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Preparation of lodinated cyclic GMP derivatives by a lactoperoxidase method
ANALYTICAL
BIOCHEMISTRY
Preparation
YUKITAKA Third
17,429-435
(1977)
of lodinated Cyclic GMP Derivatives by a Lactoperoxidase Method MIYACHI,’
Department
AKIRA
MIZUCHI,
AND KANJI
of Internal Medicine, Faculty University
of Tokyo,
Tokyo.
SATO
of Medicine,
Japan
Received March 19, 1976; accepted September 14, 1976 Preparation of rz51-labeled succinyl cyclic GMP-tyrosine methyl ester (YSCGMP-TME) was described. The method utilized lactoperoxidase and the glucose-glucose oxidase peroxide-generating system. 1251-SCGMP-TME, purified by Sephadex G-10 gel filtration, was separated from cyclic GMP. SCGMPTME, rz51, and ‘251-labeled TME and retained extremely high immunoreactivity. A cyclic GMP radioimmunoassay. based on this iodinated material and cyclic GMP antiserum, is specific, sensitive, and reliable, permitting the determination of small quantities of cyclic GMP in rat tissue without need for chromatographic steps.
Cyclic AMP is known as the intracellular mediator of the actions of a number of hormones, while the role of cyclic guanosine 3’ ,5’-monophosphate (cyclic GMP) is still obscure. This is due partly to the relative difficulty in the measurement of extremely low concentrations of cyclic GMP. The competitive binding radioassay utilizing a cyclic nucleotide-binding protein is not as sensitive or specific for cyclic GMP as for cyclic AMP (1). However, the great sensitivity and specificity of the radioimmunoassay method of Steiner et al. (2), which utilizes a specific antibody to cyclic GMP and succinyl cyclic GMP-tyrosine methyl ester (SCGMP-TME), iodinated by a chloramine-T method as a labeled antigen, permits the measurement of cyclic GMP on small quantities of tissue without the need for chromatography. By application of the iodinated 2’Osuccinyl cyclic AMP-tyrosine methyl ester (1251-SCAMP-TME) we could obtain more immunoreactive 12%SCAMP-TME by a lactoperoxidase method (9) than by a chloramine-T method. In this report we shall describe the labeling of SCGMP-TME with radioactive iodide using a lactoperoxidase method. SCGMP-TME, iodinated by this method, retains very high immunoreactivity and is, therefore, suitable for use as a labeled antigen in the cyclic GMP radioimmunoassay. r Present address: Third Department of Internal School of Medicine, Kasumi-cho, Hiroshima, Japan.
Medicine,
Hiroshima
University,
429 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN OCQ3-2697
430
MIYACHI,
MIZUCHI TABLE
PROCEDURE
FOR
IODINATION
AND SAT0 1 OF
SCGbfP-TME
Reactants
Amount
SCGMP-TME 0.4 M Acetate buffer, pH 6.0 Lactoperoxidase Glucose oxidase Nalz51 0.1% Glucose
4 Pis 30pl 100 ng/lO ~1 50 mu/5 ~1 0.5 mCi/S ~1
25/.A
Incubated at 23°C for 10 min Purified on Sephadex G-10 column (0.9
MATERIALS
X
10 cm)
AND METHODS
Production of antisera to Cyclic GMP. 2’-0-Succinyl cyclic GMP (SCGMP) was prepared by using the succinic anhydride method of Falbriard et al. (4). SCGMP was coupled with human serum albumin by the carbodiimide method (2). Rabbits were immunized by multiple intradermal injections of 1 mg of cyclic nucleotides-protein conjugates, emulsified in complete Freund’s adjuvant (5). Booster injections were given 8 weeks after the first immunization and then every 4 to 6 weeks. The blood was collected at appropriate intervals, and serum was stored frozen at -20°C until use. Preparation of iodinated SCGMP-TME. SCGMP-TME was prepared by the carboxylic-carbonic acid reaction (6). SCGMP-TME was iodinated with lz51 by the lactoperoxidase method of