- Email: [email protected]
Purification of radioiodinated succinyl cyclic nucleotide tyrosine methyl esters by anion-exchange thin-layer chromatography
ANALYTICAL
BIOCHEMISTRY
141,499-502 (1984)
Purification of Radioiodinated Succinyl Cyclic Nucieotide Tyrosine Esters by Anion-Exchange Thin-Layer Chromatography’ KURT
SCHMIDT~
Methyl
AND HANS P. BAER~
Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received February 15, 1984 Tyrosine methyl esters of 2’-O-succinyl cyclic AMP and 2’-0-succinyl cyclic GMP were radioiodinated, and the products were purified by anion-exchange chromatography on polyethyleneimine-cellulose thin layers. Using I .OM LiCl for development, two major, immunologically active fractions (presumably the mono and diiodo products) were separated from unreacted iodide, ScAMP-TME, or ScGMP-TME, and immunologically inactive radiolabeled fractions. When used in a radioimmunoassay, both major fractions from each nucleotide showed essentially identical binding affinities; however, assayswere more sensitive with the presumed diiodo product because of its higher content of ‘*‘I. Compared with other purification methods. this technique is faster and permits separation of defined, radioactive iodine-containing products from unreacted starting materials. KEY WORDS: radioimmunoassay; iodination; methods: cyclic AMP; cyclic GMP.
Among the many assay procedures for cyclic nucleotides, radioimmunoassays have become particularly favored since the initial reports by Steiner et al. in 1969 (1). Thus, antisera are produced in various species using protein conjugates of 2’-O-succinyl cyclic nucleotides as antigens, and the tyrosine methyl esters of 2’-O-succinyl cyclic nucleotides are used as ligands in assays after iodination with radioactive iodine isotopes. The sensitivity and specificity of assasys depends on the quality of antisera as well as on the purity of the radiolabeled ligands. While the iodination of the tyrosine methyl ester derivatives is an easy and rapid procedure (2), the separation and purification of the iodinated products with the common gel-filtration technique on ’ This work was supported by the Medical Research Council of Canada. K.S. was the recipient of a fellowship from the Alberta Heritage Foundation for Medical Research. 2 Current address: Institut fur Pharmakodynamik und Toxikologie, Karl-Franzens-Univeritat, A-3010 Grax, Austria. 3 To whom correspondence and reprint requests should lx addressed.
Sephadex G 10 or 25 ( 1), which is probably used most frequently, is time consuming. Besides the preparation of the column and the collection of a large number of fractions, the most suitable peak has to be located by assaying for appropriately high binding and low background. Recently we observed that the separation of a radioiodinated adenosine derivative from noniodinated starting material could be achieved most reproducibly by TLC on PE14impregnated cellulose, making use of the increased acidity of phenolic hydroxy groups after iodination (3). It appeared that this method might be generally applicable to the purification of compounds of low molecular weight after radioiodination, and it was applied to the cyclic nucleotide derivatives widely used as ligands in RIAs of cyclic AMP and cyclic GMP. This convenient and efficient procedure is described in this paper. 4 Abbreviations used SCAMP-TME, 2-O’-succinyl adenosine-3’,5’-monophosphate+tyrosine methyl ester; ScGMP-TME, 2-O’-succinyl guanosine-3’,5’-monophosphate+tyrosine methyl ester; PEI, polyethyleneimine: RIA, radioimmunoassay. 499
0003-2697184 $3.00 Copyright 0 1984 by Academic Press Inc. All rights of reproduction m any form reserved
500
SCHMIDT
MATERIALS
AND
METHODS
