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Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride
ANALYTICAL
BIOCHEMISTRY
87,
562-565 (1978)
Radiolabeling of Proteins by Reductive Alkylation [14C]Formaldehyde and Sodium Cyanoborohydride DIANE
DOTTAVIO-MARTIN
AND JOANNE
with
M. RAVEL
Clayton Foundation Biochemical Institute and The Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712 Received October 11, 1977; accepted February 23. 1978 A procedure is described for radiolabeling proteins in vitro by reductive alkylation. The proteins are treated with [W]formaldehyde in the presence of sodium cyanoborohydride, a reducing agent that is stable in aqueous solution at pH 7. The advantage of this procedure is that the reaction can be carried out at neutral pH for extended periods of time and over a wide range of temperatures (O-37°C). Moreover, owing to the flexibility in the reaction conditions afforded by the use of sodium cyanoborohydride, higher incorporation of radiolabel into protein can be attained.
The procedure described by Rice and Means (1) for the radiolabeling of proteins in vitro by reductive alkylations has been used by a number of investigators (2-4). This procedure utilizes [14C]formaldehyde and sodium borohydride as the reducing agent. Because sodium borohydride is unstable in aqueous solution at neutral pH, the reaction is generally carried out at 0°C in borate buffer, pH 9, and the sodium borohydride is added in small increments over a short period of time. In attempts to radiolabel initiation factor 1 (IF-l) from Escherichia coli by reductive alkylation, we found that appreciably higher amounts of [14C]methyl groups could be attached to IF-l if sodium cyanoborohydride is used as the reducing agent instead of sodium borohydride. Sodium cyanoborohydride has been used for the reductive alkylation of amino acids (5) and for the formation of p-nitrobenzyl derivatives of certain proteins (6). Sodium cyanoborohydride, unlike sodium borohydride, is stable in aqueous solution at pH 7 (5). The advantages of using sodium cyanoborohydride for radiolabeling proteins are that the reaction can be carried out at neutral pH for extended periods of time and that greater incorporation of [14C]methyl groups into protein can be attained. METHODS
Materials. [14C]formaldehyde (58 &i/pmol) was purchased from SchwarzlMann. Anhydrous sodium cyanoborohydride was purchased from SchwarzlMann, and hydrous sodium cyanoborohydride was pur0003-2697/78/0872-0562$02.00/O Copyright All rights
8 1978 by Academic Press, Inc. of reproduction in any fom reserved.
562
RADIOLABELING
PROTEINS
BY REDUCTIVE
ALKYLATION
563
chased from Aldrich Chemical Co. and stored under nitrogen. Sodium borohydride was purchased from Sigma. Proteins were obtained from the following sources: chymotrypsinogen A, cytochrome c, and bovine serum albumin were from Boehringer Mannheim; ricin and Phytolacca americana protein (PAP) were gifts from Dr. Jon Robertus; and IF-l was isolated from Escherichiu coli as previously described (7). Radiolabeling of proteins. Five microliters of [14C]formaldehyde (85 nmol, 5 &i) was added to 25 ~1 of a 1 mg/ml solution of protein in 0.04 M potassium phosphate buffer, pH 7.0, followed by the addition of 10 ~1 of a freshly prepared solution of NaBH,CN (6 mg/ml in 0.04 M phosphate buffer, pH 7.0). The 0.04-ml reaction mixture was incubated at 25°C for 1 hr, unless indicated otherwise, and was shaken gently at 15min intervals. The volume of the reaction mixture was then increased to 0.25 ml by addition of 0.04 M phosphate buffer (pH 7.0), and the low molecular weight components were removed by dialysis for 16 hr at 4°C. The amount of [“Cl incorporated into the protein was determined by counting two 10-1.1.1 aliquots of the dialyzed protein in I5 ml of Aquasol scintillation fluid. At a counting efficiency of approximately 50%, 64 cpm represents 1 pmol of [14C]formaldehyde incorporated into protein. RESULTS
AND DISCUSSION