Miyachi et al., as described previously (3). Approximately 4 pg of SCGMP-TME in 30 ~1 of 0.4 M sodium acetate buffer, pH. 6.0 (AcB), 100 ng of lactoperoxidase (Sigma) in 10 ~1 of 0.1 M ACB, SO mU/5 ~1 of a solution of glucose oxidase (Sigma), and 0.5 mCi of carrier-free Na1251 (AmershamSearle) were mixed in a glass tube (7.5 x 100 mm). The iodination reaction was initiated by adding 25 ~1 of 0.1% glucose (Table 1). After the reaction proceeded for 10 min, the reaction mixture was transferred to a column of Sephadex G-10 (0.9 x 10 cm), previously washed and equilibrated with 0.05 M AcB and 1 ml of 1% bovine serum albumin. Elution was performed with 0.05 M AcB, and l-ml fractions of eluate were collected. Chromatography of cyclic [3H]GMP and SCGMP-TME. To ascertain the elution volume of unlabeled cyclic GMP and SCGMP-TME from a Sephadex G-10 column, small amounts of cyclic C3H]GMP or SCGMPTME were applied to the column (0.9 x 10 cm). Elution was performed with 0.05 M AcB. The radioactivity of cyclic [3H]GMP in the eluate was determined by liquid scintillation counting and the concentration of
ENZYMATK
RADIOIODINATION TABLE
431
OF CYCLIC GMP
2
RADIOIMMUNOASSAY PROTOCOL FORCYCLIC GMP Amount (PO 0.05 M Acetate buffer, pH 6.0 Standard cyclic GMP (O.Ol- 10 pmol) Or Samples rz51-SCGMP-TME” (5,000-10,000 cpm) Antiserum (1:3O,OOO)
200 100 100 100
Incubated at 4°C for 18 h 1 1 ml of 20% polyethylene glycol J Centrifuge at 3000 rpm for 30 min at 4°C 1 Supematant aspirated J Bound precipitates counted 0 Diluted in 0.05 M acetate buffer, pH 6.0, containing 0.5% bovine y-globulin.
SCGMP-TME assay.
in the eluate was measured by cyclic GMP radioimmuno-
Radioimmunoassay procedure. The protocol of cyclic GMP radioimmunoassay is shown in Table 2. After the mixture was incubated at 4°C for 18 hr, 1 ml of a 20% polyethylene glycol solution was added, and then the mixture was thoroughly stirred. The tubes were centrifuged for 30 min at 3000 rpm, and the precipitates (lz51-SCGMP-TME, bound to antibody) were counted in an Autowell gamma spectrometer. A computer program was used to calculate the standard curve and the potency estimate of cyclic GMP level in each sample (7). Tissue preparation. Frozen tissues (lo-30 mg) from the testes of approximately 2-month-old rats were homogenized at 4°C in 1 ml of cold 6% trichloroacetic acid (TCA). After centrifugation at 25OOxg for 15 min, TCA supernatants were extracted three times with 3 ml of ethyl ether saturated with water. The extracted aqueous phase was evaporated under a stream of Nz gas, and the residue was redissolved in 0.5 ml of 0.05 M AcB. One-hundred-milliliter aliquots were used directly in the cyclic GMP radioimmunoassay. RESULTS
The elution pattern after gel filtration on Sephadex G-10 of the iodinated cyclic nucleotide derivatives as well as their immunoreactivity to antibody
432
MIYACHI,
10
al ELUTION
MIZUCHI
AND SAT0
l!o
40
30 VOLUME
I ml )
FIG. 1. Elution pattern of iodinated cyclic GMP derivatives on a Sephadex G-10 column. Elution was performed with 0.05 M acetate buffer. The immunoreactivity of each fraction was determined using 50$00x-diluted antiserum.
are shown in Fig. 1. Three distinct radioactive peaks, I, II, III, were found, the K,, values of which were 0, 0.743, and 3.144, respectively. Approximately 15% of the 1251was incorporated into peak III. Peaks I and II were not bound by antibody, while the 1: lOO,OOO-diluted cyclic GMP antiserum 15
% - 10 s %
Y t g * s
5
10 ELUllON
20 VOLUME
30
40
f ml )
FIG. 2. Elution pattern of [3H]GMP and SCGMP-TME Elution was performed with 0.05 M acetate buffer.
on a Sephadex G-10 column.
