DETERMINATION OF THE EXTENT OF PROTEIN BIOTINYLATION BY FLUORESCENCE BINDING ASSAY

A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumi n, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete aci d hydrolysis or by enzymatic digestion using proteinase K to release b iotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection sy stem is based on the enhancement of the fluorescence of streptavidin-F ITC by biotin, The extent of biotinylation determined by this method w as compared with the values obtained by a conventional colorimetric me thod that is based on the displacement of the dye 4-hydroxyazobenzene- 2-carboxylic acid (HABA) from the binding sites of avidin. it was foun d that, because the described method determines the amount of liberate d biotin after hydrolysis, it does not suffer from steric hindrance pr oblems associated with the ability of biotin on a protein surface to d isplace HABA from avidin, Therefore, this method can provide a more ac curate determination of the extent of biotinylation. Tt was also deter mined that the acid hydrolysis of the biotinylated protein was more ef fective in releasing the conjugated biotin compared to enzymatic diges tion by proteinase K.