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Purification of 125I-labeled succinyl cyclic nucleotide tyrosine methyl esters by high-performance liquid chromatography
ANALYTICAL
BIOCHEMISTRY
168,417-420
(1988)
Purification of 1251-Labeled Succinyl Cyclic Nucleotide Tyrosine Esters by High-Performance Liquid Chromatography AMRAT
Methyl
PATEL AND JOEL LINDEN’
Departments of Physiology University of Virginia,
and Internal Medicine (Cardiology), Charlottesville, Virginia 22908
Received June 15, 1987 Carrier free ‘251-labeled succinyl cyclic adenosine monophosphate (SCAMP) and succinyl cyclic guanosine monophosphate (ScGMP) tyrosine methyl esters (TME) were purified by reversed phase high-performance liquid chromatography (HPLC) or descending paper chromatography. Using an isocratic buffer for HPLC, mono-ScAMP-‘251-TME and mono-ScGMP‘*‘I-TME were eluted from a Cl8 column at 8.9 and 6.9 min, respectively. Both of the monoiodinated radioligands were completely separated from their noniodinated precursors and other iodinated products. The radioligands purified by HPLC or paper chromatography were used for the radioimmunoassay (RIA) of CAMP and cGMP. Cyclic AMP or cGMP inhibited binding of the HPLC purified radioligands at three- to fivefold lower concentrations than the paper chromatography purified radioligands. The sensitivity of the RIA decreased with time if paper chromatography purified radioligands were used, but remained stable for 4 months if the HPLC purified compounds were used, even with storage at 4°C. We attribute these results to better purification of radioligands by the HPLC than by the paper chromatography. Using optimal conditions the HPLC method takes only 10 min and results in a high yield (>95%) ofadded 125I into the monoiodinated products. o 1988 Academic PIW, I~C. KEY WORDS: HPLC; cyclic nucleotides; cyclic AMP; cyclic GMP, radioimmunoassay; radioligands.
The role of cyclic nucleotides as second messengers of hormone action has been extensively studied beginning with the discovery of cyclic AMP (cAMP)~ by Rall and Sutherland (1). The development of methodology for the determination of cyclic AMP has progressed from difficult and time consuming (2) to rapid, straightforward, and suitable for automation (3). Radioimmunoassay (RIA) methodology and procedures for the preparation of reagents for the assay of cyclic AMP and cyclic GMP has been de’ To whom correspondence and reprint requests should be addressed. ’ Abbreviations used: CAMP, cyclic AMP, cGMP, cyclic GMP, Cl-T, chloramine-T, PC, paper chromatography; RIA, radioimmunoassay; SCAMP and ScGMP, 2’-0succinylated tyrosine methyl ester derivatives of cyclic nucleotides; TME, tyrosine methyl ester.
417
scribed in detail by various groups (4-6). Steiner et al. (4) were the first to develop sensitive and specific RIAs for cyclic AMP and cyclic GMP. They synthesized 2’-O-succinyl cyclic nucleotides and their corresponding tyrosine methyl esters which were used to raise antibodies against cyclic nucleotides and to prepare 1251 iodinated ligands, respectively. A variety of chromatographic methods have been used to separate the iodination reaction products of cyclic nucleotide tyrosine methyl esters, such as column chromatography over Sephadex G- 10 (4) or Sephadex G-25 (5), TLC on cellulose (4), and descending paper chromatography on Whatman 3 IET paper (6). We have developed a method to separate the iodinated from the noniodinated products of succinyl tyrosine
0003-2697188 $3.00 Copyrisht Q 1988 by Academic F%s.s, Inc. All rights of reproduction in any form reserved.