Reductive alkylation of several proteins was carried out at pH 7 in the presence of [14C]formaldehyde and sodium cyanoborohydride for 1 hr at 25°C. As shown in Table 1, the amount of [‘“Cl remaining with the protein after dialysis ranged from 4 x IO6 to 20 x 10” cpm/mg of protein. To confirm that the radioactivity present in each of the samples was bound to protein, each protein was electrophoresed on 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. In each case, the majority of the radioactivity was found to coincide with the protein band. Also, precipitation of the proteins with hot trichloroacetic acid showed that the radioactivity was in the hot trichloroacetic acid-insoluble material. In separate experiments, it was found that the ability of IF-l to stimulate the binding of fMet-tRNA to E. coli ribosomes (7) and the ability of ricin and Phytolacca americana protein to inhibit protein synthesis in a rabbit reticulocyte system (Bird and Robertus, personal communication) were not significantly altered by alkylation of the protein under the conditions described. In the experiment reported in Table 1, no attempt was made to optimize the conditions for obtaining maximal labeling of the proteins. The data given in Fig. 1 show that at 25°C a three-fold increase in the amount of [‘“Cl label incorporated into chymotrypsinogen A was obtained if the reaction mixture was incubated for 6 hr instead of 1 hr. Moreover, the reaction could be carried out‘at 0 or 37°C. Although the rate of alkylation was slower at 0°C) the extent of labeling obtained in 24 hr at 0°C was close to that obtained
564
DOTT’AVIO-MARTIN
AND RAVEL
TABLE
1
RADIOLABELINGOFPROTEINSBYREDUCTIVEALKYLATION ANDSODIUMCYANOBOROHYDRIDE
WITH[T]FORMALDEHYDE
Radioactivity
Protein Bovine serum albumin Chymotrypsinogen A Cytochrome c Initiation factor 1 (E. co/i) Phytolacca
protein Ricin
[W]Methyl (pmol/pmol of protein)
Molecular weight
Lysine residues”
(cpmlmg)
67,000 25,000 12,500
59 14 17
10 a.7 21
670 217 262
10.4 3.3 4.1
9,400
10
16
150
2.3
30,000 66,000
16-18 10
9.0 3.9
270 257
4.2 4.0
(X 10-G) (cpmhmol)
americana
(1The molecular weight and number of lysine residues of the proteins listed above come from the following sources: bovine serum albumin, Ref 8; chymotrypsinogen A and cytochrome c, Ref 9; Phytolacca americana protein, J. Robertus, personal communication; and ricin, Ref 10.
in 4 to 6 hr at 25°C or in 1 hr at 37°C. Therefore, a wide range of reaction conditions are possible, and the conditions best suited for the alkylation of a particular protein can be selected. In addition, it was found that the alkylation reaction can be carried out in Tris or Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer as long as the pH remains close to neutral. A comparison of the amounts of [‘“Cl label incorporated into protein in the presence of sodium borohydride at pH 9 and in the presence of sodium cyanoborohydride at pH 7 is shown in Table 2. Depending upon the protein being labeled, 6- to 45fold greater incorporation is obtained with the
2
4 Time
6
22
24
blourr)
FIG. 1. The effect of time and temperature on the amount of [“Cl incorporation into chymotrypsinogen A. The reaction mixture described in the Methods section was increased to 300 ~1 and incubated at (0 0) O”C, (a @) 25”C, and (0 - - - 0) 37°C. Aliquots of 30 ~1 were removed at the times indicated.
RADIOLABELING
PROTEINS
BY REDUCTIVE
TABLE
565
ALKYLATION
2
COMPARISON OFTHEAMOIJNTOF[W]LABELINCORPORATED INTOPROTEIN INTHE~RESENCEOF NaBH,, AT pH9 AND NaBH,CN AT pH7 [C”] incorporated
(cpm/mg) x 10e6 Increase (-fold)
Protein
NaBH,. pH 9”
Bovine serum albumin Chymotrypsinogen A Cytochrome c Initiation factor 1 Phytotacca americana protein Ricin
1.4 0.7 1.0 2.7
10 10 20 16
7 14 20 6
0.2 0.3
9 4
45 13
NaBH,CN,
pH 7”
’ The procedure of Rice and Means (1) was modified in the following manner: 25 ~1 of a 1 mgiml solution of protein in 0.2 M sodium borate buffer, pH 9.0, was treated at 0°C with 5 ~1 of [i4C]formaldehyde, followed by three S-p1 additions of NaBH, (5 mg/ml in sodium borate buffer, pH 9) over a 2- to 3-min period. After 30 min at O”C, an additional 10 ~1 of NaBH, was added; the reaction mixture was then brought to a final volume of 250 ~1 with borate buffer and treated as described in the Methods section. ’ The reaction was carried out at 25” for 1 hr as described in the Methods section.
