Peroxidase enzymes occur widely in nature and fall into three classes [1]. The class III horseradish peroxidase isoenzyme C [HRPC] is well-characterized [2,3] and has many applications, especially in diagnostics. This laboratory has reported previously that modification with the bifunctional compound ethylene glycol bis-succinimidyl succinate stabilizes HRPC against heat [4], guanidine chloride and some water-miscible organic solvents [5]. Loss of enzyme activity following short-term (<3h) exposure to these adverse influences likely occurs via unfolding of the protein tertiary structure. However, chemical deterioration can occur also during extended storage under quite mild conditions [6] with resultant losses of biological activity. Here we describe the effects of some days' storage at acidic and alkaline pH-values on the residual activities of native HRPC and the stabilized chemical derivative (EG-HRP).

Sigma horseradish peroxidase C (RZ 3.0) was used as supplied. Treatment of HRPC with ethylene glycol bis-succinimidyl succinate and assay of activity using 3,3',5,5'-tetra-methyl benzidine (TMB) were as described in ref. [4]; recovery of starting activity exceeded 90%. Buffers for storage at acid and alkaline pH values were prepared using orthophosphate compounds at 10mM final concentration; the control was pH 7.0. Samples were passed through 0.22mm filters into sterile containers to prevent microbial growth during the experiment. Samples were assayed at time zero. Storage took place at 4°C.

The pH 7.0 controls of native and EG-HRP retained full activity over the 14-day duration of the experiment. At pH 4, native HRPC activity declined to zero within 8 days but EG-HRP retained approx. 70% activity at this time and approx. 55% activity at 14 days. At pH 11.6, EG-HRP again retained more activity than native: remaining activities of native and EG-HRP samples after 7 days were approx. 50% and 75% respectively. These results clearly show that modification of HRPC lysines with ethylene glycol bis-succinimidyl succinate improves the enzyme's storage stability at pH values removed from neutrality. The exact nature of the deleterious reaction or process resulting in loss of activity has not yet been ascertained, however.

[1] Welinder, K.G. Curr. Opin. Struc. Biol., 2, 388-393, 1992.

[2] Gajhede, M., Schuller, D.J., Henriksen, A., Smith, A.T., Poulos, T.L. Nature Struc. Biol., 4, 1032-1038, 1997.

[3] Dunford, H.B. Heme Peroxidases, Wiley-VCH, NY, 58-218, 1999.

[4] Ryan, O., Smyth, M.R., Ó'Fágáin, C. Enzyme Microb. Technol. 16, 501-505, 1994.

[5] Miland, E., Smyth, M.R., Ó'Fágáin, C. Enzyme Microb. Technol. 19, 242-249, 1996.

[6] Ó'Fágáin, C. Stabilizing Protein Function, Springer Verlag, Berlin, 23-28, 1997.