418
PATEL
AND
methyl esters of cyclic AMP (SCAMP-TME) and cyclic GMP (ScGMP-TME) by reversed-phase high-performance liquid chromatography (HPLC) in less then 10 min. The method is advantageous not only because of its rapidity, but also because of the improved recovery and purity of the reaction products. MATERIALS
AND
METHODS
2’-O-Succinyl tyrosine methyl esters of cyclic AMP and cyclic GMP were obtained from Sigma, carrier free “‘1 from New England Nuclear, and solvents for HPLC from Fisher. Stock solutions of SCAMP-TME and ScGMP-TME (1 mM) were made up in 50 mM sodium acetate buffer, pH 4.75, and kept frozen in small aliquots at -20°C. The cyclic nucleotides were iodinated by the method of Hunter and Greenwood (7). Five nanomoles of the SCAMP-TME or ScGMPTME and 0.2 mCi of 1251were dissolved in 0.3 M phosphate buffer, pH 7.0. Following the addition of 5 ~1 of chloramine-T (1 mg/ml in water) the reactants were mixed for a minute in a fume hood equipped with a charcoal filter. The iodination reaction was quenched with 50 ~1 of sodium metabisulfite (5 mg/ml in 1.0 M acetic acid). Part of the reaction products was applied directly to a 4.5 X 250-mm Cl8 (5 pm) column and purified by HPLC using an IBM LC9533 system. The isocratic elution buffer consisted of methanol/5 mM KI-IP04, pH 4.0 (35:65), at a flow rate of 1 ml/min. Ultraviolet absorbance was monitored at 254 nm and 125Idetected with an in-line Beckman Model 170 radioisotope detector. Part of the iodination reaction products was purified by descending paper chromatography as described by Brooker et al. (6). The radioligands separated by these methods were used to generate standard curves for acetylated cyclic AMP and cyclic GMP in the range 0 to 80 pmol/ml as assayed by Gammaflo automated radioimmunoassay (3).
LINDEN
RESULTS
AND
DISCUSSION
Figure 1 shows the HPLC elution profile of SCAMP-‘~~I-TME. The noniodinated starting material, SCAMP-TME and chloramine-T, were eluted at 4.1 and 7.3 min, respectively. When the ratio of starting material to ‘25I was large, i.e., >50, most of the radioiodinated product was mono-SCAMP‘251-TME which was eluted at 8.9 min. If the ratio was lowered, a second diiodinated product was eluted at 2 1 min. ScGMP-TME and its mono- and diiodinated reactions products were eluted at 3.4, 6.9, and 13.2 min, respectively (Fig. 2). Mono - SCAMP - ‘25I - TME and mono SCGMP-‘~~I-TME purified by HPLC or the major ‘251-containing peak obtained by paper chromatography were used in generating a standard curve for CAMP and cGMP RIA (Fig. 3). At the highest concentration of CAMP and cGMP (80 pmol/ml) greater than 95 and 85% of the respective bound HPLC purified radioligands were displaced, with IC5,,‘s (nM) of 4.5 and 11.2. Standard curves
SCAMP-TME
125
4
8 Elution
12 Time,
16
20
minutes
FIG. 1. HPLC of the iodination products of ScAMPTME. SCAMP-TME was iodinated with carrier free ‘*‘I and the reaction products purified by HPLC. Ultraviolet absorbance (-) and “‘1 (---) were continuously monitored as described under Materials and Methods. SCAMP-TME, SCAMP-‘*‘I-TME, and SCAMP-i2’12TME were eluted at 4.1, 8.9, and 2 1 min, respectively. Chloramine-T (Cl-T) was eluted at 7.3 min. Fractions containing the SCAMP-‘*>I-TME contained 95% of the starting ‘251. These were diluted (1:l) with MeOH and stored at -20°C.
