Zymographic Determination of Intrinsic Specific Activity of Oxidases in the Presence of Interfering Proteins.

Zymography on peroxidase-entrapped gels enables the determination of the intrinsic specific activity (ISA) of H2O2-producing oxidases in the presence of interfering enzymes including catalase (decomposing hydrogen peroxide). To reveal the searched oxidase, the zymography gel is incubated first in the developing solution containing the appropriate substrate of the oxidase and an oxidizable chromogen such as o-phenylenediamine (o-PDA) peroxidase substrate. The zymographic gel, after image analysis, can be restained by Coomassie Blue for molecular weights, for the whole electrophoretic pattern and for the protein concentration, thus allowing for the determination of oxidase ISA.

Zymographic approach to determine the intrinsic enzyme specific activity of diamine oxidase in presence of interfering enzymes.

The purpose of this investigation was to elaborate a fast zymographic assay of oxidase enzymes in the presence of interfering enzymes as catalase (which disturbs current dosages based on H2O2 detection). This method also allows the determination of intrinsic specific activity (ISA) of oxidases, such as diamine oxidase (DAO) or glucose oxidase (GOD). The SDS-PAGE gels with entrapped peroxidase have been obtained by polymerization of acrylamide and bis-acrylamide in the presence of horse-radish peroxidase. The entrapped peroxidase was uniformly distributed in the PolyacrylAmide (PAA) material and did not migrate during electrophoresis. The obtained PAA gels allow the electrophoretic separation of various oxidases from contaminating proteins. As an example, to reveal DAO, the resulting PAA-gel should be incubated after the electrophoretic run in the developing solution containing putrescine (a DAO substrate) and o-phenylenediamine (a HRP substrate) to give coloured bands on the gel in the presence of DAO-generated H2O2. The results showed that is possible to determine the DAO in the presence of interfering catalase because they migrate differently. Thus, the H2O2 released in situ by DAO is no more decomposed by catalase because of its different mobility. It was also found that the same electrophoretic gel, after zymography, can be restained by Coomassie Blue for quantitation of proteins corresponding to the zymographic bands. With the obtained enzyme units and protein concentration it is also possible to calculate the intrinsic specific activity of DAO directly from the intensities of enzyme bands in zymography and from those of protein bands (Coomassie Blue staining), quantified by densitometry.