Materials. ScAMP-TME and ScGMPTME were obtained from Boehringer-Mannheim Canada Ltd., DorvaI, Quebec, and carrier-free NalZ51 (3.7 GBq/ml) was purchased from Edmonton Radio-Pharmaceuticals. Rabbit antisera to cyclic AMP and cyclic GMP had been prepared previously in this laboratory according to the procedure of Steiner et al. (1). Zodination. SCAMP-TME and ScGMPTME were radioiodinated by the procedure of Hunter and Greenwood (2) as follows: 10 ~1 of freshly prepared chloramine T (14 mg/ ml in 50 mM potassium phosphate, pH 7.5) was rapidly mixed with a solution of 10 ~1 SCAMP-TME or ScGMP-TME (0.1 mg/ml), 10 ~1 Na’251 (40 MBq) and 10 1.c1of 0.5 M potassium phosphate (pH 7.5). Alter shaking for 1 min, the reaction was stopped by adding 10 ~1 sodium metabisulphite ( 19 mg/ml in 50 mM potassium phosphate, pH 7.5). TLC separation and autoradiography. Aliquots of the reaction mixture were applied in narrow bands to PEI-cellulose sheets cut to 20 X 10 cm, (Macherey-Nagel, Germany), previously washed with water. For analytical purposes bands were 1 cm wide and, for preparative purposes, the entire (20 cm) width of plates was used. After developing with 1.O M LiCl in the IO-cm direction, the radioactive spots were located by radioautography, carefully cut out with scissors, and extracted twice with 1 ml 0.1 M NaCl. After adding 2 ml 0.2 M Na-acetate (pH 5.8) to the extracts, the solutions were stored in small aliquots at -20°C. The extraction procedure with 0.1 M NaCl was about 70-80% effective. Separation with geljiltration. The reaction mixture was applied to a Sephadex G 25 column (1 X 30 cm) equilibrated with 0.2 M Na-acetate (pH 5.8), and l-ml fractions were collected. Radioimmunoassay. Assays were performed in the presence of 50 InM Na-acetate (pH 4.85), 50 ~1 normal rabbit serum, 1O,OOO-20,000 cpm ‘*$labeled l&and, anti-
AND
BAER
serum (diluted in 50 mM Na-acetate, pH 4.85, 30 mg/ml bovine serum albumin) and, for displacement experiments, cyclic AMP and cyclic GMP in different concentrations, in a total volume of 0.35 ml. Incubation was carried out at 4’C overnight, and the bound radioactive ligand was separated by precipitation with 0.5 ml polyethylene glycol 6000 (20% in 100 mM potassium phosphate, pH 7.5). After 15 min samples were centrifuged for 5 min (Eppendorf 3200 centrifuge) and, after removal of supematants by suction, the precipitates were counted in a gamma spectrometer. RESULTS
AND
DISCUSSION
A typical separation of iodinated ScAMPTME and ScGMP-TME is shown in columns A and D of Fig. 1, respectively. In both cases five radioactive bands could be detected, which were numbered (and accordingly will be referred to in the following) 1 to 5 in the case of the cyclic AMP derivative and 1* to 5* in the case of the cyclic GMP derivative, in the order of increasing Rf values. In both cases equivalent products (bands 1 to 4 and l* to 4*) seem to be formed, in addition to bands 5 and 5* which migrate identically to iodide (column B). The distribution of radioactivity was measured by cutting out the bands and counting directly, and the immunological reactivity of the products with the respective antisera was determined after eluting the segments with 0.1 M NaCl, using the same amount of radioactivity (10,000 cpm) in each case. As shown in Table 1, bands 2 and 3, as well as 2* and 3*, represent the major products, and bands 1 and l* minor ones. The products of all four bands were bound by the respective antisera, whereby bands 1 to 3 and l* to 3* showed about similar degrees of binding, while bands 4 and 4* were essentially inactive, yielding the same background as obtained with the iodide bands 5 and 5*. The chemical identity of bands l-4 and 1*-4* was not established directly. However,
ISOLATION
OF IODINATED
CYCLIC NUCLEOTIDE
501
DERIVATIVES
diiodo derivatives of SCAMP-TME and ScGMP-TME, respectively. The location of the noniodinated starting materials ScAMP5 5’ TME and ScGMP-TME (detected under uv light) is indicated on the autoradiograph (Fig. 1) for comparison. The relative yields of bands 2 and 3, or 2* 4 4’ and 3*, differed slightly between iodinations. 3 Furthermore, we noted that the use of the 3’ same batch of nucleotide derivatives 4 months 2 2+ later led to the occurrence of a new band I migrating between bands I and 2 in the case I* of SCAMP-TME, and bands 3 and 4 in the case of ScGMP-TME. These new products did not interfere with the separation and A B C isolation of bands 2/2* or 3/3*. It was obFIG. 