Partially folded conformations of bovine liver glutamate dehydrogenase induced by mild acidic conditions.
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.
Rooki
Hassan
H
Department of Biology, Faculty of Science, Razi University, 67149-67346, Kermanshah, Iran.
Khajeh
Khosro
K
Mostafaie
Ali
A
Kashanian
Soheila
S
Ghobadi
Sirous
S
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D005969:Glutamate Dehydrogenase