Iodine reaction with tyrosine

Directing the iodination reaction toward histidine residues in proteins, as opposed to principally tyrosine modification, is possible simply by increasing the pH of the lodobeads reaction from the manufacturer s recommended pH 7.0-8.2 (Tsomides et ai, 1991). No reducing agent is required to stop the iodination reaction as is the case with chloramine-T and other methods. 

Thioamides are reducing agents. They inhibit thyroid hormone synthesis by inhibiting the peroxidase enzymatic system, which catalyzes oxidation of iodide ions and iodine that are consumed in food, which is necessary for iodination of tyrosine derivatives. Thus they reduce the concentration of free iodine necessary to react with tyrosine derivatives, and they can also block oxidative addition reactions of mono- and diiodtyrosines, which form L-thyroxine and L-triiodothyronin. 

Two methods have been described for effecting the smooth reaction of tyrosine with iodine to form 3,5-diiodotyrosine. In one (a) a solution of iodine (320 g.) and... 

Tryptophan can also be iodinated directly. Both mono and diiodinated derivatives are known. Reaction is slower than with tyrosine and histidine, and hence occurs rarely. 

The mechanism of thyroglobufin (Tg) iodination has been intensely debated and remains an open question the specific controversy focuses on whether or not iodination of Tg tyrosyl groups occurs via an enzyme-bound intermediate, or by freely diffusing iodination equivalents released from TPO. It is clear, however, that TPO actuates nonspecific iodination reactions in the thyroid. The two chemical species of iodine that can iodinate biomolecules under physiologic conditions are HOI and I2. Dunford and Ralston (1983) demonstrated that HOI, not I2, is the primary species that iodinates tyrosine in aqueous environments that contain both I2 and HOI. I2 is likely to be the species that is responsible for the iodination of lipids. The distinct pharmacological and toxicological properties of iodide and oxidized iodide (I2) have been a topic of some interest in the literature, as some activity not associated with THs has been ascribed to I2. 

The length of the iodination reaction can be modified to increase or decrease the specific activity of the labeled protein however, there is a trade-off between achieving a protein of higher specific activity and the possibility of either protein denatur-ation or modification of key tyrosine residues that may affect the protein s activity. We have used reaction times of 5-10 min with successful results. In addition, we have performed assays under various conditions of specific activity (from 2.0 X 10 to 1.3 X 10 cpm//xg), with no apparent effect on the specificity of the interaction (see Crawford et al., 1992). 

Suppose that sequence analysis of a protein has provided the data that the given protein contains 7 residues of t5Toslne. React the protein with radioactive iodine - 131 in a sultabk reaction mixture so that tyrosine residues pick up the iodine. After this precipitate the protein with the help of trichloroacetic acid. Count the precipitate. Suppose that the count given Is 4000 cpm. Now denature amother aliquot of the protein and subject this too to iodination reaction in the presence of radioactive iodine 131. After iodination is over, precipitate the protein and count for radioactivity. Suppose now that the count is 9000 cpm. 

The reactions are parallel with respect to iodine (fi) and consecutive with respect to iodine-substituted derivatives of L-tyrosine (R). 

Each bead can iodinate up to 500 pg of tyrosine-containing protein or peptide. This translates into an oxidative capacity of about 0.55 pmol per bead. The rate of reaction can be controlled by changing the number of beads that are used and altering the sodium iodide concentration added to the reaction. Reaction volumes of 100-1,000 pi are possible per bead. The following protocol is suggested for iodinating proteins. Optimization should be done to determine the best incorporation level to obtain good radiolabel incorporation with retention of protein activity. 

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