Refolding of pyridoxine-5'-P oxidase assisted by GroEL

Oh Shin Kwon, Jorge E. Churchich

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The unfolding of brain pyridoxine-5'-P oxidase by guanidinium chloride has been investigated at equilibrium. Circular dichroism, fluorescence spectroscopy and gel exclusion chromatography were used to monitor the unfolding process. The enzyme dissociates reversibly into monomers, but the fluorescence properties of the cofactor FMN are not restored upon dilution with potassium phosphate buffer (pH 7.4). Spontaneous refolding leads to 20% recovery of the catalytic activity. Addition of GroEL to the renaturing buffer accelerates the recovery of catalytic activity that approaches a level of 80% with respect to the native enzyme. The rate of recovery of catalytic activity assisted by GroEL parallels the rate of FMN fluorescence quenching, suggesting that structural rearrangements of the catalytic domain is the last step to take place in the refolding process.

Original languageEnglish
Pages (from-to)1057-1064
Number of pages8
JournalBiochimie
Volume81
Issue number11
DOIs
StatePublished - Nov 1999

Keywords

  • Circular dichroism
  • Fluorescence
  • FMN
  • GroEL
  • PNP oxidase
  • Refolding process

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