PURIFICATION
OF “‘I-LABELED
generated with SCAMP-‘~~I-TME and SCGMP-‘~~I-TME purified by paper chromatography (a procedure which takes 8- 10 h) were less sensitive. Less than 75 and 70% of the respective bound radioligands were displaced, with IC50)s (nM) of 23.7 and 32.3. The standard curves were shallower in the latter cases than those obtained with HPLCpurified radioligands, which is indicative of some inhibitory component(s) interfering with the ligand-antibody interaction. Both the radioligands purified by HPLC were stable for over 4 months at 4°C based on the standard curves generated using new and old radioligands. In contrast, binding of the paper-chromatography-purified compounds decayed with time, possibly due to enrichment of contaminants. Chloramine-T produced a uv-absorbing peak which could not be well resolved from SCGMP-‘~~I-TME by the HPLC procedure used, but this compound did not interfere with the RIA. During the radioiodination reaction, exposure of cyclic nucleotides to chloramine-T (ca. 1 mM) for up to 5 min has no damaging effects on the cyclic nucleotides as determined by HPLC (data not shown).
ScGMP-TME
ScGMP-1251-TME \
t/ 11 ~I I’ I/ /I // ’ I ; ’ I ’ -TME
/
0
2
I
4
6 Elution
6 Time,
10
12
14
:6
minutes
FIG. 2. HPLC of the iodination products of ScGMPTME. ScGMP-TME iodination products were chromatographed as described under Materials and Methods and the legend of Fig. 1. ScGMP-TME, !%zGMP-“~ITME, and ScGMP-‘2512-TME were eluted at 3.4, 6.9, and 13.2 min, respectively. Fractions containing ScGMP-‘251-TME contained 97% of the added ‘*%
419
CYCLIC NUCLEOTIDES
0.8
0.2
1
-1
0 Log
1 CCyclic
nucleotidel.
2 nM
RG. 3. Standard curves for the radioimmunoassay of CAMP and cGMP. Standard curves for CAMP and cGMP were generated using their respective antibodies and radioligands purified by HPLC or paper chromatography (PC). The ICso’s for CAMP and cGMP to inhibit antibody binding of the HPLC-purified radioligands, mono-SCAMP-‘*‘I-TME (A) and mono-ScGMP-‘*% TME (C), were 4.5 and 11.2 nM, respectively. For paper-chromatography-purified SCAMP-‘*‘I-TME (B) and ScGMP-‘*‘I-TME (D) ICso’s were 23.7 and 32.3 nM, respectively.
We confirmed the observations of Delaage et al. (5) that a gentler radioiodination of the cyclic nucleotides with lactoperoxidase enzyme as indicated by Yukitaka et al. (8) is not necessary. In summary, carrier free radioligands for cyclic AMP and cyclic GMP RIA have been purified by HPLC in less then 10 min. The monoiodinated products were well separated from the diiodo and other minor iodinated breakdown products of SCAMP-TME and ScGMP-TME. Apparent instability of lz51-labeled succinyl cyclic nucleotide tyrosine methyl esters purified by paper chromatography can be attributed to gradual concentration of contaminants. The HPLC-purified radioligands are stable at 4°C for over 4 months. ACKNOWLEDGMENTS This work was supported by NIH Grant No HL37942. Joel Linden is an Established Investigator of the American Heart Association.
PATEL
420
AND LINDEN
REFERENCES 1. Rail, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232, 1056-1076. 2. Rail, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232,1077-1091. 3. Brooker, G., Terasaki, W. L., and Price, M. G. (1976) Science 194,270-276. 4. Steiner, A. L., Parker, C. W., and Ripnis, D. M. (1972) J. Biol. Chem. 247, 1106-l 113. 5. Delaage, M. A., Roux, D., and Cailla, H. L. ( 1978) in Molecular Biology and Pharntacology of Cy-
clic Nucleotides (Folco, G., and Paoletti, R., Eds.), pp. 155-l 7 1. Elsevier/North-Holland, New York. 6. Brooker, G., Harper, J. F., Terasaki, W. L., and Moylan, R. D. (1979) in Advances in Cyclic Nucleotide Research (Brooker, G., Greengard, P., and Robison, G. A., Eds.), Vol. 10, pp. l-33. Raven Press, New York. 7. Hunter, W. M., and Greenwood, F. C. (1962) Nature (London) 194,495-496. 8. Yukitaka, M., Akira, M., and Kanji, S. (1977) Anal. Biochem. 77,429-435.