ENZYMATIC
RADIOIODINATION
433
OF CYCLIC GMP
70 -
60 -
k
=-I ‘u 1 1:150.000
\
/f 1: m.l,anl
q\
.,?
50 Cyclic
FIG.
GMP
( pmoles / tube
I
3. Dilution curves of anti-cyclic GMP antiserum (final dilution, 1:50,000- 1:200,000).
bound more than 50% of the fractions of peak III, indicating that this peak is iodinated SCGMP-TME. Peak III or iodinated SCGMP-TME was stable; it remains highly immunoreactive after 3 months of storage at -20°C. Figure 2 shows the elution pattern of cyclic [3H]GMP and SCAMPTME from the same Sephadex G-10 column. The cyclic [3H]GMP was eluted at a K,, of 0.529 and SCGMP-TME at a K,, of 0.734. Although not shown in Fig. 2, free lz51 was eluted in the fractions of peak II, and iodinated tyrosine methyl ester (1251-labeled TME) at a point very close to peak II. These results show that lz51-SCGMP-TME (peak III) was separated from any contamination with cyclic GMP, SCGMP-TME, lz51, and 1251-labeled TME. Specific activity of 1251-SCGMP-TME was greater than 100 Ci/mmol, assuming incorporation of 1 atom of 12Y per molecule of SCGMP-TME. The optimal titer of antibody was determined by testing serial dilutions of antisera with 1251-SCGMP-TME (Fig. 3). More than 65% of 1251-SCGMPTME was bound to 1:50,000-diluted antiserum, and the antiserum, when diluted 1:200,000, bound approximately 35% of lzsI-SCGMP-TME. A dose-response curve was constructed using 1251-SCGMP-TME and diluted antiserum with different doses of unlabeled cyclic GMP (Fig. 3). The sensitivity of this cyclic GMP radioimmunoassay was 0.05 pmol/tube when 1:200,000-diluted antiserum was used (Fig. 3), and the useful range of the dose-response curve was 0.05-10 pmol/tube.
434
FIG.
MIYACHI,
4. Inhibition
MIZUCHI
AND SAT0
curves with various nucleotides in the cyclic GMP radioimmunoassay.
Cross-reactivity of other structually related nucleotides tested was less than 0.01% at 50% displacement (Fig. 4). It is remarkable that this antiserum did not react with cyclic AMP at concentrations up to 5 x 104 pmol (90% displacement). The concentration of cyclic GMP in the rat testes is 17.3 t 1.5 pmol/g of testis. The value is the same range as that reported by Shibuyaet al. (IO). The intra- and interassay variabilities of this assay were approximately 9.2 and 14.1%, respectively. DISCUSSION
We were the first to apply the enzymatic radioiodination procedure using lactoperoxidase and hydrogen peroxide to gonadotropins, and we showed that those radioiodinated gonadotropins retained biologic activities as well as immunologic activities (3). This method was applied to luteinizing hormone (LH)-releasing hormone with substitution of the hydrogen peroxide-generating glucose-glucose oxidase system for hydrogen peroxide, which was presumed to be a more gentle treatment (8). We showed in the present report that the modified lactoperoxidase method was suitable for the radioiodination of SCGMP-TME, yielding a product of a labeled antigen for radioimmunoassay. 125T-SCGMP-TME, which was highly reactive to cyclic GMP antibody, could be separated on a Sephadex G-10 column from cyclic GMP, SCGMP-TME, unreacted 1251,and 1251-labeled TME, any one of which might have interfered in the cyclic GMP radioimmunoassay. i251-SCGMP-
ENZYMATIC
RADIOIODINATION
OF CYCLIC GMP
435
TME with very high immunoreactivity permitted the use of a highly diluted antiserum, allowing a sensitive cyclic GMP radioimmunoassay system. As shown in Fig. 3, the sensitivity of the assay was less than 0.05 pmol when 1:200,000-diluted antiserum was used. Since the antiserum was specific for cyclic GMP, we obtained reasonable estimates of cyclic GMP concentrations in rat tissue without the need for chromatographic separation of cyclic GMP from cyclic AMP and ATP. We also applied this method to the iodination of SCAMP-TME and found that lz51-SCAMP-TME, labeled by this modified lactoperoxidase method, was more immunoreactive to cyclic AMP antibody