1. Autoradiogram of TLC of itination products. served previously in this laboratory that Radioiodinated, unpurified ScAMP-TME (A), tyrosine SCAMP-TME and ScGMP-TME were not methyl ester (C), and ScCMP-TME (D), as well as Na’251 chemically stable even when stored as solids (B), were applied to a PEI-cellulose TLC sheet, developed with 1 M LiCl for 10 cm, and autoradiographed as at -20°C. described under Materials and Methods. Unlabeled After separation of radioiodination prodScAMP-TME and ScGMP-TME were included in colucts with the use of column chromatography umns A and D, respectively, and were visualized under using Sephadex G 25, two radioactive peaks uv light after chromatography (hatched spots). Numbers were obtained with each nucleotide derivative. indicate the obtained radioactive bands with ScAMPTME (l-5) and ScCMP-TME (1*-5*). Tentative assign- The first one represented iodide, and the ments of the bands are: 1/ I *, unknown; 2/2*, diiodinated ScAMP-TME and ScGMP-TME, respectively; 3/3*, monoiodinated ScAMP-TME and ScGMP, respectively; 4/d*, unknown; S/5*, iodide. The autoradiograph was strongly overexposed in order to visualize the minor bands; there was complete separation of bands 2 and 3 as well as 2* and 3* (cf., width of uv-marker bands).
it is evident that a possible contamination of the unlabeled nucleotide derivatives with Ltyrosine methyl ester, and formation of respective mono- or diiodo derivatives, does not account for any of the bands (column C, Fig. 1). Assuming that the major products should be the mono and diiodo derivatives (i.e., with iodine substitution in the two positions ortho to the phenolic hydroxy group of tyrosine), and knowing that successive iodination will increase the acidity of the phenolic hydroxy group, resulting in increased binding to the PEI-impregnated plates, we assume that bands 3 and 3* represent the monoiodo products and bands 2 and 2* the
TABLE 1 YIELDOF RADIOI~DWATI~N PRODUCTS AND BINDING TO ANTISERA
Band
RI value
Percentage of total radioactivity
Percentage bound
I 2 3 4 5
0.13 0.23 0.30 0.38 0.57
3 34 40 17 6
38 54 45 6 5
1* 2* 3* 4* 5*
0.10 0.19 0.27 0.38 0.57
7 52 26 9 6
32 52 55 6 6
Note. A quantity of 10,000 cpm of each product isolated after PEI chromatography (Fig. I) was used in immunoassays,adjusting the respective antiserum dilutions to obtain about 50% binding with band 2 and 2* products. See text for details.
502
SCHMIDT
second contained immunoreactive product. Rechromatography of the second peak by PEI TLC and autoradiography showed that two bands, fully equivalent to bands 2 and 3 as well as bands 2* and 3*, were present (data not shown). Similarly, we investigated the identity of radioactive products separated by TLC on cellulose with butanolglacial acetic acid:H20 (12:3:5), a system referred to in an article by Weinryb (4) and elsewhere (5,6). Thus, three radioactive spots were located on the chromatogram which were separate from the starting materials. One consisted of unreacted iodide, and the other two contained at least three products when rechromatographed by PEI TLC, clearly indicating that chromatography on cellulose yields a mixture of labeled products. To characterize the immunoreactivity of bands 213 and 2*/3*, the respective fractions were tested in immunoassays in more detail. Assuming that the assignment of mono and diiodo derivatives, as explained above, is correct, equivalent molar concentrations of the products of bands 213 and 2*/3* were employed by using 20,000 cpm of bands 2 and 2* and 10,000 cpm of bands 3 and 3* in assays with antisera dilutions binding about 70-80% of bands 3 and 3* (i.e., the presumed monoiodo derivatives), respectively. Displacement of the ligands by increasing concentrations of unlabeled cyclic AMP and cyclic GMP, respectively, was measured. Figure 2 shows that the respective displacement curves covered the same concentration range of unlabeled nucleotides, and that the affinity of the ligands eluted from bands 2 and 3 as well as 2* and 3* to their binding sites must thus be essentially identical on a molar basis. Since bands 2 and 2”’ contain twice the amount of radiolabel per molecule, steeper “standard” curves result, suggesting that employment of these particular products in radioimmunoassays is preferable. The successful use of PEI-cellulose TLC for the separation of radioiodination products,
AND BAER
0.01
0.1 I pmol/tube
IO
loo
FIG. 2. Displacement of radioiodinated ligands by cyclic nucleotides. A RIA was performed using equimolar amounts of ligands, i.e., 20,000 cpm of the diiodo products 2 (m) and 2* (O), and 10,000 cpm of the monoiodo products 3 (0) and 3* (0) in the presence of increasing concentrations of cyclic AMP or cyclic GMP, respectively.
