteine, it is likely that this residue can be introduced at any position of the growing peptide chain, since the amino group is relatively stable under the conditions used for synthesis, and does not undergo undesirable side reactions. This second method is not of wide applicability, since it requires a manual or automatic peptide synthesizer: however, since the labeled, N-protected carboxymethylcysteine is prepared only once, and can be stored ready for use in aliquots, it permits the labeling of all peptides synthesized over a long period of time. Finally, the use of 14C offers some advantages such as the long half-life and stability of the labeled product and the use of lower amounts of a safer radioactive agent. The 14C-labeled peptide generally displays sufficiently high specific activity .
t--
N.
o
o
0 o
t 50
i
8
i 200 Dilution
o
~
0 o
] 800
o o
I
Fig. 1. Binding of 14C-labeled peptide M to antipeptide antibody 6148 (anti-M). Serial dilutions of either antiserum 6148 (e) or normal rabbit serum (o) were incubated in 1 ml PBS containing 0.05% ovalbumin, with 6 x 105 cpm ( ~ 1.2 /~g) of peptide, for 2 h at 18°C. Bound and free peptides were separated using Protein A-Sepharose (100 #1 of a 20% suspension). After incubation for 2 h, the pellet was washed twice, then suspended in 100 ~1 PBS. 100 ~1 were then measured in a beta counter.
to be employed in most conventional immunological assays; as an example the determination of antigen-antibody binding is shown in Fig. 1.
Acknowledgements
The authors thank Dr. R.A. Houghten, Scripps Clinic and Research Foundation, La Jolla, CA, for the gift of peptide M. This investigation was partly supported by the Associazione Italiana per la Ricerca sul Cancro, Milan.
References Bolton, A.E. and Hunter, W.M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a 1251 containing acylating agent. Biochem. J. 133, 529. Chersi, A., Morganti, M.C., Houghten, R.A. and Chillemi, F. (1988) Identification of linear HLA Class II amino acid sequences recognized by rabbit antisera against native molecules. Scand. J. Immunol., in press. Church. W.B., Walker, L.E., Houghten, R.A. and Reisfeld, R.A. (1983) Anti-HLA antibodies of predetermined specificity: A chemically synthesized peptide induces antibodies specific for HLA-A,B heavy chain. Proc. Natl. Acad. Sci. U.S.A. 80, 255. Ellman, G.L. (1969) Tissue sulphydryl groups. Arch. Biochem. Biophys. 82, 7077. Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963) The preparation of 131I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114. Kagnoff, M.F., Austin, R.K., Hubert, J.J., Bernardin, J.E. and Kasarda, D.D. (1984) Possible role for a human adenovirus in the pathogenesis of celiac disease. J. Exp. Med. 160, 1544. Liu, F.T., Zinnecker, M., Hamaoka, T. and Katz, D.H. (1979) New procedure for preparation and isolation of conjugates of proteins and a synthetic copolymer of amino acids and immunochemical characterization of such conjugates. Biochemistry 18, 690. Ulrich, R.G., Atassi, H., Lutz, P., Cresswell, P. and Atassi, M.Z. (1987) Immune recognition of human major histocompatibility antigens: localization by a comprehensive synthetic strategy of the continuous antigenic sites in the first domain of HLA-DR2 t9 chain. Eur. J. Immunol. 17, 497. Yajima, H., Ogawa, H., Watanabe, H., Fujii, N., Kurobe, M. and Miyamoto, S. (1975) Studies on peptides XLIII. 1.2 Application of the trifluoromethansulphonic acid procedure to the synthesis of tuftsin. Chem. Pharmacol. Bull. 23, 371.
271
Elsevier JIM04781 Technical note
14C-labeling of synthetic peptides A l b e r t o Chersi, M a r i a Luisa T r i n c a a n d M a t i l d e C a m e r a Istituto Regina Elenafor Cancer Research, Rome, Italy
(Received 19 November1987, accepted 18 January 1988)
Two methods are described for the labeling of synthetic peptides using iodo[a4C]acetic acid. The first procedure may be employed when the synthetic fragment contains a cysteine with a free sulfhydryl group. Alternatively, a commercial amino-protected cysteine may be carboxymethylated using radioactive iodoacetic acid. This derivative can be added to the growing peptide chain in the manual or automatic solid-phase synthesis of the fragment. Key words: Syntheticpeptide; 14C-labeling; Carboxymethylation
In the last 4 years, synthetic peptides have been widely used in immunology for the production and detection of antibodies. In the latter case, immune sera that are supposed to contain antibody subsets to linear antigenic determinants, are tested against synthetic peptides that correspond to known amino acid sequences of the immunogen (Chersi et al., 1987; Ulrich et al., 1987). For this assay, the synthetic peptides are generally radioiodinated at a tyrosine residue by the chloramine-T method (Greenwood et al., 1963), or, alternatively, at a lysine residue (Bolton and Hunter, 1973; Church et al., 1983). Although iodination of peptides (and proteins) is generally easy to accomplish, the technique suffers the disadvantage that the nuclide decays rapidly (half life: 60 days). Moreover, the introduction into the molecule of
Correspondence to: A. Chersi, Istituto Regina Elena for Cancer Research, Laboratory of Biochemistry, Viale Regina Elena 291, 00161 Rome, Italy. Abbreviations: DCM, dichloromethane; DCC, dicyclohexylcarbodimide; TFA, trifluoroacetic acid; MBS; m-maleimidobenzoyl-N-hydroxysuccinimide ester; EtOH, ethanol; tBoc, tert-butyloxycarbonyl; OBzl, O-benzyl.
