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Approaches to radioiodination of insect neuropeptides
Peptides 23 (2002) 2045–2051
Approaches to radioiodination of insect neuropeptides Joe W. Crim a,∗ , Stephen F. Garczynski a , Mark R. Brown b a
Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA b Department of Entomology, University of Georgia, Athens, GA 30602, USA Received 1 April 2002; accepted 18 July 2002
Abstract High quality radioiodinated neuropeptides are essential to radioimmunoassays (RIA) and receptor binding assays. Approaches of direct and indirect labeling of neuropeptides with 125 Iodine (125 I) are compared. An HPLC equipped with an in-line gamma detector and UV absorbance detector was used to evaluate selected labeling methods and products. Treatment of [Y1 ]-adipokinetic hormone-I ([Y1 ]-AKH-I) with chloramine-T caused oxidative damage, whereas enzymatic labeling with lactoperoxidase in the presence of H2 O2 produced a good yield of intact, apparently monoiodinated peptide. Labeling of the FMRFamide-related peptide (YGGFMRFa), with chloramine-T apparently formed the methionine sulfoxide, which subsequently could be reduced with dithiothreitol. Products of high specific activity typically are achievable. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Adipokinetic hormone; Chloramine-T; Insect; Radiolabel; YGGFMRFa
1. Introduction The foundation for successful binding assays with neuropeptides rests on the quality of the labeled ligand used as a trace. For radioimmunoassays (RIA) or receptor assays, ligands most often are labeled with the isotope 125 Iodine (125 I; e.g. [15,16]). A quality radioligand is prepared to high specific radioactivity, is stable, exhibits minimal non-specific binding, and retains robust activity in binding to the antibody or the receptor (see [26]). Radiolabeled ligands that are heterogeneous or low in specific activity undermine assay development and can lead to spurious conclusions (see [15]). An HPLC equipped in tandem with an in-line gamma detector and a UV absorbance detector is a widespread and long-established approach to provide a direct means for purification of radioiodination products and evaluation of reaction methods. Complete separation of reactants and products allows for purification of synthesized products of very high specific activity, a feature that improves RIA sensitivity. For products of demonstrable purity, quantification of radioactivity in turn allows precise specification of the concentration of the monoiodinated form, key information for accurate characterization of receptors based on binding assays. Methods for direct labeling of tyrosine residues in peptides with 125 I typically rely on a few basic approaches, ∗
Corresponding author. Tel.: +1-706-542-8023; fax: +1-706-542-4271. E-mail address: [email protected] (J.W. Crim).
which differ in the vigor of the reaction process. Summaries of protocols have been published (e.g. [3,11]), as have detailed considerations of radioiodination chemistry [7,21,29]. As an overview, it can be noted that stronger oxidation typically involves use of chloramine-T or solid phase synthesis via freshly prepared Iodogen® . Application of chloramine-T to iodinate hormones was pioneered by Hunter and Greenwood [13] 40 years ago. Comparatively gentle oxidation is achieved through enzymatic reactions catalyzed by peroxidase, either in the presence of added H2 O2 or with the incremental production of H2 O2 by glucose oxidase. For particular insect neuropeptides, the preferred method may involve a balance between yield and inherent instability after oxidation. As a rule, as oxidative lability increases ([Y1 ]-AKH-I below), more gentle methods are selected. Similarly, for many neuropeptides containing methionine, formation of the sulfoxide is an undesirable consequence of strong oxidation (see YGGFMRFa below). For some neuropeptides that lack tyrosine, use of Bolton–Hunter reagent for indirect labeling is a productive alternative. Iodination of some insect neuropeptides is uncomplicated. Starting material of high purity yields a predominant, monoiodinated product, that is separable from the unlabeled peptide and any diiodinated product. For such peptides, any of the procedures outlined in the following sections are suitable. Exemplary are select allatostatins of the cockroach, Diploptera punctata. Activities of allatostatin (AST)
0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 1 9 2 - 4
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peptides include inhibition of juvenile hormone secretion, and radiolabeled ASTs have been used for RIA (Dippu-AST 5 [24]; Dippu-AST 7 [30]) and as probes to characterize receptor binding (Dippu-AST 7 [5,30]). Radioiodinations of Dippu-AST 7 by chemical and enzymatic methods produce equivalent HPLC profiles (Crim, Bowser, Tobe, unpublished observations). Isolation of pure apparently monoiodinated AST 7 from the unlabeled peptide or labeled contaminants provides a label of high specific activity; this product markedly increases the sensitivity of the AST RIA (Bowser, personal communication; cf. [26]). The ability to customize a separation scheme for the HPLC depends on the ability to monitor simultaneously both the unlabeled peptide(s) via the UV detector and the radioactive products via the in-line gamma detector. Here, we report the use of such a dual detector system in purifying iodinated neuropeptides. This instrumentation has also allowed us to troubleshoot the labeling of insect peptides which are less amendable to radioiodination. In each case, the ability of the individual products to bind to a dilution series of antibodies raised against the non-iodinated peptides was used to infer the apparent monoiodinated product.
(3) Five microliters 125 I (0.5 mCi). (4) Five microliters chloramine-T (0.5 mg/ml PB, freshly prepared).
2. Adipokinetic hormone (AKH) analogs and oxidative degradation
(5) Forty-five microliters 50% acetic acid.
In insects, AKH act to mobilize lipids and sugars from fat body. The sequences of AKHs and related peptides have been determined for many insects, and these hormones comprise a large family. Quantification of AKH by RIA has been reported [8] for the tobacco hornworm, Manduca sexta. In that study, [Y1 ]-Manduca AKH (YLTFTSSWGa) was radioiodinated with chloramine-T and purified by stepwise elution from C18 Sep–Pak cartridges. Unfortunately, [Y1 ]-Manduca AKH is biologically inactive [31], and therefore unlikely to be useful as a radioiodinated ligand for receptor studies. We have studied the iodination characteristics of [Y1 ]AKH-I from Locusta migratoria, using different approaches for radiolabeling. This peptide, YLNFTPNWGTa, exemplifies the potential problems encountered when AKHs are treated with strongly oxidizing compounds such as chloramine-T or Iodogen® . An outline of the procedures followed with each of these reagents is given below as a general guide for preparing radiolabeled insect neuropeptides for purification by HPLC. The chromatogram obtained for the product resulting from chloramine-T labeling is presented to illustrate the possible consequences of strong oxidation. Chloramine-T protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially: (1) Fifteen microliters phosphate buffer (PB; 0.2 M, pH 7.4). (2) Ten microliters peptide (2–5 g/10 l PB).
Reaction time: 20 s. (5) Thirty-five microliters 50% acetic acid (v/v). Immediately inject reaction mixture onto HPLC and wash reaction vessel twice with 100 l 50% acetic acid for injection. Iodogen® protocol: Into a 12 mm × 75 mm borosilicate tube, pipette: (1) Twenty microliters Iodogen® (50 g/ml acetonitrile, no splashing). Evaporate completely under a gentle stream of dry N2 . To add reactants, pipette sequentially: (2) Twenty microliters PB (0.2 M, pH 7.4). (3) Twenty microliters peptide (2–5 g/20 l PB). (4) Five microliters 125 I (0.5 mCi). Reaction time 4 min, with occasional gentle rotation of the tube.
