ANALYTICAL
BIOCHEMISTRY
117,
136-146
(1981)
lodination of Proteins, Glycoproteins, and Peptides Using a Solid-Phase Oxidizing Agent, 1,3,4,6-Tetrachloro-3a,6a-diphenyl Glycoluril (lodogen) PHILIP Pituitary
R. P. SALACINSKI, CHARLES MCLEAN, JANE E. C. VICKY V. CLEMENT-JONES, AND PHILIP J. LOWRY
Hormone
Laboratory, 51-53
Bartholomew
Department Close.
Received
of Chemical London
March
ECIA
Pathology, 7BE.
United
SYKES,
St Bartholomew’s
Hospital,
Kingdom
24, 1981
A new, commercially available oxidizing agent, 1,3,4,6-tetrachloro-3a,6a-diphenyl glycoluril (Iodogen) was compared with chloramine-T and solid-phase Iactoperoxidase in the radioiodination of proteins, glycoproteins, and peptides. A method for performing low-level iodinations is described and was used to determine maximum ‘*‘I incorporation. Iodinated proteins were purified on analytical gel filtration columns and peptides by reverse-phase high-performance liquid chromatography. Both methods were designed to analyze the tracers for the presence of aggregate and breakdown products caused by the iodination. All tracers prepared were tested in antibody dilution and dose-response curves in their respective radioimmunoassays. Results indicate that Iodogen can be used for a wide range of proteins and peptides, can permit theoretical iodine incorporation with minimal oxidation damage, and can produce tracer stable for up to 3 months.
Since the introduction of radioimmunoassay, there has been a need for high specific activity tracers in order to measure the concentration of proteins and peptides in the picomole range. The most commonly used radiolabel is ‘25I and the mechanism of iodination must be considered if adequate preparations of tracers containing this radiolabel are to be made. Between pH 7 and 8, the ortho position in the aromatic ring of tyrosine of peptides and proteins is activated for electrophilic attack, owing to the electron-donating effect of the neighboring hydroxyl group. Similarly, at pH 9, the two nitrogen atoms in the imidazole ring of histidine have an electron-donating effect on their mutual neighboring carbon atom (C2). Iodine, in the form of iodous ion (I+), acts as the electrophilic agent to give the radiolabeled tracer ( 1). Initially, iodine monochloride (2) was used as a direct iodinating agent, but low specific activities resulted from the incorporation of nonradioactive iodine ( 1). Nearly
0003-2697/81/150136-l Copyright All
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1981
by
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inc reserved
I36
all subsequent methods have involved the prior oxidation of sodium iodide to form the iodous ion. Improved incorporation has been achieved using an excess of oxidizing agents such as chloramine-T (3), hydrogen peroxide (4-6), chlorine gas (7), and sodium hypochlorite (8). Oxidation damage, specifically the oxidation of methionine to methionine sulfoxide (9,lO) and tryptophan to the oxindole (1 l), occurs due to the use of excesses of oxidizing agents that come into intimate contact with the protein or peptide in solution. This damage affects the physicochemical integrity of the protein or peptide and results in losses of biological and immunological activity, especially when the tryptophan or methionine residues are involved in the biological or immunological sites of these molecules. The electrolytic method of Pennisi and Rosa ( 12) has not been reported to cause chemical damage, even though the protein or peptide comes into contact with the electrode. However, the method is complex and
IODINATION
OF PROTEINS
AND
time-consuming and thus inappropriate for routine laboratory use. In 1978, Fraker and Speck (13) reported the possibility of using a sparingly soluble chloramine, 1,3,4,6 - tetrachloro - 3a,6a - diphenyl glycoluril (Iodogen) as a solid-phase oxidizing agent for the radioiodination of rabbit immunoglobulin G, chicken lysozyme, and a living cell membrane. Their results indicated the mildness of this reagent, as enzyme activity was preserved and greater than 80% viability of cells was obtained. Similarly, Markwell and Fox ( 18) used the reagent for the surface-specific iodination of membrane proteins of viruses and eucaryotic cells while maintaining viral integrity and cell viability. We have previously described the use of this compound to iodinate human growth hormone and human luteinizing hormone, obtaining theoretical iodine incorporation with minimal chemical damage (14). This paper reports the development of the method for the iodination of a wide range of proteins and peptides and compares it to the solid-phase lactoperoxidase and chloramine-T methods. Also discussed are various purification procedures, including a simple purification scheme which uses reverse-phase high-performance liquid chromatography. MATERIALS
