Determination of the Extent of Protein Biotinylation by Fluorescence Binding Assay

Abstract

A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.

Fluorometric Assay for Quantitation of Biotin Covalently Attached to Proteins and Nucleic Acids

“…When we first attempted to determine the biotin DOL of our standards the calculated DOL results were consistently below the expected DOL. This was not unexpected, as previous reports in the literature (9,10) have underestimated biotin DOL of macromolecules due to steric hindrance of access to avidin-binding sites.…”

“…The effective biotin concentration is the total biotin concentration minus the cryptic and sterically hindered biotins described previously. In order to determine the total amount of biotinylation, we used micrococcal nuclease to digest biotinylated nucleic acids and the protease Pronase E to digest biotinylated proteins, similar to Rao et al (9). The total biotinylation of both the oligonucleotide and goat anti-mouse IgG were also determined via MALDI-TOF mass spectrometry (data not shown).…”

Controlled layer-by-layer immobilization of horseradish peroxidase

“…The amount of biotin in the hydrolysate was then determined by HPLC using a fluorescence-based postcolumn reaction detection system (Rao et al, 1997). The extent of biotinylation was determined by measuring the amount of biotin released by the complete acid hydrolysis of the biotinylated enzyme.…”

“…This property can be used to determine the concentration of biotin in biotinylated protein hydrolysates (Rao et al, 1997). The extent of biotinylation of HRP was thus determined as a ratio of the amount of biotin present in the hydrolyzed product and the moles of HRP used for acid hydrolysis.…”

Quantitative determination of noncovalently bound acridinium in protein conjugates by liquid chromatography/electrospray ion trap mass spectrometry

“…Due to increasing quality and regulatory demands,2 the need for additional characterization methods is required. The characterization of protein conjugates typically involves the measurement of average incorporation ratio of label to protein (hapten density),3–8 and, more recently, the distribution of label on the protein 9–12. While the degree of protein modification plays an important role in the performance of the conjugate, an evaluation of conjugate quality should include the amount of noncovalently bound label, which may remain from the conjugation procedure.…”