[67] Determination of the conversion of dethiobiotin to biotin in Aspergillus niger

Add time:08/08/2019    Source:sciencedirect.com

Publisher SummaryThis chapter describes the procedure for the assay of the conversion of dethiobiotin to biotin. The mold is grown aerobically for several days on a liquid medium to which a small amount of radioactive d-dethiobiotin is added. Mycelia are filtered off and the filtrate, which contains d-biotin- l-sulfoxide and the β-oxidative catabolites of d-dethiobiotin as principal metabolites, is subjected to a series of treatments. In this procedure, the radioactive biotin vitamers are concentrated by adsorption on and elution from charcoal, carrier biotin is added, biotin and its sulfoxide are oxidized to the sulfone by treatment with excess hydrogen peroxide, biotin sulfone is separated by anion-exchange chromatography on Dowex 1 (formate) with ammonium formate as eluent, ammonium ions are removed on Dowex 50, and the sulfone is recrystallized from water to constant specific radioactivity. The extent of the conversion of dethiobiotin to biotin can be assessed by using desthiobiotin, which is 3H(random)-, 14C(carbonyl)-, or 14C(carboxyl)-labeled. The loss of tritium from the 3H(random)-dethiobiotin can be used to approximate the number of hydrogens abstracted during conversion to biotin.

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