Tyrosine radioiodination

Figure 12.6 The immobilized glucose oxidase/lactoperoxidase system radioiodinates proteins through the intermediate formation of hydrogen peroxide from the oxidation of glucose. H2O2 then reacts with iodide anions to form reactive iodine (I2). This efficiently drives the formation of the highly reactive H2OI+ species that is capable of iodinating tyrosine or histidine residues (see Figure 12.2).

Histidine reacts more slowly thus, when both tyrosine and histidine fragments are present on a peptide, histidine reacts only in the presence of a large excess of radioiodine, and with a longer reaction time. 

A variety of tritiated ( H) steroids are available as the radiolabeled compounds for use in the RIA. The relatively low sensitivity of H can be increased by multiple labeling. Another alternative is I labeling. Radioiodinated aromatic amines or amino acids, such as tyramine, tyrosine, or histamine, are linked to the steroid by the above-mentioned reactions to obtain the label. 

Both agents are widely applied as auxiliary agents for radioiodination. The in situ formed Cl- I or Na I adds to activated phenyl residues by an electrophilic aromatic substitution reaction. Since tyrosine comprises an activated phenyl residue, the labeling of proteins and peptides is performed with this method vide infra). For smaller organic compounds like 2-hydroxy-6-methoxy-N- [(1 -ethyl-2-pyrrolidinyl)methyl]benzamide (BZM), Chloramine-T has also been applied for radioiodination (Kung et al. 1988). 

ICl rapidly undergoes electrophilic substitution predominantly with tyrosine residues of the target molecule. The reaction is fast and finishes within a few seconds. The optimum conditions for the mild incorporation of radioiodine into proteins are a low temperature of 4°C, a pH value in the range of 6-8, and a short reaction time of <1 min. Chloramine-T often degrades sensitive peptides. This has to be prevented by the addition of a reducing agent. 

Under these conditions, radioiodine incorporation of up to 99% can be achieved at labeled protein recovery rates of >90%. The reaction is more gentle than soluble Chloramine-T because there is less contact between the protein and the immobilized oxidizing agent. If the reaction is conducted at pH values above 8, it is possible to radioiodinate histidine residues. The reaction is believed to proceed in the close proximity of the surface of the polymer beads. An electrophilic iodosulfonamide intermediate species that reacts with either tyrosine or histidine residues of the protein to be labeled is formed. 

The predominant mechanism of radiolabelling of antibodies with radioiodine involves electrophilic substitution of the icxline into the activated phenolic ring of tyrosine side-chains as shown in Scheme 1. Provided that low substitution ratios are used it is unlikely that radiolabelling will take place at any other site in the large immunoglobulin molecule but in proteins lacking tyrosine residues... 

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