than that prepared by a chloramine-T method (9). In order to obtain good labeled antigens by a chloramine-T method, it is necessary to establish experimentally and maintain reproducibly the proper conditions, such as the ratio of chloramine-T to the substrate, the reaction time, and the amount of sodium metabisulfite. In contrast, lactoperoxidase-catalyzed iodination is a gentle, simple, and rapid method, allowing one to obtain with great ease iodinated cyclic nucleotide derivatives that are suitable for reproducible radioimmunoassays. REFERENCES 1. Gilman, A. G. (1972) in Advances in Cyclic Nucleotide Research Vol. 2, p. 9, Raven Press, New York. 2. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1972)J. Biol. Chem. 347, 1106- 1113. 3. Miyachi, Y., Vaitukaitis, J. L., Nieschlag, E., and Lipsett, M. B. (1972)J. Clin. Endocrinol. 34, 23-28. 4. Falbriard, J. G., Posternak, T., and Sutherland, E. W. (1967) Biochim. Biophys. Acfa 148, 99- 105. 5. Vaitukaitis, J. L., Robbins, J. B. Nieschlag, E., and Ross, G. T. (1971) J. C/in. Endocrinol. 33, 988-991. 6. Greenstein, J. P., and Winitz, M. A. (l%l) in Chemistry of the Amino Acid Vol. 2, p. 978, Wiley, New York. 7. Rodbard, D., and Hutt, D. M. (1974) In Symposium on Radioimmunoassay and Related Peptides in Medicine, p. 165, International Atomic Energy Agency, Vienna. 8. Miyachi, Y., Chrambach, A., Mecklenburg, R., and Lipsett, M. B. (1973) Endocrinology 92, 1725- 1730. 9. Sato, K., Miyachi, Y., Mizuchi, A., Ohsawa, N., and Kosaka, K., in preparation. 10. Shibuya, Y., Arai, K. and Kajiro, Y. (1975) Biochem. Biophys. Res. Commun. 62, 129.
BIOCHEMISTRY
Preparation
YUKITAKA Third
17,429-435
(1977)
of lodinated Cyclic GMP Derivatives by a Lactoperoxidase Method MIYACHI,’
Department
AKIRA
MIZUCHI,
AND KANJI
of Internal Medicine, Faculty University
of Tokyo,
Tokyo.
SATO
of Medicine,
Japan
Received March 19, 1976; accepted September 14, 1976 Preparation of rz51-labeled succinyl cyclic GMP-tyrosine methyl ester (YSCGMP-TME) was described. The method utilized lactoperoxidase and the glucose-glucose oxidase peroxide-generating system. 1251-SCGMP-TME, purified by Sephadex G-10 gel filtration, was separated from cyclic GMP. SCGMPTME, rz51, and ‘251-labeled TME and retained extremely high immunoreactivity. A cyclic GMP radioimmunoassay. based on this iodinated material and cyclic GMP antiserum, is specific, sensitive, and reliable, permitting the determination of small quantities of cyclic GMP in rat tissue without need for chromatographic steps.
Cyclic AMP is known as the intracellular mediator of the actions of a number of hormones, while the role of cyclic guanosine 3’ ,5’-monophosphate (cyclic GMP) is still obscure. This is due partly to the relative difficulty in the measurement of extremely low concentrations of cyclic GMP. The competitive binding radioassay utilizing a cyclic nucleotide-binding protein is not as sensitive or specific for cyclic GMP as for cyclic AMP (1). However, the great sensitivity and specificity of the radioimmunoassay method of Steiner et al. (2), which utilizes a specific antibody to cyclic GMP and succinyl cyclic GMP-tyrosine methyl ester (SCGMP-TME), iodinated by a chloramine-T method as a labeled antigen, permits the measurement of cyclic GMP on small quantities of tissue without the need for chromatography. By application of the iodinated 2’Osuccinyl cyclic AMP-tyrosine methyl ester (1251-SCAMP-TME) we could obtain more immunoreactive 12%SCAMP-TME by a lactoperoxidase method (9) than by a chloramine-T method. In this report we shall describe the labeling of SCGMP-TME with radioactive iodide using a lactoperoxidase method. SCGMP-TME, iodinated by this method, retains very high immunoreactivity and is, therefore, suitable for use as a labeled antigen in the cyclic GMP radioimmunoassay. r Present address: Third Department of Internal School of Medicine, Kasumi-cho, Hiroshima, Japan.
