title: Effects of phthalic anhydride modification on horseradish peroxidase stability and activity.
abstract: Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.
authors.affiliation: School of Biotechnology, Dublin City University, Dublin 9, Republic of Ireland.
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: O'Brien
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Smith
authors.forename: Ciarán
authors.initials: C
authors.lastname: O'Fágáin
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D005682:Gadolinium / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D010078:Oxazines / Q000737:chemistry; D010720:Phosphines / Q000494:pharmacology; D010796:Phthalic Anhydrides / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics
chemical_list: D004492:Edetic Acid; D005682:Gadolinium; D019791:Guanidine; D006735:Horseradish Peroxidase; D010078:Oxazines; D010720:Phosphines; D010796:Phthalic Anhydrides; C080938:tris(2-carboxyethyl)phosphine; C044808:nile red; C038958:gadolinium chloride; C043103:phthalic anhydride