1,3,4,6-Tetrachloro-3a,6a-diphenyl glycoluril (Iodogen) was obtained from Pierce and Warriner (U. K.) Ltd., Chester, England; carrier-free Na’*‘I in NaOH (100 mCi/ml) from the Radiochemical Centre, Amersham, Bucks., England; and iodination vials (No. 690 test tubes) from Starstedt (U. K.) Ltd., Leicester, England. Sephadex G-100 superfine was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden; chromatography columns from Whatman Laboratory Sales Ltd., Maidstone, Kent, England; and absorbant strips from Gelman (Inc.) Ltd., Ann Arbor, Michigan. Syringes were purchased from Becton Dickinson
PEPTIDES
USING
IODOGEN
137
(U. K.) Ltd., Wembley, Middlesex, England; and porous Teflon was obtained from Jones Chromatography Ltd., Wales. Octadecylsilyl silica (ODS)’ (10 pm) was purchased from Water Associates (U. K.) Ltd., Nantwich, Cheshire, England. Lactoperoxidase (Calbiochem Ltd., U. K.) was coupled to the solid support (methyl maleic anhydride copolymer, supplied by BDH Chemical Co. Ltd., Poole, Dorset, England) using the method of Brummer et al. ( 15). Polypep was obtained from Sigma London Chemical Company Ltd., Poole, Dorset, England. All other chemicals were of Analar grade and obtained from BDH Chemicals Ltd. Hormone
and Antibody Preparations
Human growth hormone (hGH), human luteinizing hormone (hLH), human follicle stimulating hormone (hFSH), human thyroid stimulating hormone (hTSH), free (Ysubunit, hLH-a-subunit, human prolactin (hPr), human adrenocorticotrophic hormone (h-ACTH), and human P-lipotrophin (hoLPH) were prepared in our laboratory ( 16,17). Rat growth hormone (rGH) and rat prolactin (rPr) were supplied by the National Institute of Arthritis, Metabolism, and Digestive Disease (NIAMDD), Bethesda, Maryland. Synthetic methionine enkephalin (Met-enk) and leucine enkephalin (Leu-enk), a-endorphin, and /3-endorphin were obtained from Bachem Ltd., Torrance, ’ Abbreviations used: ODS, octadecysilyl silica; hGH, human growth hormone; hLH, human luteinizing hormone; hFSH, human follicle stimulating hormone; hTSH, human thyroid stimulating hormone; hPr, human prolactin; h-ACTH, human adrenocorticotrophic hormone; h@-LPH, human (34ipotrophin; rGH, rat growth hormone; rPr, rat prolactin; NIAMDD, National Institute of Arthritis, Metabolism, and Digestive Disease; Met-enk, methionine enkephalin; Leu-enk, leutine enkephalin; a-MSH, a-melanocyte stimulating hormone; (3-MSH, &melanocyte stimulating hormone; rLHRH, rat luteinizing hormone releasing hormone; BSA, bovine serum albumin; HSA, human serum albumin; TFA, trifluoroacetic acid; HPL, human placental lactogen.
SALACINSKI
138
California; and a-melanocyte stimulating hormone (a-MSH) and @melanocyte stimulating hormone @I-MSH) from Ciba-Geigy Ltd., Horsham, Sussex, England. Arginine vasopressin was purchased from Ferring AB, Malmo, Sweden; rat luteinizing hormone releasing hormone (rLHRH) from Hoechst, Hounslow, Middlesex, England; rat neurophysin from NIAMDD, and porcine vasointestinal peptide from Dr. S. Bloom, Hammersmith Hospital, London, England. Bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin were purchased from Sigma London Chemical Company, Poole, Dorset, England. Antihuman rabbit immunogamma globulin was obtained from the Virology Department, St. Bartholomew’s Hospital, London, England. Antibody for arginine vasopressin was obtained from Dr. Tj. B. van Wimersma Greidanus, Rudolf Magnus Institute, The Netherlands; rLHRH antibody was a gift from Dr. C. H. Mortimer; rat neurophysin antibody from Dr. B. Pickering; and rGH and rPr antibody from NIAMDD. Human ACTH antibody was E4 supplied by Burroughs Wellcome Ltd., Beckenham, Kent, England. All other antibodies were raised in our own laboratories. Donkey antirabbit serum and horse serum (uninactivated) were obtained from Wellcome Reagents Ltd., Beckenham, Kent, England. Goat antibody to monkey gamma globulin and rhesus monkey serum were purchased from Calbiochem Ltd., Bishop Stortford, England. METHODS
Radioiodination Both high- and low-level iodinations were used. High-level iodinations required 1 mCi ( 10 ~1) 50 PM Na’*? for each iodination and were carried out in designated iodination rooms. In contrast, low-level iodinations were performed using a solution of 50 pM NaI to which enough Nalz51 had been added to give 22 nCi in 10 ~1. The use of such lowlevel radioactivity has obvious implications
ET AL.