Immediately inject reaction mixture onto HPLC and wash reaction vessel twice with 100 l 50% acetic acid for injection. Note: All reactions should be conducted in a properly rated fume hood. Carefully avoid aerosol formation during manipulations, and follow all institutional requirements for safe handling of radioactive materials. Sources of reagents are listed in the following sections. The iodination mixture from the chloramine-T reaction was injected onto a C8 reverse phase HPLC column (Vydac 208TP C8 , 300 Å, 5 m, 4.6 mm i.d. × 150 mm), and eluted by a linear gradient with acetonitrile (Fig. 1a). Radioactivity in the eluant was detected by an in-line gamma detector (Beckman Model 170 Radioisotope Detector). Unlabeled [Y1 ]-AKH-I eluted as a major symmetrical peak (Fig. 1a, upper trace). Efficient incorporation of 125 I was indicated by a small peak of free 125 I (Fig. 1a, lower trace), however the resultant radiolabeled products were heterogeneous and the identity of the intact monoiodinated product, if any, was uncertain. To distinguish if heterogeneity was present in the [Y1 ]-AKH-I starting material or resulted from strong oxidation, labeling was conducted enzymatically with lactoperoxidase in the presence of H2 O2 . This approach (cf. [25]) exemplifies more gentle conditions for oxidation, and an outline of the general procedure is detailed. A modified method in which H2 O2 is generated via glucose oxidase also is illustrated. Lactoperoxidase–H2 O2 protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially:
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Fig. 1. Elution profiles of [Y1 ]-AKH-I by reversed phase HPLC following radioiodination. The upper trace represents the UV absorbance (A280 , 0.05 AUFS); the lower trace, radioactivity (104 KCPM full scale); the dashed line, the elution gradient (32–40% B/50 min; B: 80% acetonitrile in 0.1% trifluoroacetic acid; flow rate 1 ml/min); numbers indicate %B read from controller; note that the beginnings of upper and lower traces from the 2-pen chart recorder are offset. (a) Iodination with chloramine-T. Note heterogeneity of labeled products. (b) Iodination with lactoperoxidase in the presence of H2 O2 . Note predominant apparent monoiodinated product, separate from the unlabeled peptide.
(1) (2) (3) (4) (5)
Fifty microliters PB (0.2 M, pH 7.5). Five microliters 125 I (0.5 mCi). Ten microliters peptide (2–5 g/10 l PB). Twenty microliters lactoperoxidase (20 g/ml PB). Ten microliters 0.006% H2 O2 .
Reaction time 10 min; cap with parafilm; occasional gentle rotation of the tube. (6) Ten microliters 0.006% H2 O2 (second addition). Reaction time 10 min; cap with parafilm; occasional gentle rotation of the tube. (7) One hundred microliters 50% acetic acid. Immediately inject reaction mixture onto HPLC and wash reaction vessel three times with 100 l 50% acetic acid for injection. Lactoperoxidase–glucose oxidase protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially: (1) Ten microliters peptide (2–5 g/10 l PB, 0.2 M, pH 7.5). (2) Twenty microliters -d-glucose (10 mg/ml PB). (3) Twenty microliters glucose oxidase (1 g/ml PB). (4) Five microliters 125 I (0.5 mCi).
(5) Twenty microliters lactoperoxidase (20 g/ml PB). Reaction time 1 h; cap with parafilm; occasional gentle rotation of the tube. (7) Seventy-five microliters 50% acetic acid. Immediately inject reaction mixture onto HPLC and wash reaction vessel three times with 100 l 50% acetic acid for injection. For [Y1 ]-AKH-I, the iodination mixture from the lactoperoxidase–H2 O2 reaction was injected onto the HPLC and eluted as before. The unlabeled [Y1 ]-AKH-I exhibited a similar elution time and appearance (Fig. 1b, upper trace) as before. In contrast, the profile of radioactivity was modified (Fig. 1b, lower trace). On the one hand, a larger peak of free 125 I suggested a reduced efficiency of incorporation. A single prominent peak comprising the apparent monoiodinated product was evident, and was wholly separate from the unlabeled peptide. Such positioning of the monoiodinated products relative to the unlabeled peptides reflects increased hydrophobicity and is characteristic of other neuropeptides. When labeled products are adequately separated, automated collection (0.5 ml/fraction) of the eluant in turn allows recovery of the apparent monoiodinated peptide free from unlabeled peptide, assuring very high specific activity (∼2000 Ci/nmol).
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Generation of apparent oxidative breakdown products during iodination of [Y1 ]-AKH-I is problematical, and at least in part may reflect lability of tryptophan residues [1,2]. The breakdown products comprised a significant proportion of the radiolabeled peptides, and their existence was revealed by use of the in-line gamma detector. The degree of heterogeneity likely would have been unrecognized with just counting of aliquots of fractions on a gamma counter. Heterogeneity itself is problematical [15] and in this instance would lead to an erroneous estimate of specific activity, as well as possibly increased non-specific binding. An early study of AKHs iodinated with 127 I reported that the diiodinated Y4 -analog was biologically inactive (activity of the monoiodo-analog was not tested); in contrast, a tritiated Y4 -analog exhibited biological activity comparable to the native peptide [19]. Subsequently, tritium labeled Manduca AKH was used to demonstrate specific, high affinity binding on fat body membranes [32]. With the recent identification of AKH receptors from Drosophila melanogaster and Bombyx mori [23], 125 I labeling of biologically active AKH analogs now is desirable for detailed characterization of receptor binding. Oxidative lability of AKHs suggests that enzymatic iodination may be the preferred approach.
Below, we outline a general procedure which can be applied to any peptide for iodination followed by reduction of the methionine sulfoxide with DTT, and use YGGFMRFa and chloramine-T for illustration. Chromatograms of products following reduction with DTT are compared to those resulting from reaction with chloramine-T alone and from lactoperoxidase–H2 O2 . Dithiothreitol reduction protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially:
3. FMRFamide-related peptides and oxidation of methionine
(7) Two hundred microliters 50% acetic acid.
Methionine, which occurs in many insect neuropeptides, may be oxidized to the sulfoxide under conditions used for radioiodination [20,22,26,27], resulting in a product which may exhibit undesirable characteristics in binding assays. Studies of vertebrate peptides have illustrated the need for caution and have provided both direct and indirect solutions to the problem of methionine oxidation. For the gut hormones gastrin and cholecystokinin (CCK), methionine occurs in the C-terminal tetrapeptide sequence required for biological activity. In an early study of the iodination of gastrin, chloramine-T was found to abolish biological activity, even in the absence of iodide [22], through formation of the sulfoxide. For CCK-8, an indirect labeling procedure (see [18]) yielded a suitable receptor ligand, thus avoiding oxidative alteration of methionines. Similarly, substance P, bombesin, and gastrin releasing peptide (GRP) each have a C-terminal methionine amide vital for biological activity (cf. [26,27]). In an important advance, Vigna et al. [27] developed a convenient method using dithiothreitol (DTT; Cleland’s reagent) for reduction of methionine after oxidative iodination of peptides. FMRFamide-related peptides (FARPs) are widely represented in insects, and other invertebrates. The sequences of FARPs often exhibit N-terminal extensions, and anti-FMRFa sera are directed towards the C-terminus. Accordingly, we found that iodination of YGGFMRFa, a commercially available FARP, provides a useful label for RIA [12,17].
