An enzymatic method for the quantitative determination of biotin has been developed. The method involves the enzymatic binding of biotin in situ T* the pyruvate carboxylase apoprotein of biotin-deficient bakers' yeast and the subsequent estimation of the pyruvate carboxylase activity by a "CO,-fixation method. The method is specific for biotin. Several biotin analogs and precursors were tested, and only biocytin was found to interfere. Biotin amounts of less than 5 pg can be estimated.
The important role of biotin as the prosthetic group of certain carboxylating, transcarboxylating, or decarboxylating enzymes is well known (see Ref. 1 for a complete review and references). Because of their sensitivity and simplicity, bioassays based on the use of fungal or bacterial biotin auxotrophs as test organisms are commonly used in routine methods of estimating biotin contents, e.g., in raw materials of the yeast industry. These methods, however, estimate not the content of biotin alone but the total biotin activity of the material; for several compounds, like dethiobiotin, biotin sulfoxide, and even aspartic and oleic acid, act like biotin in bioassays. Besides being unspecific, the methods are imprecise and time consuming (for bioassays of biotin see Ref. 2). Some physicochemical biotin assays have been reported (see, e.g., Refs. 3-.5), but they have not become widely accepted for routine biotin assays.
Bakers' yeast (Saccharomyces cerevisiae) grown under biotin limitation is known to have reduced pyruvate carboxylase activity, even though it contains a considerable amount of pyruvate carboxylase apoprotein which, in the presence of biotin, ATP, and Mg2+ ions, is enzymatically converted to the active holoenzyme (Refs. 6,7; Haarasilta and Oura, unpublished work). Similarly, the necessary synthetase is also present in biotin-deficient yeast. This report describes a precise, sensitive, and specific biotin assay that exploits the regular relationship found between the amount of available biotin and the pyruvate carboxylase activity formed when pyruvate holocarboxylase is reconstituted in situ from the apoenzyme of biotin-deficient bakers' yeast. The scheme of the assay is as follows: