295 Journal of Protein Chemistry, Vol. 22, No. 3, April 2003 (© 2003) 0277-8033/03/0400-0295/0 © 2003 Plenum Publishing Corporation Denaturation Studies by Fluorescence and Quenching of Thermophilic Protein NAD  -Glutamate Dehydrogenase from Thermus thermophilus HB8 Jose L. Ruiz, 1 Juan Ferrer, 1 Carmen Pire, 1 Francisco I. Llorca 1 , and Maria José Bonete 1,2 Received December 20, 2002 Fluorescence techniques have been used to study the structural characteristics of many proteins. The thermophilic enzyme NAD-glutamate dehydrogenase from Thermus thermophilus HB8 is found to be a hexameric enzyme. Fluorescence spectra of native and denatured protein and effect of denat- urants as urea and guanidine hydrochloride on enzyme activity of thermophilic glutamate dehydro- genase (t-GDH) have been analyzed. Native t-GDH presents the maximum emission at 338 nm. The denaturation process is accompanied by an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum. Fluorescence quenching by external quenchers, KI and acrylamide, has also been carried out. KEY WORDS: Quenching; thermophilic; glutamate dehydrogenase; Thermus thermophilus HB8. 1 División de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Alicante, Spain. 2 To whom correspondence should be addressed at División de Bio- química y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99 E-03080 Alicante, Spain. E-mail: mjbonete@ua.es 1. INTRODUCTION The study of thermophilic organisms aroused theoretical and technological interest during recent years. Thermus thermophilus HB8 is a gram-negative bacterium that can grow at temperatures up to 85°C (optimal temperature, 70°C; Oshima and Imahori, 1974). It is the most ther- mophilic bacterium known to have a system of genetic engineering (Koyama et al., 1990). The proteins from this organism are known to be extremely thermostable, as confirmed by in vitro experiments (Taguchi et al., 1991; Motohashi et al., 1994). Glutamate dehydrogenases are widely distributed enzymes that link the carbon and nitrogen metabolism, and they are involved in collection, sorting, and distribu- tion of -amino groups in the cells. This enzyme cataly- ses the interconversion of -ketoglutarate (-KG) 3 and L- glutamate, using NAD + as a cofactor (Smith et al., 1975): L-glutamate + NAD + + H 2 O ⇔ -ketoglutarate + NH 4 + + NADH + We reported the purification of a hexameric NAD- dependent glutamate dehydrogenase, from the ther- mophilic eubacteria Thermus thermophilus HB8 (Ruiz et al., 1998). The genes for the GDHs from several hyper- thermophiles such as Thermococcus litoralis (Britton et al., 1995), Sulfolobus solfataricus (Maras et al., 1992), or Thermotoga maritima (Kort et al., 1997) have been cloned and sequenced. The three-dimensional structure of the hexameric glutamate dehydrogenases from Pyrococ- cus furiosus (Yip et al., 1995) and Thermotoga maritima (Knapp et al., 1997) have been determined recently, pro- viding considerable insights for structure function rela- tionship studies of these enzymes (Baker et al., 1992). In proteins that contain all three aromatic amino acids, fluorescence is usually dominated by the contribu- tion of the tryptophan residues, because both their ab- sorbance at the wavelength of excitation and their quantum yield of emission are considerably greater than the respec- tive values for tyrosine and phenylalanine. Tryptophan flu- orescence is highly sensitive to environment; therefore, 3 Abbreviations: t-GDH, Thermus thermophilus glutamate dehydroge- nase; b-GDH, bovine glutamate dehydrogenase; pf-GDH, Pyrococcus furiosus glutamate dehydrogenase; py-GDH, Pyrobaculum is- landicum glutamate dehydrogenase; NH 4 + , ammonium ion; GdmCl, guanidine hydrochloride; -KG, -ketoglutarate.