Chloramine-T in Protein Radioiodination Methods

Introduction

Chloramine-T is a sodium salt of N-chloro-p-toluenesulfonamide and is widely used as titrimetric reagent. Both the anhydrous salt and its trihydrate are white powers. The use of chloramine-T in a number of colorimetric reactions, among which the determinations of cyanide and iodide would seem to be the most important.[1]

Preparation

Chloramine-T may be prepared by crystallization from alkaline solution, ether after the alkaline reaction of dichloramine-T with sodium hydroxide, or after the alkaline reaction of p-toluenesulfonamide with sodium hypochlorite. The possible likely impurities are, therefore, p-toluenesulfonamide, sodium chloride, and dichloramine-T. It is claimed that the possible dichloro contaminant may be removed by washing the solid with carbon tetrachloride, and that the purity may be increased to 99.5% by recrystasllization from an aqueous solution.[1]

Chloramine-T Method

Many different substances can be labeled by radioiodination. Such labeled molecules are of major importance in a variety of investigations, e.g., studies of intermediary metabolism, determinations of agonist and antagonist binding to receptors, quantitative measurements of physiologically active molecules in tissues and biological fluids, and so forth. In most of those studies, it is necessary to measure very low concentrations of the particular substance, and that in turn, implies that it is essential to produce a radioactively labeled tracer molecule of high specific radioactivity. Such tracers, particularly in the case of proteins, can often be conveniently produced by radioiodination. [2]

Many methods have been devised for labeling proteins with radioactive iodine, The chloramine-T method have been most widely used. It has the following additional advantages: (a) inexpensive, uncomplicated, readily available reagents; (b) simple execution requiring no special skills or training; (c) quick completion of reaction; (d) minimal exposure of personnel to radiation. The iodination reaction is run as a titration which uses the potential to measure the minimum amount of chloramine-T required for iodination, and there is a method depends upon the observations that the rate at which iodine combines with protein is dependent on the EMF or “redox” potential of the reaction mixture and that different proteins require different potentials for the same rate of reaction. Once the behavior of a protein has been established, the rate of iodination reaction can be controlled using potential to determine the amount of chloramine-T needed. If the reaction is run slowly, a TCT precipitation test can be run during the course of the iodination to be sure that it is going as planned. Alternatively, if one desires to label a protein rapidly or does not have immediate access to a gamma counter, the reaction may be stopped at the predicted time by the dropwise addition of enough sodium metabisulfite to reduce the potential to the starting levels. If the labeling then proves to be insufficient, the reaction can be continued by raising the potential again with chloraine-T. [3] [4]

The basic steps of the modified method of chloramine-T radioiodination of proteins are as fellowing: (1) Dissolve protein in phosphate-buffered saline or any buffer at pH 7 taking care that no reducing reagents are present. Protein concentration ordinarily is the rage of 0.5-5 mg/ml, the total volume rarely exceeding 2 ml. (2) Add desired quantity of radioiodine to the protein solution. Just prior to addition, radioiodine is neutralized with 0.1 N HCl and diluted with 0.1 M phosphate buffer, pH 7. (3) Mix protein plus radioiodine. (4) Initiate radioiodination by adding freshly prepared chloramine-T. Chloramine-T is usually prepared at a concentration of 500 μg/ml in 0.1 M phosphate buffer, pH 7. The radio of chloramine-T to protein most commonly used is 10 μg per milligram of protein. (5) Mix protein plus radioiodine plus chloramine-T. (6) Permit radioiodination to proceed for 10 min. (7) Stop radioiodination by addition of freshly prepared Na2S2O5. Na2S2O5 is usually prepared at 500μg/ml in 0.1 M phosphate buffer, pH 7. The ratio of Na2S2O5 commonly used is the same as that for chloramine-T: 10 μg per milligram of protein. (8) Immediately remove unreacted radioiodine by dialysis. Where a large number of proteins are radioiodinted, it is convenient to transfer the protein mixtures to dialysis bags and dialyze away the unreacted radioiodine plus chloramine-T plus Na2S2O5 against a continuously changing volume of 0.1 M phosphate buffer, pH 7.

References

[1] Jennings, V. J., & Bishop, E. (1973). Analytical applications of chloramine-T.

[2] Bailey, G. S. (2002). The chloramine T method for radiolabeling protein. In The Protein Protocols Handbook (pp. 963-965). Totowa, NJ: Humana Press.

[3] Hunter, R. (1970). Standardization of the chloramine-T method of protein iodination. Proceedings of the Society for Experimental Biology and Medicine, 133(3), 989-992.

[4] McConahey, P. J., & Dixon, F. J. (1980). [11] Radioiodination of Proteins by the Use of the Chloramine-T Method. In Methods in Enzymology (Vol. 70, pp. 210-213). Academic Press.