as demonstrated in this study and in a previous project (3), suggests that this method may be generally applicable to situations where one deals with compounds of low molecular weight. Apart from the convenient procedure and low risk of radioactive contamination, the major advantage of the method is that the mono and diiodo products can be isolated separately, and that these products are separated fully from their parent compounds, thus allowing quantitative antigen binding studies and assays with maximal sensitivity. REFERENCES 1. Steiner, A. L., Kipnis, D. M., Utiger, R., and Parker, C. W. (1969) Proc. Natl. Acad. Sci. USA 64,367373. 2. Hunter, W. M., and Greenwood, F. C. (1962) Nature (London) 194,495-496. 3. Munshi, R., and Baer, H. P. (1982) Canad. J. Physiol. Pharmacol. 60, 1320- 1322. 4. Weinryb, I. (1972) in Methods in Cyclic Nucleotide Research (Chasin, M., ed.), pp 29-79, Dekker, New York. 5. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1970) in Advances in Biochemical Psychopharmacology (Greengard, P., and Costa, E., eds.), Vol. 3, pp 89-l 1I, Raven Press, New York. 6. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1970) J. Biol. Chem. 247, 1106-l 113.
BIOCHEMISTRY
141,499-502 (1984)
Purification of Radioiodinated Succinyl Cyclic Nucieotide Tyrosine Esters by Anion-Exchange Thin-Layer Chromatography’ KURT
SCHMIDT~
Methyl
AND HANS P. BAER~
Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received February 15, 1984 Tyrosine methyl esters of 2’-O-succinyl cyclic AMP and 2’-0-succinyl cyclic GMP were radioiodinated, and the products were purified by anion-exchange chromatography on polyethyleneimine-cellulose thin layers. Using I .OM LiCl for development, two major, immunologically active fractions (presumably the mono and diiodo products) were separated from unreacted iodide, ScAMP-TME, or ScGMP-TME, and immunologically inactive radiolabeled fractions. When used in a radioimmunoassay, both major fractions from each nucleotide showed essentially identical binding affinities; however, assayswere more sensitive with the presumed diiodo product because of its higher content of ‘*‘I. Compared with other purification methods. this technique is faster and permits separation of defined, radioactive iodine-containing products from unreacted starting materials. KEY WORDS: radioimmunoassay; iodination; methods: cyclic AMP; cyclic GMP.
Among the many assay procedures for cyclic nucleotides, radioimmunoassays have become particularly favored since the initial reports by Steiner et al. in 1969 (1). Thus, antisera are produced in various species using protein conjugates of 2’-O-succinyl cyclic nucleotides as antigens, and the tyrosine methyl esters of 2’-O-succinyl cyclic nucleotides are used as ligands in assays after iodination with radioactive iodine isotopes. The sensitivity and specificity of assasys depends on the quality of antisera as well as on the purity of the radiolabeled ligands. While the iodination of the tyrosine methyl ester derivatives is an easy and rapid procedure (2), the separation and purification of the iodinated products with the common gel-filtration technique on ’ This work was supported by the Medical Research Council of Canada. K.S. was the recipient of a fellowship from the Alberta Heritage Foundation for Medical Research. 2 Current address: Institut fur Pharmakodynamik und Toxikologie, Karl-Franzens-Univeritat, A-3010 Grax, Austria. 3 To whom correspondence and reprint requests should lx addressed.
