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Horseradish Peroxidase:Isolation,Preparation and Purification

Fan Jun Li Chun Ji Yaoqing Cheng Beijiu Jiang Like (The Biotechnoligical Research Center,Anhui Agricultural University,Hefei 230036)  
Peroxidase (HRP,EC 1 11 1 7) was isolated and purified from horseradish tubes.The purification steps included centrafuge,ammonium sulphate frationing,heating 20 min at 50℃,acetone fractioning and CM-23 cellous anion exchange chromatography.The purified HRP has a K 4 of 2 5×10 5 and a RZ of 3 19.Its specific activity was 406 U/mg protein.SDS-PAGE shown one band.It also revealed that HRP subunit has a molecular of 40 KD.IEF-PAGE conformed three bands,which meant there existed three isozymes at least.The HRP activity dye indicated more isozymes existed.Some improving methods were made such as controlling pH with NH 3·H 2O,adding 5 mmol/L Fe 3+ and changing elution buffer.
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