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Journal of Biochemistry Advance Access originally published online on December 6, 2008
Journal of Biochemistry 2009 145(2):259-265; 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 Aspergillus terreus

Adepu Kiran Kumar and Pranab Goswami*

Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, India

*To whom correspondence should be addressed. Tel: + 91-361-25-82-202, Fax: + 91-361-25-82-249, E-mail: pgoswami{at}iitg.ernet.in

Received September 22, 2008; Accepted November 25, 2008


  Abstract

A multimeric alcohol oxidase from Aspergillus 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. On 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

Abbreviations: ABTS, 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio] propane-1-sulfonic acid; CLSM, confocal laser scanning microscopy; CPS, counts per second; DMSO, dimethyl sulfoxide; DLS, dynamic light scattering; FAD, flavin adenine dinucleotide; HRP, horseradish peroxidase; β-ME, β-mercaptoethanol; TCA, trichloroacetic acid


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