Denaturing behavior of glutathione reductase from cyanobacterium Spirulina maxima in guanidine hydrochloride.
The influence of guanidine hydrochloride (Gdn-HCl) on glutathione reductase from Spirulina maxima has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, thiol groups accessibility, and gel filtration chromatography. It was found that the denaturation process involves several intermediate states. At low, Gdn-HCl concentrations (Cm = 0.4 M), reductase activity was fully lost. However, below 3 M Gdn-HCl, this inhibition was freely reversible upon removal of the denaturing agent. Gel filtration experiments revealed that this reversible inhibition was not due to dissociation of the tetrameric enzyme. Structural studies strongly suggest that the conformation of this intermediate state is similar to that of native enzyme. A model in which a local region of the polypeptide chain assumes an extended conformation (D. T. Haynie, and E. Freire, Proteins 16,115-140) is proposed for the reversibly inactivated enzyme. Between 3 and 4 M Gdn-HCl (Cm = 3.5), the enzyme activity was irreversibly lost, this inhibition being concomitant with the loss of ellipticity, changes in both wavelength and intensity at the maximum of fluorescence emission, and dissociation of the enzyme into unfolded monomers; these results reveal that gross changes in the protein conformation occur under these conditions. At 4 M Gdn-HCl an equilibrium exists between the denatured forms of dimer and monomer, which is completely shifted toward the unfolded monomers at 5 M Gdn-HCl. Irreversibility in the Gdn-HCl-induced denaturation of S. maxima glutathione reductase was not due to aggregation of the unfolded enzyme.
Rendón
J L
JL
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, México, DF.
Pardo
J P
JP
Mendoza-Hernández
G
G
Rojo-Domínguez
A
A
Hernández-Arana
A
A
D002850:Chromatography, Gel; D002942:Circular Dichroism; D000458:Cyanobacteria / Q000201:enzymology*; D005980:Glutathione Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D046911:Macromolecular Substances; D005980:Glutathione Reductase; D019791:Guanidine