Biotinylation of biotinidase following incubation with biocytin

Jeanne Hymes, Kristin Fleischhauer, Barry Wolf*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

Human serum biotinidase, purified to homogeneity (1920 units/mg protein), was incubated with biocytin prior to electrophoresis and transblotting with avidin-peroxidase. Avidin reacted with biotinidase maximally when incubated at pH 7.5-9, less at pH 7 and none below pH 7. No avidin reactivity occurred when biotinidase was incubated with biotin or in the absence of biocytin. Inclusion of the nucleophilic acceptors, ethanolamine or hydroxylamine, to the incubation mixture with biocytin and biotinidase resulted in loss of avidin reactivity. High concentrations of mercaptoethanol also prevented avidin reactivity. These results suggest that biotinidase can be biotinylated in the presence of biocytin at neutral to alkaline pH probably through a thioester bond formed with a cysteine residue in the active site of the enzyme. Biotinidase may then function as a biotinylating enzyme when incubated with appropriate nucleophilic acceptors.

Original languageEnglish (US)
Pages (from-to)39-45
Number of pages7
JournalClinica Chimica Acta
Volume233
Issue number1-2
DOIs
StatePublished - Jan 16 1995

Keywords

  • Avidin
  • Biocytin
  • Biotin
  • Biotinidase
  • Biotinylation
  • Thioester

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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