(1) (2) (3) (4)
Fifteen microliters PB (0.2 M, pH 7.4). Ten microliters peptide (2–5 g/10 l PB). Five microliters 125 I (0.5 mCi). Five microliters chloramine-T (0.5 mg/ml PB, freshly prepared).
Reaction time: 20 s. (5) Four hundred and fifteen microliters 1 M ammonium bicarbonate. Transfer to new 12 mm×75 mm borosilicate tube. (6) Fifty microliters 7.25 M dithiothreitol. Reaction time 1 h; 80 ◦ C; cap with aluminum foil. Caution: Cool prior to next addition to prevent bumping.
Immediately inject reaction mixture onto HPLC and wash reaction vessel twice with 200 l 50% acetic acid for injection. For radioiodinations of YGGFMRFa, products of various iodination mixtures were injected separately onto the C8 reverse phase HPLC column, and eluted by a linear gradient of acetonitrile (Fig. 2). Following oxidation with chloramine-T alone (Fig. 2a), unlabeled YGGFMRFa eluted as a small distinctive peak (Fig. 2a, upper trace). The chromatogram of radioactivity exhibited a prominent peak of apparent monoiodinated product (Fig. 2a, lower trace), which was well separated both from the likely diiodinated product and from the unlabeled peptide. Use of DTT to reduce products of the chloramine-T reaction resulted in a different pattern of elutions (Fig. 2b). Unlabeled YGGFMRFa eluted as two distinctive peaks (Fig. 2b, upper trace), one oxidized and a later one reduced, by comparison to the results before. Similarly, the elution profile of radioactive products (Fig. 2b, lower trace) exhibited oxidized and reduced forms of both the major apparent monoiodinated products and of the minor likely diiodinated products. Quantitative analysis of an aliquot of each apparent monoiodinated product indicated concentrations of 29 nM for the oxidized form and 19 nM for the reduced form, suggesting a reduction efficiency of approximately 40% in this instance. Enzymatic iodination of YGGFMRFa with lactoperoxidase in the presence of H2 O2 produced a complex elution profile (Fig. 2c), the components of which could be compared to the earlier results. In this chromatogram, unlabeled
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Fig. 2. Elution profiles of YGGFMRFa by reversed phase HPLC following radioiodination. The upper trace represents the UV absorbance (A280 , 0.05 AUFS); the lower trace, radioactivity (104 KCPM full scale); the dashed line, the elution gradient (0–100% B/80 min; B: 80% acetonitrile in 0.1% trifluoroacetic acid; flow rate 1 ml/min); numbers indicate %B read from controller; note that the beginnings of upper and lower traces from the 2-pen chart recorder are offset. (a) Iodination with chloramine-T. Note elution pattern of oxidized products. (b) Iodination with chloramine-T followed by reduction of products with dithiothreitol. Note oxidized and reduced forms of both labeled and unlabeled peptides. (c) Iodination with lactoperoxidase in the presence of H2 O2 . Note the reduced, apparent monoiodinated product is predominant.
YGGFMRFa appeared as a prominent peak (Fig. 2c, upper trace) with an elution time corresponding to the reduced form (cf. Fig. 2b). Similarly, the elution profile for radioactive products (Fig. 2c, lower trace) exhibited an abundance of the apparent reduced monoiodinated product compared to the oxidized one. These observations suggest that enzymatic oxidation under these conditions is more rapid for iodination of the tyrosyl residue than for sulfoxide formation of the methionine residue. For the FMRFamide RIA, reduced 125 I-YGGFMRFa bound to the antiserum with greater affinity than oxidized 125 I-YGGFMRFa (Brown and Crim, unpublished observations; cf. [26]), and a higher dilution of the antiserum in turn markedly enhanced assay sensitivity. As for bombesin, chemical reduction with DTT [27] and mild enzymatic oxidation [28] both yielded useful products. For tachykinins, metcaptoethanol also has been used for reduction of labeled products [20,26]. For other peptides, the choice of approach may hinge on either oxidative breakdown (see previous sections) or yield, which for some peptides may be relatively low with enzymatic methods [14]. For peptides containing both methionine residues and disulfide bonds, the enzymatic approach may be the only alternative, even if imperfect. Again, the utility of the in-line gamma detector is evident, for optimization of iodination procedures for a particular peptide, as well as for isolation of the demonstrably pure, apparently monoiodinated product.
4. Peptides lacking tyrosine and indirect labeling Many insect neuropeptides lack tryosine. For others, iodination of tryosines in the active site may diminish biological activity unacceptably (cf. [19]). Often, the preferred alternative involves indirect labeling methods [3,11]. The most common of these labels primary amines via an iodinated acylating agent 3-(4-hydroxyphenyl)proprionic acid N-hydroxysuccinimide ester, which forms the Bolton–Hunter reagent [4]. For peptides containing methionine, such as tachykinins and CCK, the use of the Bolton–Hunter reagent has provided a useful alternative [18,26] to oxidative labeling. Even for proteins such as hemoglobin, Bolton–Hunter labeling may reduce damage compared to oxidation with chloramine-T [9]. However, like other animals, many insect neuropeptides have a pyroglutamyl residue at the N-terminal, which would preclude labeling with 125 I-Bolton–Hunter reagent at this location. Recent developments in Bolton–Hunter labeling are worth noting. Gaudriault and Vincent [10] demonstrated conditions for selective labeling of ␣- or ⑀-amino groups of neuropeptides. Such alternative strategies could be pivotal for labeling of peptides with an active site at the N-terminal versus an active site containing a lysine residue. For isolation of the purified 125 I-Bolton–Hunter reagent from HPLC fractions, these authors suggested extraction with toluene [10], a welcome alternative to the use of benzene. In a modification
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expected to increase yield, Miller et al. [18] demonstrated a promising approach in which CCK-8 was dissolved in dimethylformamide containing 2% (v/v) triethylamine prior to addition to the reaction vial containing the 125 I-Bolton–Hunter reagent. In our experience, small volumes of dimethylformamide dissolves peptides not readily soluble in water, and is compatible with all of the methods outlined herein. In addition to having pyroglutamyl residues at the N-terminal, many insect neuropeptides lack tyrosine or lysine, or these residues are positioned in active sites where modification with a bulky halogen or an acylating agent would present insoluble difficulties. In such instances, design of analogs for radioiodination should incorporate known information from structure–activity studies. For RIA or receptor studies, suitable analogs may be synthesized with the addition of tyrosine, d-tyrosine, des-amino-tyrosine, or tyrosine-amide at either the N- or C-terminal (e.g. [6,12]). An ideal analog would exhibit robust biological activity when labeled by a convenient method. Selection of the iodination method, in turn, should involve thorough analysis of the reaction products. Use of reverse phase HPLC with an in-line gamma counter is of evident value in obtaining a homogeneously pure, monoiodinated product of maximum specific activity. For either RIA or for analysis of receptor binding, such a product is the “gold standard.”
