Purification and inactivation of 3-hydroxyanthranilic acid 3,4-dioxygenase from beef liver

Dhirendra Nandi, Eric S. Lightcap, Yumee Kim Koo, Xingliang Lu, Jean Quancard, Richard B. Silverman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

3-Hydroxyanthranilic acid 3,4-dioxygenase (EC 1.13.11.6; HADO) was purified to homogeneity from beef liver with the use of two dye columns (Cibacron Blue and Reactive Green 19) and hydroxyapatite. Two active peaks of enzyme were isolated from the hydroxyapatite column or by nondenaturing chromatofocusing of the enzyme prior to hydroxyapatite. The two active forms moved with different electrophoretic mobilities when they were subjected to nondenaturing polyacrylamide gel electrophoresis, regardless of the method of isolation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), however, these species had apparently identical mobilities and have, therefore, close molecular mass. Analysis by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry gave them a molecular mass of 32,566 and 32,515Da, respectively, for the species with apparent pI values of 5.60 and 4.98, respectively, suggesting that they differ only in the presence or absence of the iron cofactor. The N-terminal group appears to be blocked as no amino-terminal sequence was possible from direct Edman degradation. A new inactivator of the enzyme, 6-chloro-3-hydroxyanthranilic acid, was synthesized and was shown to exhibit time-dependent inactivation. A possible mechanism for inactivation is proposed.

Original languageEnglish (US)
Pages (from-to)1085-1097
Number of pages13
JournalInternational Journal of Biochemistry and Cell Biology
Volume35
Issue number7
DOIs
StatePublished - Jul 1 2003

Keywords

  • 3-Hydroxyanthranilic acid 3,4-dioxygenase
  • Beef liver
  • Inactivation
  • Purification

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Fingerprint Dive into the research topics of 'Purification and inactivation of 3-hydroxyanthranilic acid 3,4-dioxygenase from beef liver'. Together they form a unique fingerprint.

Cite this