Immobilization and stabilization of horseradish peroxidase isoforms

Purified anionic and cationic isoforms of horseradish peroxidase (HRP) immobilized by coupling the amino acid side-chain amino groups and/or carbohydrate moieties to Sepharose have been studied for their resistance to denaturation. The isoforms were treated with periodate followed by ethylenediamine to generate additional amino groups in the glycosyl residues. The immobilized preparations were: Preparation I (Sp-aHRP, Sp-cHRP), in which HRP was covalently immobilized via side-chain amino groups exclusively; Preparation II (Sp-NHaHRP, Sp-NHcHRP), in which periodate and ethylenediamine-treated HRP was covalently immobilized via side-chain amino groups and amino groups incorporated into glycosyl residues. HRP isoforms in preparation II lacked about 33-55% carbohydrate. Both strategies of immobilization induced significant stabilization against denaturation. Inclusion of 2 mM calcium enhanced isoenzyme stability significantly.