Medicine,
Hiroshima
University,
429 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN OCQ3-2697
430
MIYACHI,
MIZUCHI TABLE
PROCEDURE
FOR
IODINATION
AND SAT0 1 OF
SCGbfP-TME
Reactants
Amount
SCGMP-TME 0.4 M Acetate buffer, pH 6.0 Lactoperoxidase Glucose oxidase Nalz51 0.1% Glucose
4 Pis 30pl 100 ng/lO ~1 50 mu/5 ~1 0.5 mCi/S ~1
25/.A
Incubated at 23°C for 10 min Purified on Sephadex G-10 column (0.9
MATERIALS
X
10 cm)
AND METHODS
Production of antisera to Cyclic GMP. 2’-0-Succinyl cyclic GMP (SCGMP) was prepared by using the succinic anhydride method of Falbriard et al. (4). SCGMP was coupled with human serum albumin by the carbodiimide method (2). Rabbits were immunized by multiple intradermal injections of 1 mg of cyclic nucleotides-protein conjugates, emulsified in complete Freund’s adjuvant (5). Booster injections were given 8 weeks after the first immunization and then every 4 to 6 weeks. The blood was collected at appropriate intervals, and serum was stored frozen at -20°C until use. Preparation of iodinated SCGMP-TME. SCGMP-TME was prepared by the carboxylic-carbonic acid reaction (6). SCGMP-TME was iodinated with lz51 by the lactoperoxidase method of Miyachi et al., as described previously (3). Approximately 4 pg of SCGMP-TME in 30 ~1 of 0.4 M sodium acetate buffer, pH. 6.0 (AcB), 100 ng of lactoperoxidase (Sigma) in 10 ~1 of 0.1 M ACB, SO mU/5 ~1 of a solution of glucose oxidase (Sigma), and 0.5 mCi of carrier-free Na1251 (AmershamSearle) were mixed in a glass tube (7.5 x 100 mm). The iodination reaction was initiated by adding 25 ~1 of 0.1% glucose (Table 1). After the reaction proceeded for 10 min, the reaction mixture was transferred to a column of Sephadex G-10 (0.9 x 10 cm), previously washed and equilibrated with 0.05 M AcB and 1 ml of 1% bovine serum albumin. Elution was performed with 0.05 M AcB, and l-ml fractions of eluate were collected. Chromatography of cyclic [3H]GMP and SCGMP-TME. To ascertain the elution volume of unlabeled cyclic GMP and SCGMP-TME from a Sephadex G-10 column, small amounts of cyclic C3H]GMP or SCGMPTME were applied to the column (0.9 x 10 cm). Elution was performed with 0.05 M AcB. The radioactivity of cyclic [3H]GMP in the eluate was determined by liquid scintillation counting and the concentration of
ENZYMATK
RADIOIODINATION TABLE
431
OF CYCLIC GMP
2
RADIOIMMUNOASSAY PROTOCOL FORCYCLIC GMP Amount (PO 0.05 M Acetate buffer, pH 6.0 Standard cyclic GMP (O.Ol- 10 pmol) Or Samples rz51-SCGMP-TME” (5,000-10,000 cpm) Antiserum (1:3O,OOO)
200 100 100 100
Incubated at 4°C for 18 h 1 1 ml of 20% polyethylene glycol J Centrifuge at 3000 rpm for 30 min at 4°C 1 Supematant aspirated J Bound precipitates counted 0 Diluted in 0.05 M acetate buffer, pH 6.0, containing 0.5% bovine y-globulin.
SCGMP-TME assay.
in the eluate was measured by cyclic GMP radioimmuno-
Radioimmunoassay procedure. The protocol of cyclic GMP radioimmunoassay is shown in Table 2. After the mixture was incubated at 4°C for 18 hr, 1 ml of a 20% polyethylene glycol solution was added, and then the mixture was thoroughly stirred. The tubes were centrifuged for 30 min at 3000 rpm, and the precipitates (lz51-SCGMP-TME, bound to antibody) were counted in an Autowell gamma spectrometer. A computer program was used to calculate the standard curve and the potency estimate of cyclic GMP level in each sample (7). Tissue preparation. Frozen tissues (lo-30 mg) from the testes of approximately 2-month-old rats were homogenized at 4°C in 1 ml of cold 6% trichloroacetic acid (TCA). After centrifugation at 25OOxg for 15 min, TCA supernatants were extracted three times with 3 ml of ethyl ether saturated with water. The extracted aqueous phase was evaporated under a stream of Nz gas, and the residue was redissolved in 0.5 ml of 0.05 M AcB. One-hundred-milliliter aliquots were used directly in the cyclic GMP radioimmunoassay. RESULTS
The elution pattern after gel filtration on Sephadex G-10 of the iodinated cyclic nucleotide derivatives as well as their immunoreactivity to antibody
432
MIYACHI,
10
al ELUTION
MIZUCHI
AND SAT0
l!o
40
30 VOLUME
I ml )
FIG. 1. Elution pattern of iodinated cyclic GMP derivatives on a Sephadex G-10 column. Elution was performed with 0.05 M acetate buffer. The immunoreactivity of each fraction was determined using 50$00x-diluted antiserum.
are shown in Fig. 1. Three distinct radioactive peaks, I, II, III, were found, the K,, values of which were 0, 0.743, and 3.144, respectively. Approximately 15% of the 1251was incorporated into peak III. Peaks I and II were not bound by antibody, while the 1: lOO,OOO-diluted cyclic GMP antiserum 15
% - 10 s %
Y t g * s
5
10 ELUllON
20 VOLUME
30
40
f ml )
FIG. 2. Elution pattern of [3H]GMP and SCGMP-TME Elution was performed with 0.05 M acetate buffer.
on a Sephadex G-10 column.
ENZYMATIC
RADIOIODINATION
433
OF CYCLIC GMP
70 -
60 -
k
=-I ‘u 1 1:150.000
\
/f 1: m.l,anl
q\
.,?
50 Cyclic
FIG.
GMP
( pmoles / tube
I
3. Dilution curves of anti-cyclic GMP antiserum (final dilution, 1:50,000- 1:200,000).
bound more than 50% of the fractions of peak III, indicating that this peak is iodinated SCGMP-TME. Peak III or iodinated SCGMP-TME was stable; it remains highly immunoreactive after 3 months of storage at -20°C. Figure 2 shows the elution pattern of cyclic [3H]GMP and SCAMPTME from the same Sephadex G-10 column. The cyclic [3H]GMP was eluted at a K,, of 0.529 and SCGMP-TME at a K,, of 0.734. Although not shown in Fig. 2, free lz51 was eluted in the fractions of peak II, and iodinated tyrosine methyl ester (1251-labeled TME) at a point very close to peak II. These results show that lz51-SCGMP-TME (peak III) was separated from any contamination with cyclic GMP, SCGMP-TME, lz51, and 1251-labeled TME. Specific activity of 1251-SCGMP-TME was greater than 100 Ci/mmol, assuming incorporation of 1 atom of 12Y per molecule of SCGMP-TME. The optimal titer of antibody was determined by testing serial dilutions of antisera with 1251-SCGMP-TME (Fig. 3). More than 65% of 1251-SCGMPTME was bound to 1:50,000-diluted antiserum, and the antiserum, when diluted 1:200,000, bound approximately 35% of lzsI-SCGMP-TME. A dose-response curve was constructed using 1251-SCGMP-TME and diluted antiserum with different doses of unlabeled cyclic GMP (Fig. 3). The sensitivity of this cyclic GMP radioimmunoassay was 0.05 pmol/tube when 1:200,000-diluted antiserum was used (Fig. 3), and the useful range of the dose-response curve was 0.05-10 pmol/tube.
434
FIG.
MIYACHI,
4. Inhibition
MIZUCHI
AND SAT0
curves with various nucleotides in the cyclic GMP radioimmunoassay.
