chloramine-t and fluorescein-5-maleimide

A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing conditions and may be particularly useful with mutant proteins carrying engineered single cysteine residues at sites that are not functionally critical.

Topics: Bacterial Proteins; Chloramines; Chromatography, Gel; Complement Hemolytic Activity Assay; Cysteine; Erythrocytes; Fluoresceins; Iodine Radioisotopes; Polymers; Protein Binding; Proteins; Sensitivity and Specificity; Spectrophotometry; Streptolysins; Thiosulfates; Tosyl Compounds