Isolation and properties of immunoperoxidase complexes
Gavrilova, E.M.; Dzantiev, B.B.; Egorov, A.M.
Biokhimiia 44(9): 1614-1622
1979
ISSN/ISBN: 0320-9725 PMID: 508866 Document Number: 143127
The properties of horseradish peroxidase(HRP)- G complexes obtained by covalent binding at the carbohydrate residues of the enzyme and oxidized by sodium periodate were studied. The composition of the complexes depended on the molar ratio of the protein components and on concentration of NaIO4. The immunoperoxidase complexes differed in their immunological and enzymatic activities depending on the composition. The complexes containing 1 or 2 molecules of enzyme/1 molecule of IgG retained up to 90-100% of activity of the original antibodies and about 70% of activity of the native enzyme. The maximum yield of these complexes was observed at initial molar concentrations of HRP/IgG equal to 2:1. The Km for the HRP complex and for native enzyme towards o-dianizidine coincide and are equal to (2.0 .+-. 0.2) .cntdot. 10-5 M. The value of kcat for the complex was about 200 s-1 (pH 7.4). The oxidation of the carbohydrate moiety of HRP affected the thermal stability of the enzyme within the complex. Thermal inactivation of the complex at 60.degree. C occurred in 2 steps, the inactivation rate at the 1st step (.apprx. 30% of HRP inactivation) being 3 times as high as that for the native enzyme. During storage at 4.degree. C the complex retained its immunological and enzymatic properties for 3 mo., while the lyophilized preparations of the enzyme in the presence of 1% albumin were preserved for 2 yr.