for safety and, furthermore, allows numerous experiments to be performed at any one time. It is applicable to any of the three iodination methods described below, and the products of the reactions can be investigated in a normal laboratory. Zodogen Method Iodogen (1 mg) was dissolved in dichloromethane (25 ml) and 50 ~1 of this Iodogen solution dispersed in the bottom of a polypropylene iodination vial and evaporated to dryness at room temperature under nitrogen ( 18). This removes the dichloromethane, produces a film of Iodogen, and ensures that the Iodogen does not form a suspension which may give variable iodinations. These dried vials may be stored for up to 6 months at -20°C. Sodium phosphate buffer (0.05 M, 10 ~1, pH 7.4) was added to the iodination vial, followed by Na”‘I (1 mCi, 50 PM, 10 ~1) and 10 pg hGH (0.5 nmol) in 0.05 M sodium phosphate buffer. The iodination was allowed to proceed for 10 min, ensuring that the reactants were in contact with the Iodogen film in the bottom of the vial, and terminated by adding 500 ~1 protein-free 0.05 M sodium phosphate. The diluted mixture was left standing for a further 15 min at room temperature prior to chromatography to allow any unincorporated iodous ions to return to molecular iodine. This precaution prevents iodination of the column buffer. Chloramine- T Method The method used was that of Greenwood et al. (3) but modified by substantially limiting the amount of chloramine-T used in an effort to prevent excessive oxidation. Rat GH ( 10 pg, 0.5 nmol) in 0.05 M sodium phosphate (pH 7.4, 10 ~1) was placed in an iodination vial and 1 mCi carrier-free 50 PM sodium iodide (‘251) in NaOH and 400 PM chloramine-T in Hz0 (20 ~1) were successively added. This reaction was termi-
IODINATION
OF
PROTEINS
nated after 30 s by the addition of 500 sodium metabisulfate in Hz0 (40 ~1). Solid-Phase
AND
pM
The method used was that of Marchalonis (4) modified by the use of solid-phase lactoperoxidase. Human GH (10 ~1, 0.5 nmol) in 0.05 M sodium phosphate (pH 7.4, 10 ~1) was placed in an iodination vial and 1 mCi carrier-free 50 PM sodium iodide (‘251), lactoperoxidase ( 10 ~1) (1 pg/pl), and 50 PM hydrogen peroxide (10 ~1) were added. The reaction was left for 10 min, another 10 ~1 hydrogen peroxide added, and after a further 10 min the reaction was terminated by the addition of 500 ~10.05 M sodium phosphate (pH 7.4). The iodination mixture was centrifuged for 15 min at 15OOg and the supernatant aspirated and purified. of
The final mixture ( 10 ~1) from each of the iodination methods used was spotted onto the origin of absorbant strips (1 x 14 cm) and developed in an ascending manner in trichloroacetic acid ( 10% w/v, 1 ml). The strips were cut into l-cm squares and the radioactivity determined. The protein precipitated at the origin and the free iodine (“‘I) chromatographed with the solvent front. Puri$cation
USING
IODOGEN
139
Fractions (1 ml) were collected and counted on a gamma counter. High-performance liquid chromatography. A tuberculine syringe ( 1 ml) containing
Lactoperoxidase
Rapid Assessment of Incorporation Iodide into Proteins
PEPTIDES
of Tracer
Gel filtration. A column of Sephadex G100 ( 1 X 100 cm) in 0.05 M sodium phosphate was washed with 0.9 M formaldehyde ( 1 bed vol) to remove bacteria (19) followed by 0.05 M sodium phosphate until the eluate was formaldehyde-free as determined by the chromotropic acid test (20). The column was finally equilibrated in 0.05 M sodium phosphate containing BSA (1% w/v). Iodination mixtures were loaded onto the column, which was developed at 3 ml/h.
