Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone

G.A. Posthuma-Trumpie, W.A.M. van den Berg, D.F.M. van de Wiel, W.M.M. Schaaper, J. Korf, W.J.H. van Berkel

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)

Abstract

The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50 ± 10%), reconstitutable dimers (20 ± 10%) and irreversibly aggregated oligomers (30 ± 20%). Incubation of monomeric apoGOx with FAD or N6-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N6-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25¿50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.
Original languageEnglish
Pages (from-to)803-812
JournalBiochimica et Biophysica Acta. Proteins & Proteomics
Volume1774
Issue number7
DOIs
Publication statusPublished - 2007

Keywords

  • flavin-adenine-dinucleotide
  • apoenzyme reactivation immunoassay
  • glucose-oxidase
  • aspergillus-niger
  • colorimetric immunoassays
  • enzyme-electrode
  • binding
  • label
  • milk
  • intermediate

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