BIOCHEMISTRY
168,417-420
(1988)
Purification of 1251-Labeled Succinyl Cyclic Nucleotide Tyrosine Esters by High-Performance Liquid Chromatography AMRAT
Methyl
PATEL AND JOEL LINDEN’
Departments of Physiology University of Virginia,
and Internal Medicine (Cardiology), Charlottesville, Virginia 22908
Received June 15, 1987 Carrier free ‘251-labeled succinyl cyclic adenosine monophosphate (SCAMP) and succinyl cyclic guanosine monophosphate (ScGMP) tyrosine methyl esters (TME) were purified by reversed phase high-performance liquid chromatography (HPLC) or descending paper chromatography. Using an isocratic buffer for HPLC, mono-ScAMP-‘251-TME and mono-ScGMP‘*‘I-TME were eluted from a Cl8 column at 8.9 and 6.9 min, respectively. Both of the monoiodinated radioligands were completely separated from their noniodinated precursors and other iodinated products. The radioligands purified by HPLC or paper chromatography were used for the radioimmunoassay (RIA) of CAMP and cGMP. Cyclic AMP or cGMP inhibited binding of the HPLC purified radioligands at three- to fivefold lower concentrations than the paper chromatography purified radioligands. The sensitivity of the RIA decreased with time if paper chromatography purified radioligands were used, but remained stable for 4 months if the HPLC purified compounds were used, even with storage at 4°C. We attribute these results to better purification of radioligands by the HPLC than by the paper chromatography. Using optimal conditions the HPLC method takes only 10 min and results in a high yield (>95%) ofadded 125I into the monoiodinated products. o 1988 Academic PIW, I~C. KEY WORDS: HPLC; cyclic nucleotides; cyclic AMP; cyclic GMP, radioimmunoassay; radioligands.
The role of cyclic nucleotides as second messengers of hormone action has been extensively studied beginning with the discovery of cyclic AMP (cAMP)~ by Rall and Sutherland (1). The development of methodology for the determination of cyclic AMP has progressed from difficult and time consuming (2) to rapid, straightforward, and suitable for automation (3). Radioimmunoassay (RIA) methodology and procedures for the preparation of reagents for the assay of cyclic AMP and cyclic GMP has been de’ To whom correspondence and reprint requests should be addressed. ’ Abbreviations used: CAMP, cyclic AMP, cGMP, cyclic GMP, Cl-T, chloramine-T, PC, paper chromatography; RIA, radioimmunoassay; SCAMP and ScGMP, 2’-0succinylated tyrosine methyl ester derivatives of cyclic nucleotides; TME, tyrosine methyl ester.
417
scribed in detail by various groups (4-6). Steiner et al. (4) were the first to develop sensitive and specific RIAs for cyclic AMP and cyclic GMP. They synthesized 2’-O-succinyl cyclic nucleotides and their corresponding tyrosine methyl esters which were used to raise antibodies against cyclic nucleotides and to prepare 1251 iodinated ligands, respectively. A variety of chromatographic methods have been used to separate the iodination reaction products of cyclic nucleotide tyrosine methyl esters, such as column chromatography over Sephadex G- 10 (4) or Sephadex G-25 (5), TLC on cellulose (4), and descending paper chromatography on Whatman 3 IET paper (6). We have developed a method to separate the iodinated from the noniodinated products of succinyl tyrosine
0003-2697188 $3.00 Copyrisht Q 1988 by Academic F%s.s, Inc. All rights of reproduction in any form reserved.
