ABSTRACT
Conformational changes at the active site of pantetheine hydrolase (EC3.5.1.-) during guanidine hydrochloride (GndHCl) denaturation were investigated by UV and circular dichroism spectroscopy and by electron spin resonance spectroscopy, following the spectral behaviour of the nitroxide radicals (N-(1-oxyl-2,2,5,5,-tetramethyl-3-pyrrolidinyl) iodacetamide) covalently linked to the two active site cysteine residues. At low denaturant concentrations (0.2 M) no conformational changes may be observed, whereas the catalytic activity, is strongly affected. The results indicate that the active site of pantetheine hydrolase is labile and unfolds under conditions in which no global tertiary structure modifications can be observed.
Subject(s)
Amidohydrolases/chemistry , Guanidine/chemistry , Protein Denaturation , Binding Sites , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , GPI-Linked Proteins , Protein Conformation , Spin LabelsABSTRACT
A method of obtaining accurate values for the optical parameters of thin film substrates is described. Calculations for reflected and transmitted fluxes in substrate sets are developed allowing for double reflection. A simple procedure to fit measurements is also illustrated. Values for substrate parameters are extracted and used to correct for substrate influence in thin film spectrophotometric measurements. Corrected thin film measurements and data calculated with obtained optical constants are compared.