Sephadex G 10 or 25 ( 1), which is probably used most frequently, is time consuming. Besides the preparation of the column and the collection of a large number of fractions, the most suitable peak has to be located by assaying for appropriately high binding and low background. Recently we observed that the separation of a radioiodinated adenosine derivative from noniodinated starting material could be achieved most reproducibly by TLC on PE14impregnated cellulose, making use of the increased acidity of phenolic hydroxy groups after iodination (3). It appeared that this method might be generally applicable to the purification of compounds of low molecular weight after radioiodination, and it was applied to the cyclic nucleotide derivatives widely used as ligands in RIAs of cyclic AMP and cyclic GMP. This convenient and efficient procedure is described in this paper. 4 Abbreviations used SCAMP-TME, 2-O’-succinyl adenosine-3’,5’-monophosphate+tyrosine methyl ester; ScGMP-TME, 2-O’-succinyl guanosine-3’,5’-monophosphate+tyrosine methyl ester; PEI, polyethyleneimine: RIA, radioimmunoassay. 499
0003-2697184 $3.00 Copyright 0 1984 by Academic Press Inc. All rights of reproduction m any form reserved
500
SCHMIDT
MATERIALS
AND
METHODS
Materials. ScAMP-TME and ScGMPTME were obtained from Boehringer-Mannheim Canada Ltd., DorvaI, Quebec, and carrier-free NalZ51 (3.7 GBq/ml) was purchased from Edmonton Radio-Pharmaceuticals. Rabbit antisera to cyclic AMP and cyclic GMP had been prepared previously in this laboratory according to the procedure of Steiner et al. (1). Zodination. SCAMP-TME and ScGMPTME were radioiodinated by the procedure of Hunter and Greenwood (2) as follows: 10 ~1 of freshly prepared chloramine T (14 mg/ ml in 50 mM potassium phosphate, pH 7.5) was rapidly mixed with a solution of 10 ~1 SCAMP-TME or ScGMP-TME (0.1 mg/ml), 10 ~1 Na’251 (40 MBq) and 10 1.c1of 0.5 M potassium phosphate (pH 7.5). Alter shaking for 1 min, the reaction was stopped by adding 10 ~1 sodium metabisulphite ( 19 mg/ml in 50 mM potassium phosphate, pH 7.5). TLC separation and autoradiography. Aliquots of the reaction mixture were applied in narrow bands to PEI-cellulose sheets cut to 20 X 10 cm, (Macherey-Nagel, Germany), previously washed with water. For analytical purposes bands were 1 cm wide and, for preparative purposes, the entire (20 cm) width of plates was used. After developing with 1.O M LiCl in the IO-cm direction, the radioactive spots were located by radioautography, carefully cut out with scissors, and extracted twice with 1 ml 0.1 M NaCl. After adding 2 ml 0.2 M Na-acetate (pH 5.8) to the extracts, the solutions were stored in small aliquots at -20°C. The extraction procedure with 0.1 M NaCl was about 70-80% effective. Separation with geljiltration. The reaction mixture was applied to a Sephadex G 25 column (1 X 30 cm) equilibrated with 0.2 M Na-acetate (pH 5.8), and l-ml fractions were collected. Radioimmunoassay. Assays were performed in the presence of 50 InM Na-acetate (pH 4.85), 50 ~1 normal rabbit serum, 1O,OOO-20,000 cpm ‘*$labeled l&and, anti-
AND
BAER
serum (diluted in 50 mM Na-acetate, pH 4.85, 30 mg/ml bovine serum albumin) and, for displacement experiments, cyclic AMP and cyclic GMP in different concentrations, in a total volume of 0.35 ml. Incubation was carried out at 4’C overnight, and the bound radioactive ligand was separated by precipitation with 0.5 ml polyethylene glycol 6000 (20% in 100 mM potassium phosphate, pH 7.5). After 15 min samples were centrifuged for 5 min (Eppendorf 3200 centrifuge) and, after removal of supematants by suction, the precipitates were counted in a gamma spectrometer. RESULTS
