Chloramine labelling with

Labelling with 1 was carried out following a procedure similar to that for 2 1. However, to evaluate the yield and radiochemical purity of the labelled peptide as a function of the reaction parameters, the amounts of DOTATATE and chloramine T were varied. Briefly, 10-15 pL of Na I in O.OIM NaOH (pH9, 3.8 GBq/mL of 1) was added to 5 pL of aqueous solution of peptide (Ipg/pL) in 10 pL of 0.5M phosphate buffer at pH7.5. To this mixture was added 10 pL of freshly prepared solution of chloramine T (0.13-60 pg/pL), followed by vortexing [16.4]. After 1 min of reaction time at room temperature, the labelled peptide was isolated from the reaction mixture by SepPak purification or by reversed phase HPLC. 

GAM-Fab is labeled with 125I by the chloramine-T method (a standard procedure used to radiolabel the secondary antibody for in vitro assays) (60). 

Antimyosin Fab was labeled with 125I and 123I by the Iodogen iodination method (61) because of its gentler nature of compared to chloramine T. Free and AM-bound radioactivity were separated as described above. 

N-hydroxysuccinimide ester )but not by the chloramine-T or lactoperoxidase procedures (24, 25). This observation suggested to us that VPg does not contain accessible Tyr residues, (iii) VPg-RNA can be labeled with ( h)tyrosine 5 vivo. Digestion of ( tyrosyl-5fi)VPg-pUp with Pronase yielded (tyrosyl-5H)Y with almost no loss of radioactivity (24). These results.are at odds with the iodination data. It is known, that 0 - derivatized tyrosine residues cannot be iodinated by the chloramine-T procedure (26). Failure to iodinate YPg by using chloramine-T or lactoperoxidase can be explained if one assumes that "VPg contains only one t osine residue whose phenolic group is blocked, (iv) Digestion of ( P ) TPg-pUp with 5.6 M HC1 at IIO C for 2 hr yields (52p o4-(3t phospho-51-uridylyl)tyrosine (Tyr- 4 pUp) whose structure was identified by degradation with enzymes and thin-layer chromatography (24). 

Due to the ease of handling and efficiency, the Chloramine-T method is still the most widely used radioiodination procedure and represents an effective way to label a variety of proteins and peptides such as albumins, globulins, neuropeptides, and chemokines. Several other methods, such as the enzymatically catalyzed iodination with lactoperoxidase, have been developed. These methods allow the labeling with mild reaction conditions. In the case of extreme oxidation, sensitive proteins such as pituitary hormones and reactive pre-iodinated compounds such as the Bolton and Hunter reagent can be used to incorporate radioiodine. 

Live worms incubated with, 26l-labelled reagents (Chloramine T, Bolton-Hunter, lodogen) to label proteins on surface... 

An aqueous solution of di(triethylamonium) dodecahydro-ctoio-dodecaborate(2-), (EtjNH)2Bl2H12, (10 pi, lmg/ml) and an astatine solution in methanol (5 pi) were added to 30 pi of buffer solution (pH 7.5). Labelling was started by addition of 10 pi of solution of the sodium salt of A chloro-p-toluenesulfonamide, Chloramine T, (5 mg/ml in water). The mixture was vortexed for a few seconds and left to react for 5 min. The reaction was quenched with 20 pi of solution of sodium metabisulfite, Na Oj, (4 mg/ml in water). Sodium iodide, Nal, (5 pi, 20 mg/ml in water) was added as carrier before analysis. 

The Bolton-Hunter reagent is an I-labeled acylating agent prepared by the chloramine-T method that reacts spontaneously and under mild conditions primarily with lysine residues. Use of this reagent supple-... 

In the other method, I-labeled aniline was prepared by the chlora-mine-T method, diazotized, and allowed to react with the protein under basic conditions (Fig. 1). Presumably it couples to the phenol moiety of tyrosine residues. If this is the case, the product is analogous to that obtained by direct iodination by the chloramine-T or lactoperoxidase procedures. Papain and certain plant lectins in which cysteine residues are part of the active site were labeled without loss of activity. When variations of the chloramine-T or lactoperoxidase methods were used, 64-83% of the activity was lost. 

Procedure. To the solution of aniline in a 3-ml tapered glass tube add the Na I followed by chloramine-T. Agitate the tube for 1 min then adjust the pH to 11 with NaOH (indicator paper). Extract the labeled aniline with three 1-ml portions of chloroform and evaporate the combined chloroform extracts to dryness under a stream of dry nitrogen. The residual product is dissolved in 100 /aI of 0.1 M HCl. 

Fig. 2. Gel filtration profile of I-labeled human prolactin, [ I]hPRL (VLS No. 3) io-dinated by the chloramine-T method and purified on a 1.5 x 30 cm Sephadex G-100 column, precoated with bovine serum albumin. Elution solvent was 10 mM phosphate buffer, pH 7.5, containing 0.15 M NaCl and 1 10,000 Merthiolate. The labeled hormone was purified just prior to use. The talc-resin-trichloroacetic acid (TCA) test results showed that peak III material was suitable for use in radioimmunoassay (Tower et al. ).

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