5. Sources of reagents 125 I,
carrier free, was from Amersham; synthetic [Y1 ]-AKH-I and YGGFMRFa were from Peninsula Labs; chloramine-T, sodium phosphate (monobasic and dibasic), DTT, -d-glucose, and ammonium bicarbonate were from Sigma; lactoperoxidase and glucose oxidase were from Calbiochem; Iodogen® and TFA were from Pierce; glacial acetic acid and hydrogen peroxide were from Baker; and, Bolton–Hunter reagent and acetonitrile (HPLC grade) were from Aldrich.
Acknowledgments The authors are grateful to Dr. Steven R. Vigna (Duke University) for helpful discussions. Supported by a grant from USDA-CSREES to MRB. References [1] Alexander NM. Oxidation and oxidative cleavage of tryptophanyl peptide bonds during iodination. Biochem Biophys Res Commun 1973;54:614–21. [2] Alexander NM. Oxidative cleavage of tryptophanyl peptide bonds during chemical- and peroxidase-catalyzed iodinations. J Biol Chem 1974;249:1946–52. [3] Bolton AE. Comparative methods for the radiolabeling of peptides. Methods Enzymol 1986;124:18–29.
[4] Bolton AE, Hunter WM. The labelling of proteins to high specific radioactivities by conjugation to a 125 I-containing acylating agent. Biochem J 1973;133:529–39. [5] Bowser PRF, Tobe SS. Partial characterization of a putative allatostatin receptor in the midgut of the cockroach Diploptera punctata. Gen Comp Endocrinol 2000;119:1–10. [6] Brown MR, Crim JW, Arata RC, Cai HN, Chun C, Shen P. Identification of a Drosophila brain–gut peptide related to the neuropeptide Y family. Peptides 1999;20:1035–42. [7] Dent AH, Aslam M. The preparation of protein-small molecule conjugates. In: Aslam M, Dent AH, editors. Bioconjugation. London: Macmillan Reference Ltd.; 1998, p. 364–482. [8] Fox AM, Reynolds SE. Quantification of Manduca adipokinetic hormone in nervous and endocrine tissue by a specific radioimmunoassay. J Insect Physiol 1990;36:683–9. [9] Frantzen F, Heggli D-E, Sundrehagen E. Radiolabelling of human haemoglobin using the 125 I-Bolton–Hunter reagent is superior to oxidative iodination for conservation of the native structure of the labelled protein. Biotechnol Appl Biochem 1995;22:161–7. [10] Gaudriault G, Vincent J-P. Selective labeling of ␣- or ⑀-amino groups in peptides by the Bolton–Hunter reagent. Peptides 1992;13:1187–92. [11] Hermanson GT. Bioconjugate techniques. San Diego, CA: Academic Press; 1996. [12] Huang Y, Brown MR, Lee TD, Crim JW. RF-amide peptides isolated from the midgut of the corn earworm, Helicoverpa zea, resemble pancreatic polypeptide. Insect Biochem Mol Biol 1998;28:345–56. [13] Hunter WM, Greenwood FC. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (London) 1962;194:495–6. [14] Karonen S-L, Mörsky P, Siren M, Seuderling U. An enzymatic solid-phase method for trace iodination of proteins and peptides with 125 iodine. Anal Biochem 1975;67:1–10. [15] Keen M. The problems and pitfalls of radioligand binding. In: Kendall DA, Hill SJ, editors. Methods in molecular biology, vol. 41. Signal transduction protocols. Totowa, NJ: Humana Press; 1995, p. 1–16. [16] Lauffenburger DA, Linderman JJ. Receptors. New York, NY: Oxford University Press; 1993. [17] Matsumoto S, Brown MR, Crim JW, Vigna SR, Lea AO. Isolation and primary structure of neuropeptides from the mosquito, Aedes aegypti, immunoreactive to FMRFamide antiserum. Insect Biochem 1989;19:277–83. [18] Miller LJ, Rosenzweig SA, Jamieson JD. Preparation and characterization of a probe for the cholecystokinin octapeptide receptor N␣ (125 I-desaminotyrosyl) CCK-8, and its interactions with pancreatic acini. J Biol Chem 1981;256:12417–23. [19] Muramoto K, Ramachandran J, Moshitzky P, Applebaum SW. Preparation of a specifically tritiated locust adipokinetic hormone analog with full biological potency. Int J Pept Protein Res 1984;23:443–6. [20] Rissler K, Cramer H, Engelmann P. Application of [125 I]-[Tyr8 ]substance P prepared by the chloramine-T method to receptor-binding experiments after subsequent reduction with mercaptoethanol and purification by reversed-phase liquid chromatography. J Chromatogr 1997;698:17–26. [21] Seevers RH, Counsell RE. Radioiodination techniques for small organic molecules. Chem Rev 1982;82:575–90. [22] Stagg BH, Temperley JM, Rochman H, Morley JS. Iodination and the biological activity of gastrin. Nature (London) 1970;228:58–9. [23] Staubli F, Jørgensen TJD, Cazzamali G, Williamson M, Lenz C, Søndergaard L, et al. Molecular identification of the insect adipokinetic hormone receptors. Proc Natl Acad Sci USA 2002;99:3446–51. [24] Stay B, Bachmann JAS, Stoltzman CA, Sairbairn SE, Yu CG, Tobe SS. Factors affecting allatostatin release in a cockroach (Diploptera punctata): nerve section, juvenile hormone analog and ovary. J Insect Physiol 1994;40:365–72. [25] Thorell JI, Johansson BJ. Enzymatic iodination of polypeptides with 125 I to high specific activity. Biochim Biophys Acta 1971;251:363–9.
J.W. Crim et al. / Peptides 23 (2002) 2045–2051 [26] Too H-P, Maggio JE. Radioimmunoassay of tachykinins. In: Cohn PM, editor. Methods in neurosciences, vol. 6. Neuropeptide technology synthesis, assay, purification, and processing. San Diego, CA: Academic Press; 1991, p. 232–47. [27] Vigna SR, Giraud AS, Reeve Jr JR, Walsh JH. Biological activity of oxidized and reduced iodinated bombesins. Peptides 1988;9:923–6. [28] Westendorf JM, Schonbrunn A. Characterization of bombesin receptors in a rat pituitary cell line. J Biol Chem 1983;558:7527–35. [29] Wilbur DS. Radiohalogenation of proteins: an overview of radionuclides, labeling methods, and reagents for conjugate labeling. Bioconjug Chem 1992;3:433–70.
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[30] Yu CG, Hayes TK, Strey A, Bendena WG, Tobe SS. Identification and partial characterization of receptors for allatostatins in brain and corpora allata of the cockroach Diploptera punctata using a binding assay and photoaffinity labeling. Regul Pept 1995;57: 347–58. [31] Ziegler R, Eckart K, Jasensky RD, Law JH. Structure–activity studies on adipokinetic hormones in Manduca sexta. Arch Insect Biochem Physiol 1991;18:229–37. [32] Ziegler R, Jasensky RD, Horimoto H. Characterization of the adipokinetic hormone receptor from the fat body of Manduca sexta. Regul Pept 1995;57:329–38.
