Quantitation of Biotin Residues in Proteins using a Fluorescence-based Assay
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Biotinylated proteins are a common tool in biotechnology. Current methods to determine the biotinylation degree in proteins either consume large amounts of sample due to lack of sensitivity or require laborious procedures. Biotinylated proteins were hydrolyzed prior to measurement to prevent steric hindrance. Two digestion methods were tested: protein hydrolyzation by acid treatment and digestion by proteinase K. The degree of biotinylation was measured with a recently proposed method, the competitive binding assay, and was verified with a previously reported strategy (fluorometric dye replacement). The degree of biotinylation was successfully determined by the competitive binding assay with acid treated or proteinase K digested protein. Values obtained for acid treated proteins were in good agreement with dye replacement measurements. Proteins hydrolyzed with 6 M HCl for 2 h at water boiling temperature gave a lower biotinylation degree, probably due to biocytin degradation under these conditions. Measurements of native biotinylated proteins gave a value markedly lower than hydrolyzed proteins due to steric hindrance. Protein digestion with proteinase K prior to measurement of biotinylation degree was found to be simple and effectively abolished steric hindrance bias in competitive binding assays. Knowledge about available biotin residues could be deduced from measurements with native proteins. Using the competitive binding assay, determination of the biotinylation degree of proteins is possible in a single measurement with a subnanomolar amount of protein.