ODS (10 pm; 15 X 5mm) packed between two porous Teflon disks was equilibrated with an aqueous solution of trifluoroacetic acid (TFA) (1% w/v) and primed with Polypep (1% w/v in 1% TFA, 500 ~1). The column was washed with methanol-H20-TFA (80: 19: 1 v/v) and reequilibrated with TFA (1% v/v) prior to use. The iodination mixture was applied and the column washed with TFA ( 1% v/v) to elute the radioactivity representing unincorporated iodine ( 1.6 ml). The column was developed using 10% stepwise increases in methanol concentration in 1% TFA. The column was washed with each concentration until the radioactivity returned to baseline. The final step was methanol-H20-TFA (80: 19: 1 v/v). Elution was simply performed by using the plunger of the syringe. Drying of the column between each application did not appear to have deleterious effects on the elution profile (2 1). High-pressure
liquid
chromatography.
High-pressure liquid chromatography was performed using a Pye Unicam Ltd. system with a Spherisorb ODS column ( 10 cm X 4.6 mm). A linear gradient from aqueous TFA (1% v/v) to methanol-H,O-TFA (80: 19: 1 v/v) in 20 min at a flow rate of 120 ml/h was used and the eluate monitored on a uv detector at 280 nm. Methionine sulfoxide enkephalin elutes at 33% MeOH and Met-enk at 39% MeOH in this system. The quantity present was calculated by the approximation of each peak area by multiplying the width at half-height by the peak height. Radioimmunoassay. All tracers prepared were tested using excess antibody and in dose-response curves in their respective assays. Human GH, hLH, hFSH, hTSH, hasubunit, h/3-subunit, and hPr were incubated for 16 h at room temperature with antibody and tracer; ACTH, Met-enk, Leu-enk, crMSH, ovalbumin, BSA, HSA, arginine va-
140
SALACINSKI
sopressin, neurophysin, rLHRH, /3-MSH, P-LPH, Lu-endorphin, and @-endorphin were incubated for 24 h at 4°C with antibody alone and then tracer added, followed by a further 24-h incubation at 4°C. Rat GH and rPr were incubated for 3 days at 4°C HPL was incubated for 24 h at room temperature. Precipitation of antibody-bound tracer was achieved using donkey antirabbit serum, except for hGH, ACTH, a-endorphin, ,&endorphin, a-MSH, fi-MSH, ,&LPH, rLHRH, and arginine vasopressin, when 500 ~1 of polyethleneglycol (20% w/v) with 200 ~1 horse serum carrier was used. In the case of rGH, the antibody-bound tracer was precipitated using goat antimonkey gamma globulin serum, and for HPL, precipitation was achieved using methanol (50% v/v). Investigation of Distribution of Iodine and Protein during Iodogen Iodination Distribution of iodine. Low-level jz51 (10 ~1) was added to (i) 12 empty iodination vials, (ii) 12 vials coated with Iodogen (50 ~1 of a 92.5 PM solution in dichloromethane), and (iii) 12 vials coated with Iodogen containing hGH (10 ~1 of a 50 PM solution in 0.05 M sodium phosphate, pH 7.4). After 10 min at room temperature, 0.05 M sodium phosphate (1 ml, pH 7.4) was added to each vial, the mixture transferred to another vial, and the reaction vial, pipette tip, and reaction mixture measured for radioactivity. Distribution of protein, using hGH as a model protein. Human GH (10 ~1 of a 50 PM solution in 0.05 M sodium phosphate pH
7.4) was added to (i) 12 routine radioimmunoassay tubes (Luckhams LP3), (ii) 12 empty iodination vials, (iii) 12 iodination vials coated with Iodogen (50 ~1 of a 92.5 pM solution in dichloromethane), and (iv) 12 iodination vials coated with Iodogen containing sodium iodide (10 ~1 of a 50 PM solution in 0.05 M sodium phosphate, pH 7.4). After 10 min, 0.05 M sodium phosphate (1 ml) containing BSA (0.25% w/v) was added to each vial and radioimmunoassay per-
ET AL.