AND
DISCUSSION
A typical separation of iodinated ScAMPTME and ScGMP-TME is shown in columns A and D of Fig. 1, respectively. In both cases five radioactive bands could be detected, which were numbered (and accordingly will be referred to in the following) 1 to 5 in the case of the cyclic AMP derivative and 1* to 5* in the case of the cyclic GMP derivative, in the order of increasing Rf values. In both cases equivalent products (bands 1 to 4 and l* to 4*) seem to be formed, in addition to bands 5 and 5* which migrate identically to iodide (column B). The distribution of radioactivity was measured by cutting out the bands and counting directly, and the immunological reactivity of the products with the respective antisera was determined after eluting the segments with 0.1 M NaCl, using the same amount of radioactivity (10,000 cpm) in each case. As shown in Table 1, bands 2 and 3, as well as 2* and 3*, represent the major products, and bands 1 and l* minor ones. The products of all four bands were bound by the respective antisera, whereby bands 1 to 3 and l* to 3* showed about similar degrees of binding, while bands 4 and 4* were essentially inactive, yielding the same background as obtained with the iodide bands 5 and 5*. The chemical identity of bands l-4 and 1*-4* was not established directly. However,
ISOLATION
OF IODINATED
CYCLIC NUCLEOTIDE
501
DERIVATIVES
diiodo derivatives of SCAMP-TME and ScGMP-TME, respectively. The location of the noniodinated starting materials ScAMP5 5’ TME and ScGMP-TME (detected under uv light) is indicated on the autoradiograph (Fig. 1) for comparison. The relative yields of bands 2 and 3, or 2* 4 4’ and 3*, differed slightly between iodinations. 3 Furthermore, we noted that the use of the 3’ same batch of nucleotide derivatives 4 months 2 2+ later led to the occurrence of a new band I migrating between bands I and 2 in the case I* of SCAMP-TME, and bands 3 and 4 in the case of ScGMP-TME. These new products did not interfere with the separation and A B C isolation of bands 2/2* or 3/3*. It was obFIG. 1. Autoradiogram of TLC of itination products. served previously in this laboratory that Radioiodinated, unpurified ScAMP-TME (A), tyrosine SCAMP-TME and ScGMP-TME were not methyl ester (C), and ScCMP-TME (D), as well as Na’251 chemically stable even when stored as solids (B), were applied to a PEI-cellulose TLC sheet, developed with 1 M LiCl for 10 cm, and autoradiographed as at -20°C. described under Materials and Methods. Unlabeled After separation of radioiodination prodScAMP-TME and ScGMP-TME were included in colucts with the use of column chromatography umns A and D, respectively, and were visualized under using Sephadex G 25, two radioactive peaks uv light after chromatography (hatched spots). Numbers were obtained with each nucleotide derivative. indicate the obtained radioactive bands with ScAMPTME (l-5) and ScCMP-TME (1*-5*). Tentative assign- The first one represented iodide, and the ments of the bands are: 1/ I *, unknown; 2/2*, diiodinated ScAMP-TME and ScGMP-TME, respectively; 3/3*, monoiodinated ScAMP-TME and ScGMP, respectively; 4/d*, unknown; S/5*, iodide. The autoradiograph was strongly overexposed in order to visualize the minor bands; there was complete separation of bands 2 and 3 as well as 2* and 3* (cf., width of uv-marker bands).