the heavy iodine atom may sometimes result in a partial loss of the biological activity of the labeled compound. According to our experience, the labeling of peptides by iodine can be advantageously replaced, in many cases, by aac-labeling using iodo[14C] acetic acid, provided that the peptide contains a cysteine. Carboxymethylation of proteins by this reagent has been widely employed over the last 20 years; cysteines are converted into stable, labeled derivatives, and carboxy-methyl cysteine-containing peptides can be easily identified. Carboxymethylation of a cysteine-containing peptide is easy and safe. Alternatively, one can prepare N-protected [carboxy-14C]methylcysteine, and introduce this labeled amino acid directly during synthesis of a polypeptide, to obtain a labeled fragment. Both of the above methods will be described here. The first, which is more obvious, can be associated with a device for separating reacted, labeled peptide from the unreacted material, in order to obtain higher specific activity. The second needs a manual or automatic synthesizer, and is suitable for labeling all peptides produced over a long period of time.
0022-1759/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
272 TABLE I A M I N O ACID SEQUENCE OF SYNTHETIC PEPTIDES SELECTED F O R 14C-LABELING The peptides correspond to sequences 61-73 and 133-143 of human histocompability antigens HLA-DR 2 and HLA-DQ fl chains. (M) Y N S Q K D I L E E A R A C (V) R N D Q E E T T G V V - P A M RESIN
(1) Peptide M (Table I) was kindly provided by Dr. R.A. Houghten, Scripps Clinic, La Jolla, CA. The peptide included a C-terminal cysteine at position 1. The determination of free - S H groups in the peptide by Ellman's reagent (Ellman, 1969) revealed that approximately 93% of those groups were available for reaction. For the labeling of the peptide, 300 /~1 of a 1 m g / m l solution of peptide in 0.05 M Tris-HC1 buffer, pH 8.2 (0.20 ttmol) were reacted for 2 h at room temperature with 5 #Ci (0.07 /~mol) of iodo[2-14C]acetic acid, in 200/~1 of the same buffer. The mixture of reacted and unreacted peptide was then recovered by chromatography on a 0.8 × 40 cm column of Sephadex G-25 superfine in 0.05 M acetic acid. The yield was approximately 270 ~g, as revealed by the absorbance of the peak at 206 nm; the total cpm were 6 × 106, the specific activity was 0.018/~Ci//~g. In order to obtain a higher specific activity, the sample was added to 500 /~1 of a 10% suspension of a gel prepared by reacting Sepharose-AH (1 g in 5 ml PBS) with 1 mg MBS (Liu et al., 1979) in 100 #1 dimethylformamide. The gel bound covalently the unreacted peptide with free - S H groups and this was then discarded by centrifugation. The peptide in the supernatant was 110/~g, cpm 5.8 × 10 6 (0.042/~Ci//zg). (2) As an alternative approach, one may introduce a radiolabeled carboxymethylcysteine directly at the N-terminal end of a peptide during synthesis. ~4C-labeled, N-protected amino acids have occasionally been successfully introduced into the growing chain of synthetic peptides (Kagnoff et al., 1984); however those derivatives are not generally available and are very expensive. We have developed therefore a procedure suitable for the preparation of N - B o c - [ c a r b o x y - 1 4 C ] m e t h y l c y s teine and its coupling to the N-terminal end of a synthetic fragment during synthesis. For this ex-
periment, we dissolved 0.66 mg (3/~mol) of commercial N-Boc-cysteine (Inalco) in 0.4 ml of 0.05 M N-ethylmorpholin-HC1 buffer, p H 8.2. 3 ttmol of iodo[2-14C]acetic acid (180 /~Ci) were then added in 0.2 ml of the same buffer; the reaction was allowed to proceed for 2 h. The sample was then taken to dryness in three aliquots. The specific activity of this protected amino acid was approximately 56/~Ci//tmol. The ll-residue peptide to be labeled (Table I) was prepared with the aid of a Vega Coupler, model 1000, starting with 0.3 g of Boc-Val-PAM resin (0.1 mmol of amino acid), and in conjunction with OBzl protecting groups for Asp, Glu, Thr. The guanidino group of arginine was left unprotected, since this was the last residue to be coupled. Details of the synthesis will be reported in detail elsewhere (Chersi et al., 1988). When the last residue (Arg) was coupled, the Boc-protecting group was cleaved by 40% T F A in DCM, the resin was neutralized and washed and then removed from the reservoir. Only 2% of the resin particles were then used for labeling: 6 mg (approximately 2 /~mol of peptide) were suspended in I ml DCM, and an aliquot of the previously prepared a4C-labeled N-Boc-S-carboxymethylcysteine (1 /~mol, 280 #g, 56 /~Ci) were added together with a six-fold