formed to produce dose-response curves for hGH content for each vial. Storage of Tracer
Fractions chosen after chromatography and containing monomeric tracer were pooled and diluted with solutions of BSA in 20% mannitol to give final concentrations of 1% BSA-10% mannitol, so that 200-~1 samples of diluted tracer contained approximately 10 &i of activity. Samples of “‘1-rGH were stored at 4°C at 4°C in 2% thiodiglycol, at 4°C in 40% methanol (or 40% ethanol), frozen at -20°C or freeze dried. Samples were stored for 8 weeks and then tested for immunogenicity in antibody dilution curves and for percentage binding of tracer in the presence of excess antibody (Table 3). The chromatographic profile of reconstituted stored tracer was also examined by gel filtration on Sephadex G- 100. Neurophysin tracer was prepared, purified on Sephadex G-100 stored at 4°C freeze dried, flash frozen, stored at -20°C stored in 40% methanol at -20°C and stored in 0.05 M sodium phosphate (pH 7.4) at 4°C. Freeze-dried samples were stored at 4°C. After 8 weeks, antibody binding was compared with the freshly prepared tracer. RESULTS AND DISCUSSION
Initially, experiments were performed to establish an ideal iodination procedure for the Iodogen method. Samples of Iodogen ( 10, 20, 30, 40, and 50 ~1) were dispensed into polypropylene vials, made up to 100 ~1, with the further addition of dichloromethane, and allowed to evaporate to dryness at room temperature. Low-level iodinations were performed using hGH ( 10 pIO.5 nmol) in 0.05 M sodium phosphate (pH 7.4). The reactions were allowed to proceed for I5 min at room temperature (20°C). The incorporation at the various concentrations of Iodogen are plotted in Fig. 1. For full
IODINATION
OF
PROTEINS
AND
80 %IZSI 40 20 0 1
2
3
4
lodogen hmoles)
FIG. 1. The incorporation of “‘1 using increasing amounts of Iodogen. Iodinations were performed using I:1 M ratios of human GH (0.5 nmol) and lz51 (0.5 nmol). Incorporation was determined using absorbant strips developed in an ascending manner in 10% trichloroacetic acid.
incorporation, a twofold molar excess of Iodogen over iodine was required. With a twofold molar excess of Iodogen, the minimum time required for maximum iodine incorporation was determined. Figure 2 shows that for hGH, 5 min was the minimum reaction time required for 95% incorporation of the iodine remaining in solution at 20°C. When iodinations were performed at 0 and 37°C the incorporations obtained were 70 and 60%, respectively. Subsequent iodinations were performed at room tem-
%
IZI
PEPTIDES
USING
IODOGEN
141
perature (20°C). It is recommended, therefore, that time-course experiments be performed whenever iodinating a protein or peptide for the first time. Once conditions for maximal iodine incorporation were established, the iodination “damage” caused by the Iodogen method was investigated. To do this, 1 ml of a hGH solution (0.1% w/v) was loaded onto a column of Sephadex G- 100 ( 1 X 100 cm), equilibrated in protein-free 0.05 M sodium phosphate, and the column eluate monitored for absorbance at 280 nm (Fig. 3). Subsequently, 10 ~1 of the original hGH solution was iodinated by the Iodogen method and the reactants diluted to 1 ml with 0.05 M sodium phosphate and chromatographed on the same Sephadex column, this time equilibrated with BSA in 0.05 M sodium phosphate (1% w/v). A comparison of the profiles obtained (Fig. 3) strongly indicates that the Iodogen iodination itself did not cause aggregation or produce breakdown products; rather, when these products were present, they were also iodinated. Distribution of iodine. The distribution of iodine between the Iodogen film and the buffer, in the absence of or in the presence of an iodine acceptor (hGH) (see Methods), is shown in Table 1 and indicates that with the protein acceptor used, less iodinated hormone was lost in the presence of Iodogen
I
o5
10
15
20
Time (minutesl
FIG. 2. The time course of the lodogen reaction to determine maximum ‘*‘I incorporation. Human GH was iodinated with an equimolar amount of sodium “‘1 for various times in the presence of a twofold molar excess of Iodogen. Iodine incorporation was determined using absorbant strips developed in an ascending manner in 10% trichloroacetic acid.
FIG. 3. A comparison of the chromatographic protile on Sephadex G-100 (1 X 100 cm) of hGH (OD 280 nm, -, before iodination) and (cpm, , after iodination) using Iodogen under the standard conditions. The column was eluted in 0.05 M sodium phosphate (pH 7.4) at 3 ml/h; 1 ml fractions were collected.
SALACINSKI
142 TABLE COMPARISON IODINATION
OF THE DISTRIBUTION OF ‘251 DURING BY THREE DIFFERENT METHODS
Iodination vial Uncoated vial lodogen-coated vial Iodogen-coated vial + hGH Lactoperoxidase solid phase Chloramine-T”
I
Pipet tip after transfer
Receptor vial
3
rtl
3+-l
94 *
I
52
t2
3*1
45 -+ 1
16
?I
3k-2
81 *
30 21.5
+I z!z 1
3+1 3*1
61 + I 15 2 1
2
Note: Values are expressed as percentages of the original amount of radioactivity present in 10 ~1 (0.5 nmol) Na’*‘l removed from the supplied stock of iodine and treated as described under methods. y Data from Greenwood et al.