sodium cyanoborohydride treatment. Thus, use of sodium cyanoborohydride as a reducing agent not only affords greater flexibility in the reaction conditions but also provides a means of obtaining more highly labeled proteins. ACKNOWLEDGMENTS This work was supported in part by USPHS Grant GM 18775. The authors wish to thank Dr. Evan Kyba and Dr. Stephen Martin for suggesting the use of sodium cyanoborohydride and Dr. Jon Robertus and Ms. Sandra Bird for supplying and determining the activity of ricin and Phytolacca americana protein.
REFERENCES 1. 2. 3. 4. 5.
Rice, R. H., and Means, G. E. (1971) J. Biol. Chem. 246, 831-832. Gualerzi, C., Wabl, M. R., and Pon, C. L. (19733 FEBS tett. 35, 313-316. Moore, G., and Crichton, R. R. (1973) FEBS Left. 37, 74-78. Suttle, D. P. (1974) Ph.D. dissertation, University of Texas, Austin, Tex. Borch, R. F., Bernstein, M. D., and Durst, H. D. (1971) J. Amer. Chem. Sot. 93, 2897-2904. 6. Friedman, M., Williams, L. D.. and Masri, M. S. (1974) ht. J. Pept. Prof. Res. 6, 183-185. 7. Suttle, D. P., Haralson, M. A., and Ravel, J. M. (1973)Biochem. Biophys. Res. Commun. 51, 376-382.
8. Brown, J. R. (1975) Fed. Proc. 34, 591. 9. Sober, H. A., ed. (1970) Handbook of Biochemistry, 2nd ed.. pp. C-229 and C-235, Chemical Rubber Co., Cleveland, Ohio. 10. Olsnes, S., Refsnes, K., Terje. B. C.. and Pihl. A. (1975) Biochim. Biophys. Acta. 405, l-10.
BIOCHEMISTRY
87,
562-565 (1978)
Radiolabeling of Proteins by Reductive Alkylation [14C]Formaldehyde and Sodium Cyanoborohydride DIANE
DOTTAVIO-MARTIN
AND JOANNE
with
M. RAVEL
Clayton Foundation Biochemical Institute and The Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712 Received October 11, 1977; accepted February 23. 1978 A procedure is described for radiolabeling proteins in vitro by reductive alkylation. The proteins are treated with [W]formaldehyde in the presence of sodium cyanoborohydride, a reducing agent that is stable in aqueous solution at pH 7. The advantage of this procedure is that the reaction can be carried out at neutral pH for extended periods of time and over a wide range of temperatures (O-37°C). Moreover, owing to the flexibility in the reaction conditions afforded by the use of sodium cyanoborohydride, higher incorporation of radiolabel into protein can be attained.
The procedure described by Rice and Means (1) for the radiolabeling of proteins in vitro by reductive alkylations has been used by a number of investigators (2-4). This procedure utilizes [14C]formaldehyde and sodium borohydride as the reducing agent. Because sodium borohydride is unstable in aqueous solution at neutral pH, the reaction is generally carried out at 0°C in borate buffer, pH 9, and the sodium borohydride is added in small increments over a short period of time. In attempts to radiolabel initiation factor 1 (IF-l) from Escherichia coli by reductive alkylation, we found that appreciably higher amounts of [14C]methyl groups could be attached to IF-l if sodium cyanoborohydride is used as the reducing agent instead of sodium borohydride. Sodium cyanoborohydride has been used for the reductive alkylation of amino acids (5) and for the formation of p-nitrobenzyl derivatives of certain proteins (6). Sodium cyanoborohydride, unlike sodium borohydride, is stable in aqueous solution at pH 7 (5). The advantages of using sodium cyanoborohydride for radiolabeling proteins are that the reaction can be carried out at neutral pH for extended periods of time and that greater incorporation of [14C]methyl groups into protein can be attained. METHODS
Materials. [14C]formaldehyde (58 &i/pmol) was purchased from SchwarzlMann. Anhydrous sodium cyanoborohydride was purchased from SchwarzlMann, and hydrous sodium cyanoborohydride was pur0003-2697/78/0872-0562$02.00/O Copyright All rights
8 1978 by Academic Press, Inc. of reproduction in any fom reserved.