it is evident that a possible contamination of the unlabeled nucleotide derivatives with Ltyrosine methyl ester, and formation of respective mono- or diiodo derivatives, does not account for any of the bands (column C, Fig. 1). Assuming that the major products should be the mono and diiodo derivatives (i.e., with iodine substitution in the two positions ortho to the phenolic hydroxy group of tyrosine), and knowing that successive iodination will increase the acidity of the phenolic hydroxy group, resulting in increased binding to the PEI-impregnated plates, we assume that bands 3 and 3* represent the monoiodo products and bands 2 and 2* the
TABLE 1 YIELDOF RADIOI~DWATI~N PRODUCTS AND BINDING TO ANTISERA
Band
RI value
Percentage of total radioactivity
Percentage bound
I 2 3 4 5
0.13 0.23 0.30 0.38 0.57
3 34 40 17 6
38 54 45 6 5
1* 2* 3* 4* 5*
0.10 0.19 0.27 0.38 0.57
7 52 26 9 6
32 52 55 6 6
Note. A quantity of 10,000 cpm of each product isolated after PEI chromatography (Fig. I) was used in immunoassays,adjusting the respective antiserum dilutions to obtain about 50% binding with band 2 and 2* products. See text for details.
502
SCHMIDT
second contained immunoreactive product. Rechromatography of the second peak by PEI TLC and autoradiography showed that two bands, fully equivalent to bands 2 and 3 as well as bands 2* and 3*, were present (data not shown). Similarly, we investigated the identity of radioactive products separated by TLC on cellulose with butanolglacial acetic acid:H20 (12:3:5), a system referred to in an article by Weinryb (4) and elsewhere (5,6). Thus, three radioactive spots were located on the chromatogram which were separate from the starting materials. One consisted of unreacted iodide, and the other two contained at least three products when rechromatographed by PEI TLC, clearly indicating that chromatography on cellulose yields a mixture of labeled products. To characterize the immunoreactivity of bands 213 and 2*/3*, the respective fractions were tested in immunoassays in more detail. Assuming that the assignment of mono and diiodo derivatives, as explained above, is correct, equivalent molar concentrations of the products of bands 213 and 2*/3* were employed by using 20,000 cpm of bands 2 and 2* and 10,000 cpm of bands 3 and 3* in assays with antisera dilutions binding about 70-80% of bands 3 and 3* (i.e., the presumed monoiodo derivatives), respectively. Displacement of the ligands by increasing concentrations of unlabeled cyclic AMP and cyclic GMP, respectively, was measured. Figure 2 shows that the respective displacement curves covered the same concentration range of unlabeled nucleotides, and that the affinity of the ligands eluted from bands 2 and 3 as well as 2* and 3* to their binding sites must thus be essentially identical on a molar basis. Since bands 2 and 2”’ contain twice the amount of radiolabel per molecule, steeper “standard” curves result, suggesting that employment of these particular products in radioimmunoassays is preferable. The successful use of PEI-cellulose TLC for the separation of radioiodination products,
AND BAER
0.01
0.1 I pmol/tube
IO
loo
FIG. 2. Displacement of radioiodinated ligands by cyclic nucleotides. A RIA was performed using equimolar amounts of ligands, i.e., 20,000 cpm of the diiodo products 2 (m) and 2* (O), and 10,000 cpm of the monoiodo products 3 (0) and 3* (0) in the presence of increasing concentrations of cyclic AMP or cyclic GMP, respectively.
as demonstrated in this study and in a previous project (3), suggests that this method may be generally applicable to situations where one deals with compounds of low molecular weight. Apart from the convenient procedure and low risk of radioactive contamination, the major advantage of the method is that the mono and diiodo products can be isolated separately, and that these products are separated fully from their parent compounds, thus allowing quantitative antigen binding studies and assays with maximal sensitivity. REFERENCES 1. Steiner, A. L., Kipnis, D. M., Utiger, R., and Parker, C. W. (1969) Proc. Natl. Acad. Sci. USA 64,367373. 2. Hunter, W. M., and Greenwood, F. C. (1962) Nature (London) 194,495-496. 3. Munshi, R., and Baer, H. P. (1982) Canad. J. Physiol. Pharmacol. 60, 1320- 1322. 4. Weinryb, I. (1972) in Methods in Cyclic Nucleotide Research (Chasin, M., ed.), pp 29-79, Dekker, New York. 5. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1970) in Advances in Biochemical Psychopharmacology (Greengard, P., and Costa, E., eds.), Vol. 3, pp 89-l 1I, Raven Press, New York. 6. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1970) J. Biol. Chem. 247, 1106-l 113.