Optimization of conditions for radioiodination of Bovine Serum Albumin (BSA)

Radioiodination of BSA employing Chloramine-T method developed by Hunter and Greenwood (1962) serves as a model for variety of applications. The procedure is widely employed for tagging radioactive 125I with BSA. Chloramine-T is a mild oxidant widely used for the labeling studies. However, damage to the protein may occur if the conditions are not standardized resulting in lower yield. The objective of the present study is to optimize the reagent concentration, Incubation time and Temperature for Radioiodination of BSA and to characterize using paper chromatography. The methodology involves the addition of buffer followed by 0.3 mCi 125I along with 10ul of BSA, chloramine-T and the mixture was incubated for a brief period followed by addition of Sodium metabisulphite (MBS) and potassium iodide. Different concentrations of chloramines-T and MBS were tried for studying damage caused due to oxidation. The optimal concentration of the reagents was studied for their incubation timings of 30, 60 and 120 seconds and temperature of 28℃ and 2℃. All the optimization studies were characterized by spotting the reaction mixture on paper chromatography strips (1x30 cm) and allowed to run at 250 V for 45 minutes. The strips were then cut into 1 cm and inserted into tubes and quantified on a gamma counter for 1 minute. The yield was calculated from the graph obtained. We found that with chloramine T and MBS concentration of 10 and 20 ug per reaction respectively the yield was 60.5%. Incubation time of 60 seconds gave 70.9% and temperature of 2℃ gave 75.4% yield. We conclude that the optimized method could be used for this radioiodination of larger proteins like BSA for use in radioimmunoassay and for assays involving precipitation of tannin using BSA. (author)