Bolton and Hunter reagent

If the antibody is immobilised on Sepharose , the supernatant containing the free, radioactive peptide can be separated easily and assayed in a gamma counter. With a standard curve drawn for known amounts of peptide subjected to assay under exactly the same conditions, unknown amounts of peptide can be determined by interpolation on the standard curve. There are two potential problems with this type of radioimmunoassay. First, the peptide to be assayed perhaps does not contain Tyr. If it contains His, however, this may suffice since His can be iodinated, especially by an enzymic procedure described below. Alternatively, the peptide is allowed to react with the Bolton and Hunter reagent (Bolton and Hunter, 1973), prepared by iodina-tion of the ester of 3-(4 -hydroxyphenyl)propionic acid and /V-hydroxysuccinimide. Any free amino group can be acylated by this reagent. Secondly, reaction of a peptide with Nal and chloramine-T can cause oxidation of Met, Cys and even Tyr residues, which can interfere with complexation of the iodinated peptide with antibodies raised to the un-iodinated peptide. An alternative method (Holohan et al., 1973) of iodination uses lactoperoxidase in the presence of H202. As pointed out above, this procedure is applicable to the iodination of His residues. This method avoids modification of the side-chains of Met, Cys and Tyr. 

The most common method of radiolabeling employs Chloramine-T, the sodium salt of the N-monochloro derivative of p-toluenesulfonamide (Fig. 17). It breaks down slowly in aqueous solution to hypochlorous acid and is used as a mild oxidizing agent in radioiodination reactions. Other oxidation reagents used in radioiodination include l,3,4,6-tetrachloro-3a,6a-diphenylglycoluril (known commercially as lodo-gen. Fig. 17) or Bolton and Hunter reagent (104). 

Both, Y and Y can be cleaved to pUp (and unidentified peptides) by snake venom 3exonuclease. These results exclude phosphoesters like Ser-P or phosphoramidates like nucleotidyl-(5 -N )-Arg or nucleotidyl-(5 -N )-lys as the bond between VPg and ENA. (ii) VPg linked to genome ENA can be iodinated by the Bolton and Hunter reagent ( iodinated 3-(4 hydroxyphenyl) proprionic acid... 

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. 

Chemical structures of representative heterobifunctional reagents for the radioiodination of proteins, (a) Bolton and Hunter reagent, (b) water-soluble Bolton and Hunter reagent,... 

Bolton and Hunter reagent Nisuccimidyl-3<4 hydroxy-3-p I]iodophenyl)propiQnate (IM5861, Amersham Pharmacia Biotech, or equivalent]... 

Into a 1.5 ml microcentrifiige tube (e.g. Eppendori) pipette the required radioactivity of Bolton and Hunter reagent. Evaporate the solvent, ideally with a Speed-Vac or alternative under a gentle stream of nitrogen. 

Separation of radiolabelled antibody from free iodide 211 Determination of radiochemical purity by TLC 212 Measurement of immunoreactive fraction of radiolabelled antibody 214 lodination of antibody with Bolton and Hunter reagent 216... 

First prepared by Rudinger and Ruegg the Bolton-Hunter reagent is one of the most frequently used conjugation reagents. Its iodinated form has been prepared by Bolton and Hunter . Chemically it is N-succinimidyl-3 (4-hydroxy,5- I iodo-... 

An alternative procedure for labelling proteins to high specific activities by acylation with a I-containing reagent has been described by Bolton and Hunter (1973). 

Bolton-Hunter Reagent Although not the first to be synthesized, the reagent developed by Bolton and Hunter (Bolton and Hunter, 1973), A -succinimidyl-3-(4-hydroxyphenyl)propionate (Figure 76.6), has found prominent use for the labeling of peptides and small molecules (biotin, melatonin, clonazepam, and so on). 

The following procedure describes the iodination process for the Bolton-Hunter reagent and its subsequent use for the radiolabeling of protein molecules. Modification of other macromolecules can be done using the same general method. For particular labeling applications, optimization of the level of iodine incorporation may have to be done to obtain the best specific radioactivity with retention of biological activity. 

Add to the iodination reaction 5 pi of DMF and 100 pi of benzene. Mix to extract the iodinated Bolton-Hunter reagent into the organic phase. 

Davies, C.E., and Palek, J. (1981) 1251-labeling of platelet proteins with Bolton-Hunter reagent. Anal. 

Salmain, M., Vessieres, A., Butler, I., and Jaouen, G. (1991) N-Succinimidyl (4-pentynoate)hexacarbon yldicobalt A transition-metal carbonyl complex having similar uses to the Bolton-Hunter reagent. Bioconjugate Chem. 2, 13-15. 

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 ... 

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