Iodination with Bolton-Hunter Reagent

In vivo, deiodination is thought to be due to enzymes, deiodases. Indeed, due to a structural analogy with thyroid hormones, molecules labeled with Bolton-Hunter reagent or iodinated on a tyrosine moiety are recognized and quickly metabolized. Three classes of deiodases are known. Type I enzymes are mainly located in the liver, kidneys, and skeletal muscle, and deiodinate in the 5 and 5 positions of the tyrosine ring. Type II enzymes, in the brain, pituitary tissue and brown fat, and type III, localized in the central nervous system, both catalyze deiodination on the tyrosine 5 position. 

No incorporation of iodine If you did not forget anything in the iodination mix and did not add anything wrong, the protein either does not contain any tyrosine residue or the oxidation conditions were too weak. If other or stronger oxidizers (e.g., more chloramine T) do not help, you could try to convert with Bolton-Hunter reagent or H-marked N-succinimidyl compounds or you could attempt to iodize a histidine of the protein. 

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. 

The introduction of I into molecules containing tyrosyl or histidyl moieties can be carried out chemically by the Chloramine T procedure or enzymatically by lactoperoxidase." The prelabeled Bolton-Hunter reagent [A-succinimidyl 3-(4-hydroxyl-5-[ I]iodophenylpropionate] reacts under mild conditions with molecules containing amino groups (e.g., proteins and haptens) to introduce an iodinated propyltyrosyl moi-... 

Procedure. In a small glass vial, add the Na I solution at room temperature to crystalline SHPP. Immediately add sodium metabisulfite to reduce the oxidizing agent and iodide, stopping the reaction. Carrier KI is added and the iodinated ester ( I-labeled Bolton-Hunter reagent) isolated by addition of DMF (5 pX) followed by extraction with two portions of reagent grade benzene. To prevent hydrolysis (i.e., inactivation) of the labeled ester, the entire procedure must be carried out as quickly as possible (<1 min). 

If the experimenter wishes for monoiodized proteins, however, just a single iodine can keep the protein from binding. In this case, the experimenter changes the reaction conditions to try to iodize another tyrosine residue or a histidine residue, in the hope that iodine in the new position does not interfere with the binding. If this hope turns out to be unfulfilled, or you do not want to get involved in endless screening of reaction conditions, Bolton-Hunter reagent or marking remain an option. 

The bane of indirect iodination methods is the low specific activity of the product. For a good yield of the conjugation reaction, you need to add large amounts of the molecule to be derivatized. However, if the specific activity is too low, you have to separate iodized from uniodized species, which can be tricky (recommendation make the iodine product with Bolton Hunter and a trace of Bolton Hunter and use it to calibrate the separation method beforehand). Bolton Hunter reagent, for example, is expensive and unstable in watery solution, and the incorporation of the large lipophile group can have even more disastrous effects on the activity of a compound than the conversion of a phenyl residue into a l-phenyl residue. 

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. 

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. 

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