than in the presence of either chloramine-T or lactoperoxidase. Distribution of protein. The distribution of protein, using hGH as an example, in the Iodogen iodinations was calculated by radioimmunoassay (see Methods). In all doseresponse curves examined, 97% ( * 1) of the hGH was found to be present in solution after iodination. Thus, less than 4% of the protein is lost on the vial during iodination. High-level iodinations were performed on rGH and hLH using both chloramine-T and Iodogen as oxidizing agents. The profiles obtained are shown in Fig. 4. For both hormones, iodination using chloramine-T produced large amounts of aggregated material and many breakdown products and did not achieve expected incorporation. The profiles obtained confirmed that rGH and hLH tracers prepared using chloramine-T require extensive repurification and not merely the separation of iodinated protein from free iodine. Stevenson and Spalding (22) showed that the apparent single peak of hLH eluted from Sephadex G-50 contained 50% nonimmunologically reactive material when
ET
AL.
chloramine-T was used as the oxidizing agent. The rGH was iodinated according to the recommended procedure (NIAMDD rat pituitary hormone distribution program sheet), which stresses that, after initial separation on Sephadex G-50 (0.9 X 20 cm), the stored tracer should be repurified by chromatography on a column of Sephadex G-100 ( 1.5 x 50 cm) immediately before use. When this is done, we have confirmed that three peaks of radioactivity are obtained, the first peak representing aggregated hormone, which is poorly immunoreactive, a second peak of highly immunoreactive rGH, and a third peak of smaller but undetermined molecular weight probably representing breakdown products containing iodinated tyrosine and/ or histidine. In our experience, the use of Iodogen for the iodination of both of these hormones has produced radiolabeled homogenous peaks at the expected elution positions of the monomeric hormones as the major product of the iodination. The absence of significant amounts of radioactivity representing aggregate, breakdown products or free iodine confirmed the almost expected incorporation of iodine into representative tracer. Similarly, in our laboratory, h-ACTH tracer has been prepared using chloramineT and the Vycor glass-purification procedure (23). When this tracer was repurified using the new ODS column method, a profile that demonstrated several radioactive peaks was obtained. When tracer was prepared using Iodogen and purified on ODS, a single peak was obtained. These results demonstrate that Iodogen apparently causes less damage than the chloramine-T procedure, the most commonly used oxidizing method. With the solid-phase lactoperoxidase for hGH and rGH, similar purification profiles to those obtained with Iodogen were achieved, but the percentage ‘25I incorporation was much lower. However, when solid-phase lactoperoxidase was used to io-
IODINATION
OF PROTEINS
AND
40
m
PEPTIDES
60
USING
m
143
IODOGEN
40
60
40
60
iii 6ooo
count*
6om
w-1
4ooo
b 6ow 4m
ii ”
Moo
2wO
i20
40
m
60
Fraction number
Fraction number
FIG. 4. Chromatographic profileon Sephadex G-100 of rGH and hLH after iodination with chloramineT and Iodogen. (a) Chloramine-T iodinated rGH; (b) Iodogen iodinated rGH; (c) chloramine-T iodinated hLH; (d) Iodogen iodinated hLH. All iodinations were carried out as described in the text. Sephadex G-100 column was eluted at 3 ml/h in 0.05 M sodium phosphate (pH 7.4); 1 ml fractions were collected. In each case, the desired product monomeric rGH or intact hLH corresponds to peak (iii); peak (i) is aggregated hormone, peak (ii) dimeric hormone, peak (iv) iodinated fragments of (a) rGH or (c) subunits -- of hLH, and peak (v) unincorporated iodine.
dinate the peptide Leu-enk and the iodination mixture purified on ODS (Fig. 5a), only 40% ‘251 incorporation was achieved, and 50% of this material had been polymerized. In contrast, when Iodogen was used as the oxidizing agent, 95% of the ‘25I was incorporated into the peptide (Fig. 5b). The immunogenicity of the prepared tracers was tested using excess antibody. Tracer (100 ~1) and antisera (1: 10, 100 ~1) were added to normal radioimmunoassay tubes (in triplicate), and after 4 h incubation at room temperature, horse serum carrier (200 ~1) and polyethylene glycol (20% w/v, 400 ~1) were added. The tubes were centrifuged at 2500 rpm, the supernatants aspirated, and the remaining radioactivity determined. Percentages of excess antibody binding are shown in Table 2. As can be seen, more than 80% immunogenicity was retained in all cases. All tracers produced dose-response curves equivalent to those obtained using lactoperoxidase tracer. Similarly, when hGH tracer was used prior to purification, a dose-response curve with
maximum binding comparable to that produced using purified tracer was obtained (Fig. 6).
a
L?mo CW”t5 SC -1
iv ii
zccm 1m
iii /I
Co”“ll M -1
FIG. 5. Comparison of “‘1 Leu-enk prepared by (a) lactoperoxidase or (b) Iodogen. Purification was by high-performance liquid chromatography on ODS. In each case, peak (ii) is monoiodinated peptide, and peak (iii) is di-iodinated peptide, confirmed as described in Ref. (21). Peak (i) is unincorporated iodine, and peak (iv) is polymerized product.
