Add time:07/26/2019         Source:sciencedirect.com

Publisher SummaryThe two-step synthesis of 2-iminobiotin hydrobromide involves the alkaline hydrolysis of biotin with barium hydroxide to yield 5-(3,4-diaminothiophan-2-yl)pentanoic acid. The diaminocarboxylic acid sulfate (2.5 g) is treated with barium carbonate (5 g) and dissolved in 30 ml of hot water and the solution is immediately filtered through a prewarmed fritted glass funnel. The precipitate is washed with an additional 10 ml of hot water. Cyanogen bromide (1.9 g) is added to the filtrate and crystals of 2-iminobiotin start to form within five minutes. Avidin is readily iodinated by I-labeled Bolton–Hunter reagent and retains full biological activity as judged by its ability to bind to and be specifically eluted from a 2-iminobiotin affinity column. The 2-iminobiotin affinity column is also suitable for purifying rhodamine-conjugated streptavidin and fluorescein-conjugated avidin/streptavidin derivatives. The horseradish peroxidase and the alkaline phosphatase conjugates of avidin/streptavidin can also be purified by chromatography on 2-iminobiotin columns. For these derivatives, the affinity column is equilibrated at pH 10 rather than pH 11 and specific elution is achieved with pH 6 buffer.

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