562
RADIOLABELING
PROTEINS
BY REDUCTIVE
ALKYLATION
563
chased from Aldrich Chemical Co. and stored under nitrogen. Sodium borohydride was purchased from Sigma. Proteins were obtained from the following sources: chymotrypsinogen A, cytochrome c, and bovine serum albumin were from Boehringer Mannheim; ricin and Phytolacca americana protein (PAP) were gifts from Dr. Jon Robertus; and IF-l was isolated from Escherichiu coli as previously described (7). Radiolabeling of proteins. Five microliters of [14C]formaldehyde (85 nmol, 5 &i) was added to 25 ~1 of a 1 mg/ml solution of protein in 0.04 M potassium phosphate buffer, pH 7.0, followed by the addition of 10 ~1 of a freshly prepared solution of NaBH,CN (6 mg/ml in 0.04 M phosphate buffer, pH 7.0). The 0.04-ml reaction mixture was incubated at 25°C for 1 hr, unless indicated otherwise, and was shaken gently at 15min intervals. The volume of the reaction mixture was then increased to 0.25 ml by addition of 0.04 M phosphate buffer (pH 7.0), and the low molecular weight components were removed by dialysis for 16 hr at 4°C. The amount of [“Cl incorporated into the protein was determined by counting two 10-1.1.1 aliquots of the dialyzed protein in I5 ml of Aquasol scintillation fluid. At a counting efficiency of approximately 50%, 64 cpm represents 1 pmol of [14C]formaldehyde incorporated into protein. RESULTS
AND DISCUSSION
Reductive alkylation of several proteins was carried out at pH 7 in the presence of [14C]formaldehyde and sodium cyanoborohydride for 1 hr at 25°C. As shown in Table 1, the amount of [‘“Cl remaining with the protein after dialysis ranged from 4 x IO6 to 20 x 10” cpm/mg of protein. To confirm that the radioactivity present in each of the samples was bound to protein, each protein was electrophoresed on 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. In each case, the majority of the radioactivity was found to coincide with the protein band. Also, precipitation of the proteins with hot trichloroacetic acid showed that the radioactivity was in the hot trichloroacetic acid-insoluble material. In separate experiments, it was found that the ability of IF-l to stimulate the binding of fMet-tRNA to E. coli ribosomes (7) and the ability of ricin and Phytolacca americana protein to inhibit protein synthesis in a rabbit reticulocyte system (Bird and Robertus, personal communication) were not significantly altered by alkylation of the protein under the conditions described. In the experiment reported in Table 1, no attempt was made to optimize the conditions for obtaining maximal labeling of the proteins. The data given in Fig. 1 show that at 25°C a three-fold increase in the amount of [‘“Cl label incorporated into chymotrypsinogen A was obtained if the reaction mixture was incubated for 6 hr instead of 1 hr. Moreover, the reaction could be carried out‘at 0 or 37°C. Although the rate of alkylation was slower at 0°C) the extent of labeling obtained in 24 hr at 0°C was close to that obtained
564
DOTT’AVIO-MARTIN
AND RAVEL
TABLE
1
RADIOLABELINGOFPROTEINSBYREDUCTIVEALKYLATION ANDSODIUMCYANOBOROHYDRIDE
WITH[T]FORMALDEHYDE
Radioactivity
Protein Bovine serum albumin Chymotrypsinogen A Cytochrome c Initiation factor 1 (E. co/i) Phytolacca
protein Ricin
[W]Methyl (pmol/pmol of protein)
Molecular weight
Lysine residues”
(cpmlmg)
67,000 25,000 12,500
59 14 17
10 a.7 21
670 217 262
10.4 3.3 4.1
9,400
10
16
150
2.3
30,000 66,000
16-18 10
9.0 3.9
270 257
4.2 4.0
(X 10-G) (cpmhmol)
americana
(1The molecular weight and number of lysine residues of the proteins listed above come from the following sources: bovine serum albumin, Ref 8; chymotrypsinogen A and cytochrome c, Ref 9; Phytolacca americana protein, J. Robertus, personal communication; and ricin, Ref 10.