418
PATEL
AND
methyl esters of cyclic AMP (SCAMP-TME) and cyclic GMP (ScGMP-TME) by reversed-phase high-performance liquid chromatography (HPLC) in less then 10 min. The method is advantageous not only because of its rapidity, but also because of the improved recovery and purity of the reaction products. MATERIALS
AND
METHODS
2’-O-Succinyl tyrosine methyl esters of cyclic AMP and cyclic GMP were obtained from Sigma, carrier free “‘1 from New England Nuclear, and solvents for HPLC from Fisher. Stock solutions of SCAMP-TME and ScGMP-TME (1 mM) were made up in 50 mM sodium acetate buffer, pH 4.75, and kept frozen in small aliquots at -20°C. The cyclic nucleotides were iodinated by the method of Hunter and Greenwood (7). Five nanomoles of the SCAMP-TME or ScGMPTME and 0.2 mCi of 1251were dissolved in 0.3 M phosphate buffer, pH 7.0. Following the addition of 5 ~1 of chloramine-T (1 mg/ml in water) the reactants were mixed for a minute in a fume hood equipped with a charcoal filter. The iodination reaction was quenched with 50 ~1 of sodium metabisulfite (5 mg/ml in 1.0 M acetic acid). Part of the reaction products was applied directly to a 4.5 X 250-mm Cl8 (5 pm) column and purified by HPLC using an IBM LC9533 system. The isocratic elution buffer consisted of methanol/5 mM KI-IP04, pH 4.0 (35:65), at a flow rate of 1 ml/min. Ultraviolet absorbance was monitored at 254 nm and 125Idetected with an in-line Beckman Model 170 radioisotope detector. Part of the iodination reaction products was purified by descending paper chromatography as described by Brooker et al. (6). The radioligands separated by these methods were used to generate standard curves for acetylated cyclic AMP and cyclic GMP in the range 0 to 80 pmol/ml as assayed by Gammaflo automated radioimmunoassay (3).
LINDEN
RESULTS
AND
DISCUSSION
Figure 1 shows the HPLC elution profile of SCAMP-‘~~I-TME. The noniodinated starting material, SCAMP-TME and chloramine-T, were eluted at 4.1 and 7.3 min, respectively. When the ratio of starting material to ‘25I was large, i.e., >50, most of the radioiodinated product was mono-SCAMP‘251-TME which was eluted at 8.9 min. If the ratio was lowered, a second diiodinated product was eluted at 2 1 min. ScGMP-TME and its mono- and diiodinated reactions products were eluted at 3.4, 6.9, and 13.2 min, respectively (Fig. 2). Mono - SCAMP - ‘25I - TME and mono SCGMP-‘~~I-TME purified by HPLC or the major ‘251-containing peak obtained by paper chromatography were used in generating a standard curve for CAMP and cGMP RIA (Fig. 3). At the highest concentration of CAMP and cGMP (80 pmol/ml) greater than 95 and 85% of the respective bound HPLC purified radioligands were displaced, with IC5,,‘s (nM) of 4.5 and 11.2. Standard curves
SCAMP-TME
125
4
8 Elution
12 Time,
16
20
minutes
FIG. 1. HPLC of the iodination products of ScAMPTME. SCAMP-TME was iodinated with carrier free ‘*‘I and the reaction products purified by HPLC. Ultraviolet absorbance (-) and “‘1 (---) were continuously monitored as described under Materials and Methods. SCAMP-TME, SCAMP-‘*‘I-TME, and SCAMP-i2’12TME were eluted at 4.1, 8.9, and 2 1 min, respectively. Chloramine-T (Cl-T) was eluted at 7.3 min. Fractions containing the SCAMP-‘*>I-TME contained 95% of the starting ‘251. These were diluted (1:l) with MeOH and stored at -20°C.
