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.

Effects of mutations in the helix G region of horseradish peroxidase

“…His) are targeted, the results are stabilityneutral or -negative [25,42]. Chemical modification can also influence catalytic properties: marginally improved catalytic activity, but little K' m alteration, occurred with ABTS and other substrates following chemical modification of HRP with PA and EGNHS [27].…”

“…Proteins precipitated by ammonium sulphate from both the culture supernatant and the periplasmic preparation were collected via centrifugation, resuspended in 50mM phosphate buffer pH 7.5, pooled and dialysed versus the same buffer overnight at 4 o C. Sodium chloride (1M) and GnCl (200mM) were added to the dialysed fractions (10 mL total volume), and these latter were purified via nickel affinity chromatography at room temperature. Sodium acetate (25mM, pH 4.5) was Thermal stabilities of recombinant HRP and mutant variants were determined as for plant HRP [25,27] using 50 mM phosphate buffer, pH 7.0, in which renaturation of unfolded HRP does not occur. For t ½ estimation, a single HRP stock solution (room temperature) was plunged into a 50 o C waterbath.…”

“…Among non-genetic methods, chemical reactions such as crosslinking [21,22,23,24,25] and surface modification [26,27,28,29] have successfully increased HRP's stability. Notably, these beneficial modifications target HRP's solvent-exposed lysine residues.…”

Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

“…The stability parameters of recombinant HRP and mutant variants were determined as described for plant HRP [3,9] except that thermoinactivation time courses used 50 o C. Samples were removed periodically onto ice and their residual activities determined upon re-warming to room temperature; this procedure gives apparent half-life, t ½app ). A constant protein concentration of 0.1 mg/mL was used for all thermoinactivations to control for possible effects of protein concentration on stability.…”

“…Although moderately stable, the availability of a stabilized form of HRP would increase its applicability still further. Previous stabilisation studies have focused on the plant-derived protein, with several reports describing chemical procedures such as crosslinking [3][4][5][6], surface modification [7][8][9], attachment of PEG [10] and modification of carbohydrate residues [11]. Immobilisation of HRP [12,13] and addition of stabilising reagents [14,15] have also led to enhanced stability.…”