Chemical modification of Horseradish peroxidase with carboxylic anhydrides: Effect of negative charge and hydrophilicity of the modifiers on thermal stability

HRP has achieved a prominent position in the pharmaceutical, chemical, and biotechnological industries and therefore methods improving the stability and functionality of HRP will clearly broaden the range of its present and future applications. In the present study, chemical modification of HRP was carried out using citraconic anhydride and trimellitic anhydride introducing one and two carboxylic groups respectively, per each amino group. As is well known, chemical modification provides a rapid and inexpensive method to stabilize enzymes. Thermoinactivation, kinetic parameters and activation energy of catalysis and structural changes of HRP were studied using spectroscopic techniques. The results indicated that both modifiers stabilized HRP in the range of 50–60 °C but citraconic anhydride was the only stabilizer at higher temperatures. Structural study implied that HRP became less compact upon modification. In addition, catalytic efficiency and activation energy did not change remarkably following reaction of the enzyme with carboxylic anhydrides. We concluded that the effect of chemical modification with carboxylic anhydrides on the thermal stability depends on temperature condition probably, due to a competition between hydrophilization of the protein surface and repulsion of the negative charges occurring following modification.

Figure optionsDownload as PowerPoint slideHighlights
► Modification of HRP with the carboxylic anhydrides leads to its thermal stability.
► The higher number of carboxylic groups leads to higher stability.
► A slight decrease in compactness of HRP's structure occurs upon modification.
► A competition between hydrophilization and repulsion occurs upon the modification.