Approaches to radioiodination of insect neuropeptides Joe W. Crim a,∗ , Stephen F. Garczynski a , Mark R. Brown b a
Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA b Department of Entomology, University of Georgia, Athens, GA 30602, USA Received 1 April 2002; accepted 18 July 2002
Abstract High quality radioiodinated neuropeptides are essential to radioimmunoassays (RIA) and receptor binding assays. Approaches of direct and indirect labeling of neuropeptides with 125 Iodine (125 I) are compared. An HPLC equipped with an in-line gamma detector and UV absorbance detector was used to evaluate selected labeling methods and products. Treatment of [Y1 ]-adipokinetic hormone-I ([Y1 ]-AKH-I) with chloramine-T caused oxidative damage, whereas enzymatic labeling with lactoperoxidase in the presence of H2 O2 produced a good yield of intact, apparently monoiodinated peptide. Labeling of the FMRFamide-related peptide (YGGFMRFa), with chloramine-T apparently formed the methionine sulfoxide, which subsequently could be reduced with dithiothreitol. Products of high specific activity typically are achievable. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Adipokinetic hormone; Chloramine-T; Insect; Radiolabel; YGGFMRFa
1. Introduction The foundation for successful binding assays with neuropeptides rests on the quality of the labeled ligand used as a trace. For radioimmunoassays (RIA) or receptor assays, ligands most often are labeled with the isotope 125 Iodine (125 I; e.g. [15,16]). A quality radioligand is prepared to high specific radioactivity, is stable, exhibits minimal non-specific binding, and retains robust activity in binding to the antibody or the receptor (see [26]). Radiolabeled ligands that are heterogeneous or low in specific activity undermine assay development and can lead to spurious conclusions (see [15]). An HPLC equipped in tandem with an in-line gamma detector and a UV absorbance detector is a widespread and long-established approach to provide a direct means for purification of radioiodination products and evaluation of reaction methods. Complete separation of reactants and products allows for purification of synthesized products of very high specific activity, a feature that improves RIA sensitivity. For products of demonstrable purity, quantification of radioactivity in turn allows precise specification of the concentration of the monoiodinated form, key information for accurate characterization of receptors based on binding assays. Methods for direct labeling of tyrosine residues in peptides with 125 I typically rely on a few basic approaches, ∗
Corresponding author. Tel.: +1-706-542-8023; fax: +1-706-542-4271. E-mail address: [email protected] (J.W. Crim).
which differ in the vigor of the reaction process. Summaries of protocols have been published (e.g. [3,11]), as have detailed considerations of radioiodination chemistry [7,21,29]. As an overview, it can be noted that stronger oxidation typically involves use of chloramine-T or solid phase synthesis via freshly prepared Iodogen® . Application of chloramine-T to iodinate hormones was pioneered by Hunter and Greenwood [13] 40 years ago. Comparatively gentle oxidation is achieved through enzymatic reactions catalyzed by peroxidase, either in the presence of added H2 O2 or with the incremental production of H2 O2 by glucose oxidase. For particular insect neuropeptides, the preferred method may involve a balance between yield and inherent instability after oxidation. As a rule, as oxidative lability increases ([Y1 ]-AKH-I below), more gentle methods are selected. Similarly, for many neuropeptides containing methionine, formation of the sulfoxide is an undesirable consequence of strong oxidation (see YGGFMRFa below). For some neuropeptides that lack tyrosine, use of Bolton–Hunter reagent for indirect labeling is a productive alternative. Iodination of some insect neuropeptides is uncomplicated. Starting material of high purity yields a predominant, monoiodinated product, that is separable from the unlabeled peptide and any diiodinated product. For such peptides, any of the procedures outlined in the following sections are suitable. Exemplary are select allatostatins of the cockroach, Diploptera punctata. Activities of allatostatin (AST)
0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 1 9 2 - 4
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peptides include inhibition of juvenile hormone secretion, and radiolabeled ASTs have been used for RIA (Dippu-AST 5 [24]; Dippu-AST 7 [30]) and as probes to characterize receptor binding (Dippu-AST 7 [5,30]). Radioiodinations of Dippu-AST 7 by chemical and enzymatic methods produce equivalent HPLC profiles (Crim, Bowser, Tobe, unpublished observations). Isolation of pure apparently monoiodinated AST 7 from the unlabeled peptide or labeled contaminants provides a label of high specific activity; this product markedly increases the sensitivity of the AST RIA (Bowser, personal communication; cf. [26]). The ability to customize a separation scheme for the HPLC depends on the ability to monitor simultaneously both the unlabeled peptide(s) via the UV detector and the radioactive products via the in-line gamma detector. Here, we report the use of such a dual detector system in purifying iodinated neuropeptides. This instrumentation has also allowed us to troubleshoot the labeling of insect peptides which are less amendable to radioiodination. In each case, the ability of the individual products to bind to a dilution series of antibodies raised against the non-iodinated peptides was used to infer the apparent monoiodinated product.
(3) Five microliters 125 I (0.5 mCi). (4) Five microliters chloramine-T (0.5 mg/ml PB, freshly prepared).
2. Adipokinetic hormone (AKH) analogs and oxidative degradation
(5) Forty-five microliters 50% acetic acid.
In insects, AKH act to mobilize lipids and sugars from fat body. The sequences of AKHs and related peptides have been determined for many insects, and these hormones comprise a large family. Quantification of AKH by RIA has been reported [8] for the tobacco hornworm, Manduca sexta. In that study, [Y1 ]-Manduca AKH (YLTFTSSWGa) was radioiodinated with chloramine-T and purified by stepwise elution from C18 Sep–Pak cartridges. Unfortunately, [Y1 ]-Manduca AKH is biologically inactive [31], and therefore unlikely to be useful as a radioiodinated ligand for receptor studies. We have studied the iodination characteristics of [Y1 ]AKH-I from Locusta migratoria, using different approaches for radiolabeling. This peptide, YLNFTPNWGTa, exemplifies the potential problems encountered when AKHs are treated with strongly oxidizing compounds such as chloramine-T or Iodogen® . An outline of the procedures followed with each of these reagents is given below as a general guide for preparing radiolabeled insect neuropeptides for purification by HPLC. The chromatogram obtained for the product resulting from chloramine-T labeling is presented to illustrate the possible consequences of strong oxidation. Chloramine-T protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially: (1) Fifteen microliters phosphate buffer (PB; 0.2 M, pH 7.4). (2) Ten microliters peptide (2–5 g/10 l PB).
Reaction time: 20 s. (5) Thirty-five microliters 50% acetic acid (v/v). Immediately inject reaction mixture onto HPLC and wash reaction vessel twice with 100 l 50% acetic acid for injection. Iodogen® protocol: Into a 12 mm × 75 mm borosilicate tube, pipette: (1) Twenty microliters Iodogen® (50 g/ml acetonitrile, no splashing). Evaporate completely under a gentle stream of dry N2 . To add reactants, pipette sequentially: (2) Twenty microliters PB (0.2 M, pH 7.4). (3) Twenty microliters peptide (2–5 g/20 l PB). (4) Five microliters 125 I (0.5 mCi). Reaction time 4 min, with occasional gentle rotation of the tube.
