Bolton-Hunter reagent preparation

Polyacetal polymers containing polyethylene glycol have been prepared which are stable at physiological pH but which readily degrade at lower pH s. Bolton Hunter reagent conjugates prepared from these materials showed a favorable biodistribution profile of the product. 

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-... 

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-... 

Radiolabeled Products Prepared with the Bolton-Hunter Reagent" (Continued)... 

Other prelabeled small molecules have been synthesized and used to prepare radioiodinated protein derivatives under conditions that circumvent exposure of the protein to oxidizing conditions. One of these compounds, I-labeled methyl p-hydroxybenzimidate, reportedly is more selective for amino groups than the Bolton-Hunter reagent - (Fig. 1). 

Alternatively, the Bolton-Hunter reagent can be used to prepare [i2 i]pa,7 xhis is the method of choice. The procedure is simple, and re-... 

Isolation of calf thymus poly(ADP-ribose) polymerase (at least 95% homogeneous, specific activity = 1500 nmol ADP-ribose incorporated mg protein in 1 min) and coenzymic DNA were carried out using published methods [8]. The synthesis of 0-amino-benzamide agarose affinity matrix, DNA-cellulose, the method of iodination of the enzyme protein (with the preparation of the Bolton-Hunter reagent adduct of... 

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. 

---