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Journal of Biochemistry Advance Access originally published online on May 21, 2007
Journal of Biochemistry 2007 142(2):193-200; doi:10.1093/jb/mvm112
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© 2007 The Japanese Biochemical Society.

Partially Folded Conformations of Bovine Liver Glutamate Dehydrogenase Induced by Mild Acidic Conditions

Hassan Rooki1, Khosro Khajeh2, Ali Mostafaie3, Soheila Kashanian4 and Sirous Ghobadi1,*

1Department of Biology, Faculty of Science, Razi University, 67149-67346, Kermanshah; 2Department of Biochemistry, Faculty of Science, Tarbiat Modares University, Tehran; 3Medical Biology Research Center, Kermanshah University of Medical Sciences; and 4Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran

*To whom correspondence should be addressed. Tel: +98-831-4274545, Fax: +98-831-4274545, E-mail: sirousghobadi{at}yahoo.com ghobadi{at}sci.razi.ac.ir

Received February 21, 2007; Accepted May 14, 2007


  Abstract

The acid-induced unfolding of bovine liver glutamate dehydrogenase (GDH) was studied using various spectroscopic methods such as far- and near-UV circular dichroism (CD), intrinsic and 1-anilino naphthalene-8-sulphonate (ANS) extrinsic fluorescence spectroscopy, light scattering and fluorescence quenching in 20 mM mixed buffer at various pHs. CD spectra show that at pH 3.5, GDH retains its secondary structure substantially, whereas its tertiary structure content is reduced considerably. Intrinsic fluorescence of GDH and ANS binding suggest that, at pH 3.5, the hydrophobic surface of enzyme is more exposed in comparison to the native form. Acrylamide quenching indicates more exposure of tryptophan residues of enzyme at pH 3.5 in comparison to pH 7.5. Another partially unfolded intermediate was detected at pH 5.0, which with its ANS binding capacity lies between the pH 3.5 intermediate and the native form of the enzyme. Gel filtration results revealed that the enzyme at pH 3.5 is dissociated into trimeric species whereas it exists as hexamer at pH 7.5 and 5.0. All the data taken together suggest the existence of two partially unfolded states of GDH at moderate acidic pHs which may be considered as molten and pre-molten globule-like states.

Key Words: acid-induced intermediate, aggregation, glutamate dehydrogenase, molten globule, refolding

Abbreviations: ANS, 1-Anilino naphthalene-8-sulphonate; GDH, glutamate dehydrogenase; NADH, nicotinamide adenine dinucleotide reduced; NAD+, nicotinamide adenine dinucleotide


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