Immediately inject reaction mixture onto HPLC and wash reaction vessel twice with 100 l 50% acetic acid for injection. Note: All reactions should be conducted in a properly rated fume hood. Carefully avoid aerosol formation during manipulations, and follow all institutional requirements for safe handling of radioactive materials. Sources of reagents are listed in the following sections. The iodination mixture from the chloramine-T reaction was injected onto a C8 reverse phase HPLC column (Vydac 208TP C8 , 300 Å, 5 m, 4.6 mm i.d. × 150 mm), and eluted by a linear gradient with acetonitrile (Fig. 1a). Radioactivity in the eluant was detected by an in-line gamma detector (Beckman Model 170 Radioisotope Detector). Unlabeled [Y1 ]-AKH-I eluted as a major symmetrical peak (Fig. 1a, upper trace). Efficient incorporation of 125 I was indicated by a small peak of free 125 I (Fig. 1a, lower trace), however the resultant radiolabeled products were heterogeneous and the identity of the intact monoiodinated product, if any, was uncertain. To distinguish if heterogeneity was present in the [Y1 ]-AKH-I starting material or resulted from strong oxidation, labeling was conducted enzymatically with lactoperoxidase in the presence of H2 O2 . This approach (cf. [25]) exemplifies more gentle conditions for oxidation, and an outline of the general procedure is detailed. A modified method in which H2 O2 is generated via glucose oxidase also is illustrated. Lactoperoxidase–H2 O2 protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially:
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Fig. 1. Elution profiles of [Y1 ]-AKH-I by reversed phase HPLC following radioiodination. The upper trace represents the UV absorbance (A280 , 0.05 AUFS); the lower trace, radioactivity (104 KCPM full scale); the dashed line, the elution gradient (32–40% B/50 min; B: 80% acetonitrile in 0.1% trifluoroacetic acid; flow rate 1 ml/min); numbers indicate %B read from controller; note that the beginnings of upper and lower traces from the 2-pen chart recorder are offset. (a) Iodination with chloramine-T. Note heterogeneity of labeled products. (b) Iodination with lactoperoxidase in the presence of H2 O2 . Note predominant apparent monoiodinated product, separate from the unlabeled peptide.
(1) (2) (3) (4) (5)
Fifty microliters PB (0.2 M, pH 7.5). Five microliters 125 I (0.5 mCi). Ten microliters peptide (2–5 g/10 l PB). Twenty microliters lactoperoxidase (20 g/ml PB). Ten microliters 0.006% H2 O2 .
Reaction time 10 min; cap with parafilm; occasional gentle rotation of the tube. (6) Ten microliters 0.006% H2 O2 (second addition). Reaction time 10 min; cap with parafilm; occasional gentle rotation of the tube. (7) One hundred microliters 50% acetic acid. Immediately inject reaction mixture onto HPLC and wash reaction vessel three times with 100 l 50% acetic acid for injection. Lactoperoxidase–glucose oxidase protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially: (1) Ten microliters peptide (2–5 g/10 l PB, 0.2 M, pH 7.5). (2) Twenty microliters -d-glucose (10 mg/ml PB). (3) Twenty microliters glucose oxidase (1 g/ml PB). (4) Five microliters 125 I (0.5 mCi).
(5) Twenty microliters lactoperoxidase (20 g/ml PB). Reaction time 1 h; cap with parafilm; occasional gentle rotation of the tube. (7) Seventy-five microliters 50% acetic acid. Immediately inject reaction mixture onto HPLC and wash reaction vessel three times with 100 l 50% acetic acid for injection. For [Y1 ]-AKH-I, the iodination mixture from the lactoperoxidase–H2 O2 reaction was injected onto the HPLC and eluted as before. The unlabeled [Y1 ]-AKH-I exhibited a similar elution time and appearance (Fig. 1b, upper trace) as before. In contrast, the profile of radioactivity was modified (Fig. 1b, lower trace). On the one hand, a larger peak of free 125 I suggested a reduced efficiency of incorporation. A single prominent peak comprising the apparent monoiodinated product was evident, and was wholly separate from the unlabeled peptide. Such positioning of the monoiodinated products relative to the unlabeled peptides reflects increased hydrophobicity and is characteristic of other neuropeptides. When labeled products are adequately separated, automated collection (0.5 ml/fraction) of the eluant in turn allows recovery of the apparent monoiodinated peptide free from unlabeled peptide, assuring very high specific activity (∼2000 Ci/nmol).
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Generation of apparent oxidative breakdown products during iodination of [Y1 ]-AKH-I is problematical, and at least in part may reflect lability of tryptophan residues [1,2]. The breakdown products comprised a significant proportion of the radiolabeled peptides, and their existence was revealed by use of the in-line gamma detector. The degree of heterogeneity likely would have been unrecognized with just counting of aliquots of fractions on a gamma counter. Heterogeneity itself is problematical [15] and in this instance would lead to an erroneous estimate of specific activity, as well as possibly increased non-specific binding. An early study of AKHs iodinated with 127 I reported that the diiodinated Y4 -analog was biologically inactive (activity of the monoiodo-analog was not tested); in contrast, a tritiated Y4 -analog exhibited biological activity comparable to the native peptide [19]. Subsequently, tritium labeled Manduca AKH was used to demonstrate specific, high affinity binding on fat body membranes [32]. With the recent identification of AKH receptors from Drosophila melanogaster and Bombyx mori [23], 125 I labeling of biologically active AKH analogs now is desirable for detailed characterization of receptor binding. Oxidative lability of AKHs suggests that enzymatic iodination may be the preferred approach.
Below, we outline a general procedure which can be applied to any peptide for iodination followed by reduction of the methionine sulfoxide with DTT, and use YGGFMRFa and chloramine-T for illustration. Chromatograms of products following reduction with DTT are compared to those resulting from reaction with chloramine-T alone and from lactoperoxidase–H2 O2 . Dithiothreitol reduction protocol: Into a 12 mm × 75 mm borosilicate tube, pipette sequentially:
3. FMRFamide-related peptides and oxidation of methionine
(7) Two hundred microliters 50% acetic acid.
Methionine, which occurs in many insect neuropeptides, may be oxidized to the sulfoxide under conditions used for radioiodination [20,22,26,27], resulting in a product which may exhibit undesirable characteristics in binding assays. Studies of vertebrate peptides have illustrated the need for caution and have provided both direct and indirect solutions to the problem of methionine oxidation. For the gut hormones gastrin and cholecystokinin (CCK), methionine occurs in the C-terminal tetrapeptide sequence required for biological activity. In an early study of the iodination of gastrin, chloramine-T was found to abolish biological activity, even in the absence of iodide [22], through formation of the sulfoxide. For CCK-8, an indirect labeling procedure (see [18]) yielded a suitable receptor ligand, thus avoiding oxidative alteration of methionines. Similarly, substance P, bombesin, and gastrin releasing peptide (GRP) each have a C-terminal methionine amide vital for biological activity (cf. [26,27]). In an important advance, Vigna et al. [27] developed a convenient method using dithiothreitol (DTT; Cleland’s reagent) for reduction of methionine after oxidative iodination of peptides. FMRFamide-related peptides (FARPs) are widely represented in insects, and other invertebrates. The sequences of FARPs often exhibit N-terminal extensions, and anti-FMRFa sera are directed towards the C-terminus. Accordingly, we found that iodination of YGGFMRFa, a commercially available FARP, provides a useful label for RIA [12,17].