144
SALACINSKI
ET AL.
TABLE IMMUNOLOGICAL
Protein
or peptide
AND PHYSICAL
hGH (6) hLH (5) hTSH (6) hFSH (6) h-a-subunit (6) hPr (8) HPL (8) rGH (6) rPr (6) hLH-B-subunit (5) Neurophysin (5) Met-enk (6) Leu-enk (6) h-@-MSH (6) h-P-LPH (6) h-ACTH (6) syn-Porcine-o-MSHd syn-Porcine-oc-endorphi& syn-Porcine-@-endorphind rLHRH (5) Argenine vasopressin Oxytocin (6) h-r-MSH (2-59) (2)
CHARACTERISTICS USING THE IODOCEN Incorporation into protein or peptide” (%I
iodinated
(7) (6) (6) (6)
Note: The majority of proteins and also Table 3) and could still be used nogenicity dropped relatively quickly 95% methionine sulfoxide enkephalin
2 OF PROTEINS METHOD
AND PEPTIDES
IODINATED
Tracer’ 6)
Yield’ (%I
Binding to excess antibody t%)
12
92
66
85
70
75
95 90
66 67
82 85
70 75
86 90
60 67
85 90
75 65
90 92
67 59
95 95
78 70 75
78 70 90
61 49 67
85 89 90
75 70 70
90 95 95
67 66 66
85 95 92
60 65
90 91
54 59
89 90
70 65
88 89
62 58
90 75
65 60 75
90 87 95
59 52 71
78 78 84
75 70 70
95 95 85
71 66 59
85 90 90
peptides iodinated with lodogen had been stored for 2 and 3 months (see in radioimmunoassays. Exceptions were hPr and rPr, both of whose immuupon storage. The iodination of Met-enk by the lodogen method produces as assessed by high-pressure liquid chromatography and amino acid analysis
(21). a These values take into account losses of iodine on the lodogen vial, pipet tip, the receptor vial (see Table I), and unincorporated iodine. They are expressed as a percentage of the original 1 mCi of stock Na’*‘I, and are the sum of lz51 incorporated in all the forms of the protein or peptide recovered after chromatography. * The percentage of a recovered as nonaggregated, nondegraded material which is usable as tracer in the radioimmunoassay. ’ The yield of iodinated tracer is calculated from the expression (a X b)/ 100. ‘syn. synthetic. Numbers in brackets are the numbers of iodinations performed.
Storage of tracer. The ability of both ‘251labeled rGH and neurophysin to bind to their specific antibodies (in excess) was impaired by storage for 8 weeks. Retention of immunogenicity as indicated by binding to antibody is shown in Table 3. Storage at 4°C or at -20°C in the presence of a free radical
acceptor such as methanol was found to preserve immunogenicity better than the other methods investigated. Methanol has two effects. First, it lowers the freezing point of the tracer solution, enabling storage at low temperatures without freezing and thus eliminating damage caused by freezing and
IODINATION
OF PROTEINS
10
100
AND
1000
hGH (rig/ml)
FIG. 6. Dose-response curve of hGH using lodogenprepared tracer before and after purification. Human GH before (. .) gel filtration and after (-) gel filtration; B, antibody binding; &, Nonspecific binding; BT. total antibody binding.
thawing. Also, methanol is a free radical “scavenger” ( 1); therefore, when radioiodine decays to tellurium-125, the free radicals TABLE
3
EFFECT OF STORAGE FOR 8 WEEKS UNDER VARIOUS CONDITIONS ON THE BINDING TO EXCESS ANTIBODY OF TRACERS PREPARED USING THE IOWGEN METHOD Percentage binding to excess antibody after storage for 8 weeks
Storage
conditions
Freeze dried 4°C 4°C in 40% ethanol 4°C in 40% methanol 4°C in 2% thiodiglycol -20°C in 40% methanol -20°C Flash frozen at -20°C in phosphate buffer
Rat growth hormone
Neurophysin
34 48 46 62 44
65 81 n.p.” n.p. n.p.
n.p. 34
90 55
n.p
60
Note; Gel filtration on Sephadex G-100 of tracer rGH stored at 4°C for 8 weeks indicated that 75% of the stored hormone rechromatographed as monomeric rGH. In contrast, only 30% of the hormone stored frozen or in the freeze-dried state rechromatographed as monomer. a np., not performed.
