Radioiodination peptides

The processes of both seed formation and fibril extension are dependent on temperature and on peptide concentration, with 37°C being required for establishing equilibrium within 24 h with 30 pM Pi 4o- A full description of the assay system may be found elsewhere [97,117], A 4 h reaction time is typically within the linear portion of the time course. This nucleus-dependent assay detects mainly inhibitors that are substoichiometric with the monomeric peptide, which is present at high concentration. It is relatively insensitive to inhibitors that target the monomeric peptide. Whether the inhibitors interact with the growing end of a seed or with a low abundance conformational form of the p peptide that is competent to add to the seed is difficult to determine at this time. Similar dose-response curves are obtained for Congo Red as an inhibitor with either thioflavin T (ThT) fluorescence or filtration of radioiodinated peptide readouts (Fig. 4) Caveats in the interpretation of both the ThT and radiometric filtration assays for the evaluation of putative inhibitors are discussed elsewhere [97]. 

The following protocol describes the use of Iodogen for the radioiodination of proteins and peptides. 

Several applications of photoreactive peptides require the presence of a radionuclide to allow specific and sensitive detection of the photo-cross-linked conjugates. In several cases, radioiodination of tyrosyl moieties and radiolabeled Bolton-Hunter reagents have been used. However, the presence of a radiolabel within the benzophenone photophore is desirable, particularly when the objective is to identify the site of photo-insertion of benzophenone. To this end some radiolabeled, benzophenone-based compounds have been developed and used in peptide synthesis, in particular tritiated Phe(4-Bz) (Scheme 24)J2161 [1-14C-carboxy]-4-benzoylbenzoic acid,1221 and 4-benzoyl-(2,3-3H2)-dihydrocinnamic acid.[154l In addition, 4-(4-hydroxybenzoyl)phenylalanine (Scheme 25) has been directly radioiodinated with Na125I and Chloramine-T)151 ... 

The labelling of TATE (AnaSpec) and DOTATATE (piCHEM R D) with [ T]NaI (Nordion) was optimized using the chloramine T method [3.1]. A solution of 10 jxg of peptide in 40 pL of phosphate buffer solution (PBS) (O.IM, pH7.5) was transferred to a reaction vial. After addition of the chloramine T solution (5 jxg/5 LxL) and 5-10 pL of radioiodine solution (37-111 MBq), the vial was carefully vortexed and the reaction was allowed to proceed for 1-3 min at room temperature. The reaction was terminated by addition of the sodium metabisulfite solution (10 pg/5 pL). Studies were carried out by varying the molar ratios of the peptide, the DOTATATE and the radionuclide. Labelling procedures with high activity [ I]NaI were also evaluated employing 1110 and 2775 MBq (30 and 75 mCi), 30 and 100 pg of the peptide, 50 and 100 pg of chloramine T and metabisulfite, respectively. The stability of these preparations was evaluated for 48 h. 

The labelling condition using a molar peptide to radionuclide ratio of 2.73 (7.4 MBq/pg of peptide) resulted in one radiochemical species (R = 22.7 min), probably the monoiodinated species (Fig. 3.1(a)). Under this labelling condition, high radiochemical purity (95.53 0.88%) was observed. After the SepPak purification procedure, the radiochemical purity of the ethanol fraction was found to be 99.32 0.09%. When a molar peptide to radionuclide ratio of 0.54 was used, a second radiochemical species R = 24.3 min) was also observed (Fig. 3.1(b)), which could be related to the diiodinated species in the radioiodination of [Tyr Joctreotide, as described by Bakker et al. [3.11]. With... 

Stability was assessed by incubating 5 pL (400 pg/mL) of the radioiodinated ior-CEAl-(scFv)2 in 500 mL of healthy donor human serum or PBS at 37°C over a 3 d period. At the designated time intervals (12, 24, 48 and 72 h), the samples were analysed by FPLC. The stabihty studies for preparations obtained by labelhng 10 pg of peptide with 1.48 MBq (40 pCi) of 4 and were carried out under similar conditions. 

The radioiodination yield was 90-95% for both peptides. Both were purified using reversed phase chromatography to remove the hydrophilic... 

A key factor in the apphcation of targeted radiotherapy is the need to maximize the tumour to normal ceU radiation dose ratio. In this study, a new series of peptides — including DOTA-Ahx-Oct (OCT), DOTA-Ahx-Ser-Val-Glu-Phe-Ala-Ahx-Oct (P3) and DOTA-Ahx-Gly-Ser-Val-Glu-Phe-Ahx-Oct (P4), where Ahx is epsilon amino hexyl — developed by Whetstone and Meares of the University of California at Davis, United States of America, were evaluated. These peptides include an additional 5 amino acid sequence, which is cleavable by cathepsin. This modification helps to improve the release and trapping of labelled catabolites within the cell [16.2]. These peptides were directly compared with radioiodinated glycated octreotate (Gluc-TOCA), which was shown to have the best internalization properties of the four peptides studied. A comparison of the binding capacity, internalization, exter-nalization and stability of each peptide was carried out under optimized conditions in order to determine their properties. 

Internalization and externahzation were determined as a function of time. Paired assays with each of the peptides labelled with Lu and radioiodinated ( I or i25i) Gluc-TOCA as a reference were done in order to provide a common point of comparison. 

The biological uptake pattern of the radioiodinated peptide indicates urinary as well as gastrointestinal excretion. In vivo metabolization is evidenced by the thyroid uptake (see Table 16.2), which reached 10.2 8.5% of the injected dose at 24 h. In the case of the Lu labelled peptide, the elimination is mainly through urinary excretion. Dose estimates for the radioiodinated peptide are shown in Table 16.4. 

The earliest somatomedin antiserum reported was that of Reber and Liske (R2), for which the antigen was a preparation of NSILAs judged to be more than 90% pure. A RIA using this antiserum, with radioiodinated NSILAs as tracer, was sensitive to about 30 pg of NSILAs per tube. The assay gave much lower values for specimens from healthy humans than those obtained by other methods (see Section 6), and it was unclear which peptides were being measured, as specificity data were not presented. 

Laburthe, M., Chenut, B., Rouyer-Fessard, C., Tatemoto, K., Couvineau, A., Servin, A. Amiranoff, B. (1986) Interaction of peptide YY with rat intestinal epithelial plasma membranes binding of the radioiodinated peptide. Endocrinology 118, 1910-1917. 

Figure 33 An early glycan photoaffinity probe. This moiecuie was used to map gaiactose-binding sites on the asialoglycoprotein receptor. The probe was prepared from a desialylated fetuin glycopeptide galactose residues were modified with aryl azides (shown in red) and the peptide was labeled by radioiodination (shown in blue).

The chemistry of biomolecule radioiodination has developed greatly over the past few years. The choice of the labehng reaction depends of the size of the peptide, its physicochemical and biological properties, as well as the presence of reactive groups. Two techniques are possible, depending on direct or indirect labehng, by a prosthetic group (Wilbur, 1992). 

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