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Journal of Biochemistry Advance Access published online on December 6, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn163
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© The authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Dissociation and reconstitution studies of a broad substrate specific multimeric alcohol oxidase protein produced by A. terreus

Kiran Kumar Adepu and Pranab Goswami*

Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India.

*Address for correspondence: Dr. Pranab Goswami, Dept. of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. Phone: (+91) 361-2582202. Fax: (+91) 361-2582249; E-Mail: pgoswami{at}iitg.ernet.in

Received September 22, 2008; Accepted November 25, 2008


  Abstract

A multimeric alcohol oxidase from A. terreus was dissociated and simultaneously deflavinated into catalytically inactive FAD-free subunits when incubated with 0.74 M β-mercaptoethanol (β-ME) for 8 h at 4 °C. This dissociation process had traversed through two FAD-associated intermediate proteins, between these one of them showed the enzyme activity. Upon removal of β-ME, the multimeric apoprotein was regenerated, which was, however, catalytically inactive. Reactivation of the FAD supplemented apoprotein was accomplished only after incubating with the substrate. This catalytic reactivation was a slow process as evident from the prolonged FAD emission quenching. The dissociation and re-association phenomena were demonstrated by using dynamic light scattering-, size exclusion chromatographic-, confocal laser scan microscopic-, and native PAGE analyses. The solvent effect caused by the high concentration of β-ME is attributed to the observed dissociation and linked deflavination of these multimeric alcohol oxidase protein particles.

Key Words: Alcohol oxidase, β-mercaptoethanol, Deflavination, Dissociation, Reactivation


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