PURIFICATION
OF “‘I-LABELED
generated with SCAMP-‘~~I-TME and SCGMP-‘~~I-TME purified by paper chromatography (a procedure which takes 8- 10 h) were less sensitive. Less than 75 and 70% of the respective bound radioligands were displaced, with IC50)s (nM) of 23.7 and 32.3. The standard curves were shallower in the latter cases than those obtained with HPLCpurified radioligands, which is indicative of some inhibitory component(s) interfering with the ligand-antibody interaction. Both the radioligands purified by HPLC were stable for over 4 months at 4°C based on the standard curves generated using new and old radioligands. In contrast, binding of the paper-chromatography-purified compounds decayed with time, possibly due to enrichment of contaminants. Chloramine-T produced a uv-absorbing peak which could not be well resolved from SCGMP-‘~~I-TME by the HPLC procedure used, but this compound did not interfere with the RIA. During the radioiodination reaction, exposure of cyclic nucleotides to chloramine-T (ca. 1 mM) for up to 5 min has no damaging effects on the cyclic nucleotides as determined by HPLC (data not shown).
ScGMP-TME
ScGMP-1251-TME \
t/ 11 ~I I’ I/ /I // ’ I ; ’ I ’ -TME
/
0
2
I
4
6 Elution
6 Time,
10
12
14
:6
minutes
FIG. 2. HPLC of the iodination products of ScGMPTME. ScGMP-TME iodination products were chromatographed as described under Materials and Methods and the legend of Fig. 1. ScGMP-TME, !%zGMP-“~ITME, and ScGMP-‘2512-TME were eluted at 3.4, 6.9, and 13.2 min, respectively. Fractions containing ScGMP-‘251-TME contained 97% of the added ‘*%
419
CYCLIC NUCLEOTIDES
0.8
0.2
1
-1
0 Log
1 CCyclic
nucleotidel.
2 nM
RG. 3. Standard curves for the radioimmunoassay of CAMP and cGMP. Standard curves for CAMP and cGMP were generated using their respective antibodies and radioligands purified by HPLC or paper chromatography (PC). The ICso’s for CAMP and cGMP to inhibit antibody binding of the HPLC-purified radioligands, mono-SCAMP-‘*‘I-TME (A) and mono-ScGMP-‘*% TME (C), were 4.5 and 11.2 nM, respectively. For paper-chromatography-purified SCAMP-‘*‘I-TME (B) and ScGMP-‘*‘I-TME (D) ICso’s were 23.7 and 32.3 nM, respectively.
We confirmed the observations of Delaage et al. (5) that a gentler radioiodination of the cyclic nucleotides with lactoperoxidase enzyme as indicated by Yukitaka et al. (8) is not necessary. In summary, carrier free radioligands for cyclic AMP and cyclic GMP RIA have been purified by HPLC in less then 10 min. The monoiodinated products were well separated from the diiodo and other minor iodinated breakdown products of SCAMP-TME and ScGMP-TME. Apparent instability of lz51-labeled succinyl cyclic nucleotide tyrosine methyl esters purified by paper chromatography can be attributed to gradual concentration of contaminants. The HPLC-purified radioligands are stable at 4°C for over 4 months. ACKNOWLEDGMENTS This work was supported by NIH Grant No HL37942. Joel Linden is an Established Investigator of the American Heart Association.
PATEL
420
AND LINDEN
REFERENCES 1. Rail, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232, 1056-1076. 2. Rail, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232,1077-1091. 3. Brooker, G., Terasaki, W. L., and Price, M. G. (1976) Science 194,270-276. 4. Steiner, A. L., Parker, C. W., and Ripnis, D. M. (1972) J. Biol. Chem. 247, 1106-l 113. 5. Delaage, M. A., Roux, D., and Cailla, H. L. ( 1978) in Molecular Biology and Pharntacology of Cy-
clic Nucleotides (Folco, G., and Paoletti, R., Eds.), pp. 155-l 7 1. Elsevier/North-Holland, New York. 6. Brooker, G., Harper, J. F., Terasaki, W. L., and Moylan, R. D. (1979) in Advances in Cyclic Nucleotide Research (Brooker, G., Greengard, P., and Robison, G. A., Eds.), Vol. 10, pp. l-33. Raven Press, New York. 7. Hunter, W. M., and Greenwood, F. C. (1962) Nature (London) 194,495-496. 8. Yukitaka, M., Akira, M., and Kanji, S. (1977) Anal. Biochem. 77,429-435.