Determination of the extent of protein biotinylation by fluorescence binding assay

Srivatsa V. Rao, Kimberly W. Anderson, Leonidas G Bachas

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)94-98
Number of pages5
JournalBioconjugate Chemistry
Volume8
Issue number1
DOIs
StatePublished - Jan 1997
Externally publishedYes

Fingerprint

Biotinylation
Biotin
Assays
Fluorescence
Proteins
Hydrolysis
Endopeptidase K
Avidin
Carboxylic Acids
Carboxylic acids
Digestion
Acids
Streptavidin
Fluorescein-5-isothiocyanate
Phosphatases
Binding sites
Horseradish Peroxidase
Bovine Serum Albumin
Alkaline Phosphatase
Membrane Proteins

ASJC Scopus subject areas

  • Chemistry(all)
  • Organic Chemistry
  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Determination of the extent of protein biotinylation by fluorescence binding assay. / Rao, Srivatsa V.; Anderson, Kimberly W.; Bachas, Leonidas G.

In: Bioconjugate Chemistry, Vol. 8, No. 1, 01.1997, p. 94-98.

Research output: Contribution to journalArticle

Rao, Srivatsa V. ; Anderson, Kimberly W. ; Bachas, Leonidas G. / Determination of the extent of protein biotinylation by fluorescence binding assay. In: Bioconjugate Chemistry. 1997 ; Vol. 8, No. 1. pp. 94-98.
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