in 4 to 6 hr at 25°C or in 1 hr at 37°C. Therefore, a wide range of reaction conditions are possible, and the conditions best suited for the alkylation of a particular protein can be selected. In addition, it was found that the alkylation reaction can be carried out in Tris or Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer as long as the pH remains close to neutral. A comparison of the amounts of [‘“Cl label incorporated into protein in the presence of sodium borohydride at pH 9 and in the presence of sodium cyanoborohydride at pH 7 is shown in Table 2. Depending upon the protein being labeled, 6- to 45fold greater incorporation is obtained with the
2
4 Time
6
22
24
blourr)
FIG. 1. The effect of time and temperature on the amount of [“Cl incorporation into chymotrypsinogen A. The reaction mixture described in the Methods section was increased to 300 ~1 and incubated at (0 0) O”C, (a @) 25”C, and (0 - - - 0) 37°C. Aliquots of 30 ~1 were removed at the times indicated.
RADIOLABELING
PROTEINS
BY REDUCTIVE
TABLE
565
ALKYLATION
2
COMPARISON OFTHEAMOIJNTOF[W]LABELINCORPORATED INTOPROTEIN INTHE~RESENCEOF NaBH,, AT pH9 AND NaBH,CN AT pH7 [C”] incorporated
(cpm/mg) x 10e6 Increase (-fold)
Protein
NaBH,. pH 9”
Bovine serum albumin Chymotrypsinogen A Cytochrome c Initiation factor 1 Phytotacca americana protein Ricin
1.4 0.7 1.0 2.7
10 10 20 16
7 14 20 6
0.2 0.3
9 4
45 13
NaBH,CN,
pH 7”
’ The procedure of Rice and Means (1) was modified in the following manner: 25 ~1 of a 1 mgiml solution of protein in 0.2 M sodium borate buffer, pH 9.0, was treated at 0°C with 5 ~1 of [i4C]formaldehyde, followed by three S-p1 additions of NaBH, (5 mg/ml in sodium borate buffer, pH 9) over a 2- to 3-min period. After 30 min at O”C, an additional 10 ~1 of NaBH, was added; the reaction mixture was then brought to a final volume of 250 ~1 with borate buffer and treated as described in the Methods section. ’ The reaction was carried out at 25” for 1 hr as described in the Methods section.
sodium cyanoborohydride treatment. Thus, use of sodium cyanoborohydride as a reducing agent not only affords greater flexibility in the reaction conditions but also provides a means of obtaining more highly labeled proteins. ACKNOWLEDGMENTS This work was supported in part by USPHS Grant GM 18775. The authors wish to thank Dr. Evan Kyba and Dr. Stephen Martin for suggesting the use of sodium cyanoborohydride and Dr. Jon Robertus and Ms. Sandra Bird for supplying and determining the activity of ricin and Phytolacca americana protein.
REFERENCES 1. 2. 3. 4. 5.
Rice, R. H., and Means, G. E. (1971) J. Biol. Chem. 246, 831-832. Gualerzi, C., Wabl, M. R., and Pon, C. L. (19733 FEBS tett. 35, 313-316. Moore, G., and Crichton, R. R. (1973) FEBS Left. 37, 74-78. Suttle, D. P. (1974) Ph.D. dissertation, University of Texas, Austin, Tex. Borch, R. F., Bernstein, M. D., and Durst, H. D. (1971) J. Amer. Chem. Sot. 93, 2897-2904. 6. Friedman, M., Williams, L. D.. and Masri, M. S. (1974) ht. J. Pept. Prof. Res. 6, 183-185. 7. Suttle, D. P., Haralson, M. A., and Ravel, J. M. (1973)Biochem. Biophys. Res. Commun. 51, 376-382.
8. Brown, J. R. (1975) Fed. Proc. 34, 591. 9. Sober, H. A., ed. (1970) Handbook of Biochemistry, 2nd ed.. pp. C-229 and C-235, Chemical Rubber Co., Cleveland, Ohio. 10. Olsnes, S., Refsnes, K., Terje. B. C.. and Pihl. A. (1975) Biochim. Biophys. Acta. 405, l-10.