PEPTIDES
USING
IODOGEN
145
released, which are a potential cause of internal damage to the tracer and a cause of iodination catastrophe (24) can be absorbed. Peptide tracers purified by high-performance chromatography are stored in a final concentration of 40% methanol with 0.05 M sodium phosphate (pH 7.4, 0.25% HSA) and Polypep to a final concentration of 0.1% (w/v). Mercaptoethanol to a final concentration of 0.5% (v/v) is added to all peptide tracers except those containing disulfide bridges. Tracer stored in this way was stable for more than 10 weeks. A list of proteins and peptides iodinated using Iodogen is given in Table 2. Human prolactin was anomalous in that an improved elution profile on Sephadex G- 100 similar to that of hGH was obtained using Iodogen, but the tracer did not store for as long as tracer prepared using lactoperoxidase, although the tracer stored longer than that obtained using chloramine-T. Iodogen may cause damage in excess of that damage caused by the oxidizing iodous ion in peptides containing methionine (e.g., Met-enk) and tryptophan residues. However, by using time-course experiments to determine maximum iodine incorporation in a minimum time, tracers of high specific activity, which can be stored for up to 3 months, have been prepared from P-LPH, ol-endorphin, and pendorphin, all of which contain the Met-enk sequence. In conclusion, we have found that Iodogen, used as an oxidizing agent as described, gives higher iodine incorporation with less iodination damage than the other commonly used reagents, chloramine-T and lactoperoxidase, for the majority of the proteins and peptides we have tested. Because the new technique generally causes minimal damage, the rapid separation of the tracer from iodine on small gelfiltration columns (1 X 5 cm) is unnecessary and can be replaced by analytical gel filtration, which produces pure, monomeric protein tracer. Small columns ( 1 X 5 cm) do not
146
SALACINSKI
resolve aggregates and breakdown products from the monomeric material and their use is not recommended. Similarly, a purer peptide tracer has resulted from the use of ODS columns in place of the previously used Vycor glass extraction. The breakdown products eventually formed in the tracer can be easily removed by rechromatography on the ODS column. The improved and simple iodination technique, together with the purification and storage methods adopted, mean that fewer iodinations need to be performed and thus that the radioiodination of proteins and peptides is safer and simpler with Iodogen than with other methods.
ET AL. 10. Alexander, 1952. I I. Shechter, (1975)
1. Bolton, A. E. (1977) Radioiodination Techniques, Review 18, The Radiochemical Centre, Amersham, England. 2. Macfarlane, A. S. (I 958) Nature (London] 182, 53-54. 3. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J. 89, I 14-123. 4. Marchalonis, J. J. (I 969) Biochem. J. 113, 299305. 5. Thorell, J. I., and Johannson, B. G. (197 1) Biochim. 251,
Biophys.
docrinol. Commun.
60,
M.
crinol.
20.
21.
22.
23,
527-528.
M. (1973) 54. 614-621.
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Y.. and Patchornik, A. 14, 4497-4503. U. (1969) J. Nucl. Biol. Med.
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14. Salacinski, P. R., Hope, J., McLean, C.. ClementJones, V. V., Sykes, J., Price, J., and Lowry, P. J. (1979) J. Endocrinol. 81, 131. 15. Brummer, W., Hennrich, N., Klockow, M., Lang, H., and Orth, H. D. (I 972) Eur. J. Biochem. 25, 129-135. 16. Scott, A. P., and Lowry, P. J. ( 1974) Biochem. J. 139, 593-602. 17. Lowry, P. J., McLean, C., Lumley-Jones, R.. and Satgunasingam, N. (1976) J. Clin. Path. 30, Suppl. Assoc. Clin. Path. No. 7, 16-21. 18. Markwell, M. A. K.. and Fox, C. F. (1978) Bio-
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6. Hubbard, A. L., and Cohn, Z. A. (1972) Cell. Bioi. 55, 390-405. 7. Butt, W. R. (1972) J. Endocrinol. 55, 453-454. 8. Redshaw, M. R., and Lynch, S. S. (1974) J. En9. Alexander,
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