Cross-reactivity of other structually related nucleotides tested was less than 0.01% at 50% displacement (Fig. 4). It is remarkable that this antiserum did not react with cyclic AMP at concentrations up to 5 x 104 pmol (90% displacement). The concentration of cyclic GMP in the rat testes is 17.3 t 1.5 pmol/g of testis. The value is the same range as that reported by Shibuyaet al. (IO). The intra- and interassay variabilities of this assay were approximately 9.2 and 14.1%, respectively. DISCUSSION
We were the first to apply the enzymatic radioiodination procedure using lactoperoxidase and hydrogen peroxide to gonadotropins, and we showed that those radioiodinated gonadotropins retained biologic activities as well as immunologic activities (3). This method was applied to luteinizing hormone (LH)-releasing hormone with substitution of the hydrogen peroxide-generating glucose-glucose oxidase system for hydrogen peroxide, which was presumed to be a more gentle treatment (8). We showed in the present report that the modified lactoperoxidase method was suitable for the radioiodination of SCGMP-TME, yielding a product of a labeled antigen for radioimmunoassay. 125T-SCGMP-TME, which was highly reactive to cyclic GMP antibody, could be separated on a Sephadex G-10 column from cyclic GMP, SCGMP-TME, unreacted 1251,and 1251-labeled TME, any one of which might have interfered in the cyclic GMP radioimmunoassay. i251-SCGMP-
ENZYMATIC
RADIOIODINATION
OF CYCLIC GMP
435
TME with very high immunoreactivity permitted the use of a highly diluted antiserum, allowing a sensitive cyclic GMP radioimmunoassay system. As shown in Fig. 3, the sensitivity of the assay was less than 0.05 pmol when 1:200,000-diluted antiserum was used. Since the antiserum was specific for cyclic GMP, we obtained reasonable estimates of cyclic GMP concentrations in rat tissue without the need for chromatographic separation of cyclic GMP from cyclic AMP and ATP. We also applied this method to the iodination of SCAMP-TME and found that lz51-SCAMP-TME, labeled by this modified lactoperoxidase method, was more immunoreactive to cyclic AMP antibody than that prepared by a chloramine-T method (9). In order to obtain good labeled antigens by a chloramine-T method, it is necessary to establish experimentally and maintain reproducibly the proper conditions, such as the ratio of chloramine-T to the substrate, the reaction time, and the amount of sodium metabisulfite. In contrast, lactoperoxidase-catalyzed iodination is a gentle, simple, and rapid method, allowing one to obtain with great ease iodinated cyclic nucleotide derivatives that are suitable for reproducible radioimmunoassays. REFERENCES 1. Gilman, A. G. (1972) in Advances in Cyclic Nucleotide Research Vol. 2, p. 9, Raven Press, New York. 2. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1972)J. Biol. Chem. 347, 1106- 1113. 3. Miyachi, Y., Vaitukaitis, J. L., Nieschlag, E., and Lipsett, M. B. (1972)J. Clin. Endocrinol. 34, 23-28. 4. Falbriard, J. G., Posternak, T., and Sutherland, E. W. (1967) Biochim. Biophys. Acfa 148, 99- 105. 5. Vaitukaitis, J. L., Robbins, J. B. Nieschlag, E., and Ross, G. T. (1971) J. C/in. Endocrinol. 33, 988-991. 6. Greenstein, J. P., and Winitz, M. A. (l%l) in Chemistry of the Amino Acid Vol. 2, p. 978, Wiley, New York. 7. Rodbard, D., and Hutt, D. M. (1974) In Symposium on Radioimmunoassay and Related Peptides in Medicine, p. 165, International Atomic Energy Agency, Vienna. 8. Miyachi, Y., Chrambach, A., Mecklenburg, R., and Lipsett, M. B. (1973) Endocrinology 92, 1725- 1730. 9. Sato, K., Miyachi, Y., Mizuchi, A., Ohsawa, N., and Kosaka, K., in preparation. 10. Shibuya, Y., Arai, K. and Kajiro, Y. (1975) Biochem. Biophys. Res. Commun. 62, 129.