excess of DCC in 1 ml DCM. The reaction was allowed to proceed in a 10 ml glass-stoppered flask, with continuous shaking, for about 2 h. Excess reagents were discarded by centrifugation in E t O H - D C M 2 : 1. Then the dry resin was treated with a 20-fold excess of trifluoromethansulfonic acid at 1 0 ° C for 30 rain (Yajima et al., 1975). After 1 in 3 dilution with water, and filtration through a Gelman Acrodisc, the free peptide was recovered by gel filtration on a 0.8 × 50 cm column of Sephadex G-25 superfine equilibrated in 0.05 M ammonia. The recovery of peptide was 1.4 ktmol (70%). The amino acid composition was within _+7% of the theoretical value and the cm-Cys content was 0.44 residues. Total cpm were 52 × 10 6 (or 30 /iCi//~mol, 0.028 /zCi//~g). Since the amino groups available for the reaction were in a 2 : 1 excess over the alkylating agent, the utilization of the radioactive compound was probably complete. No efforts were made to separate labeled from unlabeled peptide (approx. 1 : 1) since the specific
273
activity obtained was considered suitable for most purposes. Finally, if one uses for labeling N-protected, 14C-labeled carboxamidomethylcysteine, it is likely that this residue can be introduced at any position of the growing peptide chain, since the amino group is relatively stable under the conditions used for synthesis, and does not undergo undesirable side reactions. This second method is not of wide applicability, since it requires a manual or automatic peptide synthesizer: however, since the labeled, N-protected carboxymethylcysteine is prepared only once, and can be stored ready for use in aliquots, it permits the labeling of all peptides synthesized over a long period of time. Finally, the use of 14C offers some advantages such as the long half-life and stability of the labeled product and the use of lower amounts of a safer radioactive agent. The 14C-labeled peptide generally displays sufficiently high specific activity .
t--
N.
o
o
0 o
t 50
i
8
i 200 Dilution
o
~
0 o
] 800
o o
I
Fig. 1. Binding of 14C-labeled peptide M to antipeptide antibody 6148 (anti-M). Serial dilutions of either antiserum 6148 (e) or normal rabbit serum (o) were incubated in 1 ml PBS containing 0.05% ovalbumin, with 6 x 105 cpm ( ~ 1.2 /~g) of peptide, for 2 h at 18°C. Bound and free peptides were separated using Protein A-Sepharose (100 #1 of a 20% suspension). After incubation for 2 h, the pellet was washed twice, then suspended in 100 ~1 PBS. 100 ~1 were then measured in a beta counter.
to be employed in most conventional immunological assays; as an example the determination of antigen-antibody binding is shown in Fig. 1.
Acknowledgements
The authors thank Dr. R.A. Houghten, Scripps Clinic and Research Foundation, La Jolla, CA, for the gift of peptide M. This investigation was partly supported by the Associazione Italiana per la Ricerca sul Cancro, Milan.
References Bolton, A.E. and Hunter, W.M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a 1251 containing acylating agent. Biochem. J. 133, 529. Chersi, A., Morganti, M.C., Houghten, R.A. and Chillemi, F. (1988) Identification of linear HLA Class II amino acid sequences recognized by rabbit antisera against native molecules. Scand. J. Immunol., in press. Church. W.B., Walker, L.E., Houghten, R.A. and Reisfeld, R.A. (1983) Anti-HLA antibodies of predetermined specificity: A chemically synthesized peptide induces antibodies specific for HLA-A,B heavy chain. Proc. Natl. Acad. Sci. U.S.A. 80, 255. Ellman, G.L. (1969) Tissue sulphydryl groups. Arch. Biochem. Biophys. 82, 7077. Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963) The preparation of 131I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114. Kagnoff, M.F., Austin, R.K., Hubert, J.J., Bernardin, J.E. and Kasarda, D.D. (1984) Possible role for a human adenovirus in the pathogenesis of celiac disease. J. Exp. Med. 160, 1544. Liu, F.T., Zinnecker, M., Hamaoka, T. and Katz, D.H. (1979) New procedure for preparation and isolation of conjugates of proteins and a synthetic copolymer of amino acids and immunochemical characterization of such conjugates. Biochemistry 18, 690. Ulrich, R.G., Atassi, H., Lutz, P., Cresswell, P. and Atassi, M.Z. (1987) Immune recognition of human major histocompatibility antigens: localization by a comprehensive synthetic strategy of the continuous antigenic sites in the first domain of HLA-DR2 t9 chain. Eur. J. Immunol. 17, 497. Yajima, H., Ogawa, H., Watanabe, H., Fujii, N., Kurobe, M. and Miyamoto, S. (1975) Studies on peptides XLIII. 1.2 Application of the trifluoromethansulphonic acid procedure to the synthesis of tuftsin. Chem. Pharmacol. Bull. 23, 371.