(1) (2) (3) (4)
Fifteen microliters PB (0.2 M, pH 7.4). Ten microliters peptide (2–5 g/10 l PB). Five microliters 125 I (0.5 mCi). Five microliters chloramine-T (0.5 mg/ml PB, freshly prepared).
Reaction time: 20 s. (5) Four hundred and fifteen microliters 1 M ammonium bicarbonate. Transfer to new 12 mm×75 mm borosilicate tube. (6) Fifty microliters 7.25 M dithiothreitol. Reaction time 1 h; 80 ◦ C; cap with aluminum foil. Caution: Cool prior to next addition to prevent bumping.
Immediately inject reaction mixture onto HPLC and wash reaction vessel twice with 200 l 50% acetic acid for injection. For radioiodinations of YGGFMRFa, products of various iodination mixtures were injected separately onto the C8 reverse phase HPLC column, and eluted by a linear gradient of acetonitrile (Fig. 2). Following oxidation with chloramine-T alone (Fig. 2a), unlabeled YGGFMRFa eluted as a small distinctive peak (Fig. 2a, upper trace). The chromatogram of radioactivity exhibited a prominent peak of apparent monoiodinated product (Fig. 2a, lower trace), which was well separated both from the likely diiodinated product and from the unlabeled peptide. Use of DTT to reduce products of the chloramine-T reaction resulted in a different pattern of elutions (Fig. 2b). Unlabeled YGGFMRFa eluted as two distinctive peaks (Fig. 2b, upper trace), one oxidized and a later one reduced, by comparison to the results before. Similarly, the elution profile of radioactive products (Fig. 2b, lower trace) exhibited oxidized and reduced forms of both the major apparent monoiodinated products and of the minor likely diiodinated products. Quantitative analysis of an aliquot of each apparent monoiodinated product indicated concentrations of 29 nM for the oxidized form and 19 nM for the reduced form, suggesting a reduction efficiency of approximately 40% in this instance. Enzymatic iodination of YGGFMRFa with lactoperoxidase in the presence of H2 O2 produced a complex elution profile (Fig. 2c), the components of which could be compared to the earlier results. In this chromatogram, unlabeled
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Fig. 2. Elution profiles of YGGFMRFa by reversed phase HPLC following radioiodination. The upper trace represents the UV absorbance (A280 , 0.05 AUFS); the lower trace, radioactivity (104 KCPM full scale); the dashed line, the elution gradient (0–100% B/80 min; B: 80% acetonitrile in 0.1% trifluoroacetic acid; flow rate 1 ml/min); numbers indicate %B read from controller; note that the beginnings of upper and lower traces from the 2-pen chart recorder are offset. (a) Iodination with chloramine-T. Note elution pattern of oxidized products. (b) Iodination with chloramine-T followed by reduction of products with dithiothreitol. Note oxidized and reduced forms of both labeled and unlabeled peptides. (c) Iodination with lactoperoxidase in the presence of H2 O2 . Note the reduced, apparent monoiodinated product is predominant.
YGGFMRFa appeared as a prominent peak (Fig. 2c, upper trace) with an elution time corresponding to the reduced form (cf. Fig. 2b). Similarly, the elution profile for radioactive products (Fig. 2c, lower trace) exhibited an abundance of the apparent reduced monoiodinated product compared to the oxidized one. These observations suggest that enzymatic oxidation under these conditions is more rapid for iodination of the tyrosyl residue than for sulfoxide formation of the methionine residue. For the FMRFamide RIA, reduced 125 I-YGGFMRFa bound to the antiserum with greater affinity than oxidized 125 I-YGGFMRFa (Brown and Crim, unpublished observations; cf. [26]), and a higher dilution of the antiserum in turn markedly enhanced assay sensitivity. As for bombesin, chemical reduction with DTT [27] and mild enzymatic oxidation [28] both yielded useful products. For tachykinins, metcaptoethanol also has been used for reduction of labeled products [20,26]. For other peptides, the choice of approach may hinge on either oxidative breakdown (see previous sections) or yield, which for some peptides may be relatively low with enzymatic methods [14]. For peptides containing both methionine residues and disulfide bonds, the enzymatic approach may be the only alternative, even if imperfect. Again, the utility of the in-line gamma detector is evident, for optimization of iodination procedures for a particular peptide, as well as for isolation of the demonstrably pure, apparently monoiodinated product.
4. Peptides lacking tyrosine and indirect labeling Many insect neuropeptides lack tryosine. For others, iodination of tryosines in the active site may diminish biological activity unacceptably (cf. [19]). Often, the preferred alternative involves indirect labeling methods [3,11]. The most common of these labels primary amines via an iodinated acylating agent 3-(4-hydroxyphenyl)proprionic acid N-hydroxysuccinimide ester, which forms the Bolton–Hunter reagent [4]. For peptides containing methionine, such as tachykinins and CCK, the use of the Bolton–Hunter reagent has provided a useful alternative [18,26] to oxidative labeling. Even for proteins such as hemoglobin, Bolton–Hunter labeling may reduce damage compared to oxidation with chloramine-T [9]. However, like other animals, many insect neuropeptides have a pyroglutamyl residue at the N-terminal, which would preclude labeling with 125 I-Bolton–Hunter reagent at this location. Recent developments in Bolton–Hunter labeling are worth noting. Gaudriault and Vincent [10] demonstrated conditions for selective labeling of ␣- or ⑀-amino groups of neuropeptides. Such alternative strategies could be pivotal for labeling of peptides with an active site at the N-terminal versus an active site containing a lysine residue. For isolation of the purified 125 I-Bolton–Hunter reagent from HPLC fractions, these authors suggested extraction with toluene [10], a welcome alternative to the use of benzene. In a modification
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expected to increase yield, Miller et al. [18] demonstrated a promising approach in which CCK-8 was dissolved in dimethylformamide containing 2% (v/v) triethylamine prior to addition to the reaction vial containing the 125 I-Bolton–Hunter reagent. In our experience, small volumes of dimethylformamide dissolves peptides not readily soluble in water, and is compatible with all of the methods outlined herein. In addition to having pyroglutamyl residues at the N-terminal, many insect neuropeptides lack tyrosine or lysine, or these residues are positioned in active sites where modification with a bulky halogen or an acylating agent would present insoluble difficulties. In such instances, design of analogs for radioiodination should incorporate known information from structure–activity studies. For RIA or receptor studies, suitable analogs may be synthesized with the addition of tyrosine, d-tyrosine, des-amino-tyrosine, or tyrosine-amide at either the N- or C-terminal (e.g. [6,12]). An ideal analog would exhibit robust biological activity when labeled by a convenient method. Selection of the iodination method, in turn, should involve thorough analysis of the reaction products. Use of reverse phase HPLC with an in-line gamma counter is of evident value in obtaining a homogeneously pure, monoiodinated product of maximum specific activity. For either RIA or for analysis of receptor binding, such a product is the “gold standard.”
