PREPARATION AND PURIFICATION OF HIGH SPECIFIC ACTIVITY INSULIN-131IODINE

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Abstract
An iodination efficiency over 50% has been achieved by using chloramine T as the oxidising agent. 2.5 [mu]g. crystalline beef insulin in 0.025 ml. borate buffer, pH 8.0, 4.5 mc. carrier free thiosulphate free I131 in 0.05 ml. were mixed with 50 [mu]g. chloramine I in 0.025 ml. for one minute followed by the addition of 120 [mu]g. sodium metabisulphite in 0.1 ml. After removal of the free I131 by dialysis, the labelled insulin was added to 0.1 - 0.2 ml. of fresh human serum previously incubated with 0.5% iodoacetamide and stained with bromphenol blue indicator. The mixture was purified on a 1 gm. dextran (Sephadex G-75) column with barbitone buffer elution. The damaged iodocompounds came out with the distinct blue serum band followed by eluates containing relatively pure insulin I131. Insulin I131, with a specific activity of 1c/mg., with damaged fraction less than 3%, thus prepared was found suitable for its use in the immuno-assay of insulin.

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