RESUMO
The performances of different methods of quantification of protein (methods based on direct spectrophotometric and spectrofluorimetric analysis, chemical reactions and liquid chromatography) to quantify the amount of enzyme incorporated into polyalkylcyanoacrylate nanoparticles, were compared. A methodology based on size exclusion chromatography was selected. The performances of the analytical method to quantify the enzymes L-asparaginase and superoxide dismutase in different polymerization media of poly-isobutilcyanoacrylate, were evaluated. The quantification of superoxide dismutase in the presence of esterase, enzyme used to solubilize nanoparticles, was attempted. An adequate separation between enzyme and the other components of polymerization media was achieved, so the selectivity of the method is adequate to the quantification of an enzyme in polymerization medium, either before or after polymerization. Although lack of selectivity of the column to separate enzymes was observed. The retention time of L-asparaginase and superoxide dismutase in polymerization medium are, respectively, 7.4 and 7.5. Linear correlation between peak area and enzyme concentration were observed for both enzymes in the concentration range from 10 to 80 micrograms ml-1, either before or after polymerization and in different polymerization media. This SE-HPLC analytical methodology is adequate to determine the degree of incorporation of enzymes in polyalkylcyanoacrylate nanoparticles as evidenced by the linear responses of the chromatographic method, the reproductibility of repeated sample injections and the precision of the quantification of enzyme concentration.
