title: Intrinsic fluorescence spectroscopy of glutamate dehydrogenase: Integrated behavior and deconvolution analysis.
abstract: In this paper, we present a deconvolution method aimed at spectrally resolving the broad fluorescence spectra of proteins, namely, of the enzyme bovine liver glutamate dehydrogenase (GDH). The analytical procedure is based on the deconvolution of the emission spectra into three distinct Gaussian fluorescing bands G(j). The relative changes of the G(j) parameters are directly related to the conformational changes of the enzyme, and provide interesting information about the fluorescence dynamics of the individual emitting contributions. Our deconvolution method results in an excellent fitting of all the spectra obtained with GDH in a number of experimental conditions (various conformational states of the protein) and describes very well the dynamics of a variety of phenomena, such as the dependence of hexamers association on protein concentration, the dynamics of thermal denaturation, and the interaction process between the enzyme and external quenchers. The investigation was carried out by means of different optical experiments, i.e., native enzyme fluorescence, thermal-induced unfolding, and fluorescence quenching studies, utilizing both the analysis of the "average" behavior of the enzyme and the proposed deconvolution approach.
authors.affiliation: National Nanotechnology Laboratories of INFM, Biomolecular Electronics Division, Department of Innovation Engineering, University of Lecce, Via per Arnesano 73100 Lecce, Italy.
authors.forename: P P
authors.initials: PP
authors.lastname: Pompa
authors.forename: R
authors.initials: R
authors.lastname: Cingolani
authors.forename: R
authors.initials: R
authors.lastname: Rinaldi
mesh_terms: D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D005969:Glutamate Dehydrogenase