title: ESR study of free and immobilized elastase.
abstract: Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.
mesh_terms: D001665:Binding Sites; D003300:Copper / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004800:Enzymes, Immobilized; D006358:Hot Temperature; D007700:Kinetics; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011487:Protein Conformation; D013113:Spin Labels
chemical_list: D004800:Enzymes, Immobilized; D013113:Spin Labels; D003300:Copper; D010196:Pancreatic Elastase