5. Sources of reagents 125 I,
carrier free, was from Amersham; synthetic [Y1 ]-AKH-I and YGGFMRFa were from Peninsula Labs; chloramine-T, sodium phosphate (monobasic and dibasic), DTT, -d-glucose, and ammonium bicarbonate were from Sigma; lactoperoxidase and glucose oxidase were from Calbiochem; Iodogen® and TFA were from Pierce; glacial acetic acid and hydrogen peroxide were from Baker; and, Bolton–Hunter reagent and acetonitrile (HPLC grade) were from Aldrich.
Acknowledgments The authors are grateful to Dr. Steven R. Vigna (Duke University) for helpful discussions. Supported by a grant from USDA-CSREES to MRB. References [1] Alexander NM. Oxidation and oxidative cleavage of tryptophanyl peptide bonds during iodination. Biochem Biophys Res Commun 1973;54:614–21. [2] Alexander NM. Oxidative cleavage of tryptophanyl peptide bonds during chemical- and peroxidase-catalyzed iodinations. J Biol Chem 1974;249:1946–52. [3] Bolton AE. Comparative methods for the radiolabeling of peptides. Methods Enzymol 1986;124:18–29.
[4] Bolton AE, Hunter WM. The labelling of proteins to high specific radioactivities by conjugation to a 125 I-containing acylating agent. Biochem J 1973;133:529–39. [5] Bowser PRF, Tobe SS. Partial characterization of a putative allatostatin receptor in the midgut of the cockroach Diploptera punctata. Gen Comp Endocrinol 2000;119:1–10. [6] Brown MR, Crim JW, Arata RC, Cai HN, Chun C, Shen P. Identification of a Drosophila brain–gut peptide related to the neuropeptide Y family. Peptides 1999;20:1035–42. [7] Dent AH, Aslam M. The preparation of protein-small molecule conjugates. In: Aslam M, Dent AH, editors. Bioconjugation. London: Macmillan Reference Ltd.; 1998, p. 364–482. [8] Fox AM, Reynolds SE. Quantification of Manduca adipokinetic hormone in nervous and endocrine tissue by a specific radioimmunoassay. J Insect Physiol 1990;36:683–9. [9] Frantzen F, Heggli D-E, Sundrehagen E. Radiolabelling of human haemoglobin using the 125 I-Bolton–Hunter reagent is superior to oxidative iodination for conservation of the native structure of the labelled protein. Biotechnol Appl Biochem 1995;22:161–7. [10] Gaudriault G, Vincent J-P. Selective labeling of ␣- or ⑀-amino groups in peptides by the Bolton–Hunter reagent. Peptides 1992;13:1187–92. [11] Hermanson GT. Bioconjugate techniques. San Diego, CA: Academic Press; 1996. [12] Huang Y, Brown MR, Lee TD, Crim JW. RF-amide peptides isolated from the midgut of the corn earworm, Helicoverpa zea, resemble pancreatic polypeptide. Insect Biochem Mol Biol 1998;28:345–56. [13] Hunter WM, Greenwood FC. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (London) 1962;194:495–6. [14] Karonen S-L, Mörsky P, Siren M, Seuderling U. An enzymatic solid-phase method for trace iodination of proteins and peptides with 125 iodine. Anal Biochem 1975;67:1–10. [15] Keen M. The problems and pitfalls of radioligand binding. In: Kendall DA, Hill SJ, editors. Methods in molecular biology, vol. 41. Signal transduction protocols. Totowa, NJ: Humana Press; 1995, p. 1–16. [16] Lauffenburger DA, Linderman JJ. Receptors. New York, NY: Oxford University Press; 1993. [17] Matsumoto S, Brown MR, Crim JW, Vigna SR, Lea AO. Isolation and primary structure of neuropeptides from the mosquito, Aedes aegypti, immunoreactive to FMRFamide antiserum. Insect Biochem 1989;19:277–83. [18] Miller LJ, Rosenzweig SA, Jamieson JD. Preparation and characterization of a probe for the cholecystokinin octapeptide receptor N␣ (125 I-desaminotyrosyl) CCK-8, and its interactions with pancreatic acini. J Biol Chem 1981;256:12417–23. [19] Muramoto K, Ramachandran J, Moshitzky P, Applebaum SW. Preparation of a specifically tritiated locust adipokinetic hormone analog with full biological potency. Int J Pept Protein Res 1984;23:443–6. [20] Rissler K, Cramer H, Engelmann P. Application of [125 I]-[Tyr8 ]substance P prepared by the chloramine-T method to receptor-binding experiments after subsequent reduction with mercaptoethanol and purification by reversed-phase liquid chromatography. J Chromatogr 1997;698:17–26. [21] Seevers RH, Counsell RE. Radioiodination techniques for small organic molecules. Chem Rev 1982;82:575–90. [22] Stagg BH, Temperley JM, Rochman H, Morley JS. Iodination and the biological activity of gastrin. Nature (London) 1970;228:58–9. [23] Staubli F, Jørgensen TJD, Cazzamali G, Williamson M, Lenz C, Søndergaard L, et al. Molecular identification of the insect adipokinetic hormone receptors. Proc Natl Acad Sci USA 2002;99:3446–51. [24] Stay B, Bachmann JAS, Stoltzman CA, Sairbairn SE, Yu CG, Tobe SS. Factors affecting allatostatin release in a cockroach (Diploptera punctata): nerve section, juvenile hormone analog and ovary. J Insect Physiol 1994;40:365–72. [25] Thorell JI, Johansson BJ. Enzymatic iodination of polypeptides with 125 I to high specific activity. Biochim Biophys Acta 1971;251:363–9.
J.W. Crim et al. / Peptides 23 (2002) 2045–2051 [26] Too H-P, Maggio JE. Radioimmunoassay of tachykinins. In: Cohn PM, editor. Methods in neurosciences, vol. 6. Neuropeptide technology synthesis, assay, purification, and processing. San Diego, CA: Academic Press; 1991, p. 232–47. [27] Vigna SR, Giraud AS, Reeve Jr JR, Walsh JH. Biological activity of oxidized and reduced iodinated bombesins. Peptides 1988;9:923–6. [28] Westendorf JM, Schonbrunn A. Characterization of bombesin receptors in a rat pituitary cell line. J Biol Chem 1983;558:7527–35. [29] Wilbur DS. Radiohalogenation of proteins: an overview of radionuclides, labeling methods, and reagents for conjugate labeling. Bioconjug Chem 1992;3:433–70.
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[30] Yu CG, Hayes TK, Strey A, Bendena WG, Tobe SS. Identification and partial characterization of receptors for allatostatins in brain and corpora allata of the cockroach Diploptera punctata using a binding assay and photoaffinity labeling. Regul Pept 1995;57: 347–58. [31] Ziegler R, Eckart K, Jasensky RD, Law JH. Structure–activity studies on adipokinetic hormones in Manduca sexta. Arch Insect Biochem Physiol 1991;18:229–37. [32] Ziegler R, Jasensky RD, Horimoto H. Characterization of the adipokinetic hormone receptor from the fat body of Manduca sexta. Regul Pept 1995;57:329–38.