Papain As Peroxidase Mimetics And Their Applications In Analytical Chemistry

Enzymes are involved in nearly all life activities in nature. Natural enzymes can catalyze chemical reactions specifically and efficiently in the condition of mild conditions. Owing to their high catalytic efficiency, strong substrate specificity and mild reaction conditions, natural enzymes are applied widely in the medicine, agriculture and food industry. Encouraged by their superior property, a lot of efforts have been to extend the natural enzymes to enzyme mimetics. Very recently, poorphyrin, host reagents,molecular lmprinting polymers and membrane systems as enzyme mimetics have been used and developed. At the same time, nanomaterials have attracted considerable attention due to their surface effect, quantum size effect, little size effect, macroscopic quantum tunneling effect. However, the catalytic activity of nanomaterials has been ignored for a long time. Since the first report on the intrinsic peroxidase-like activity of Fe3O4 magnetic nanopartieles by Yan’ research group in 2007, nanopaticles as peroxidase-mimetics have received great interest. Various nanostructures including metal oxide nanomaterials, carbon nanomaterials, noble metal nanoparticles and other nanomaterials have also been found to possess an intrinsic enzyme mimetics catalytic activity which can catalyze the oxidation of the typical chromogenic substrates using hydrogen peroxide or other peroxides. Compared with natural enzyme, nanomaterials as enzyme mimetics own several advantages including high catalytic activity, high stability against stringent conditions and high utilization rate and so on. Nevertheless, the enzyme-like activity of nanoparticles relates to the nature, the composition, the size, the shape and the surface coating of the nanoparticles. The preparation of these nanomaterials requires laborious procedures which result in low reproducibility. The surface of nanoparticles is often needed to introduce a stabilizer to reduce the problem of aggregation. However, the existence of surface stabilizer could suppress the catalytic activity. Additionally, the preparation procedures and modification steps are complicated and time-consuming. Therefore, it still remains a great challenge to findnew peroxidase-like materials for target analysis. Based on this, we found that papain possess peroxidase-like activity, and demonstrated the catalytic mechanism. On the basis of these findings, the application of papain for colorimetric sensing of glucose and glutathione was explored.(1) It was found that papain possess an intrinsic peroxidase mimetics catalytic activity and catalyze H2O2 to oxidize peroxidase substrate TMB, ABTS and OPD. And papain’s catalytic activity is dependent on pH, temperature, H2O2 concentration and reaction time. The optimal conditions for the maximum catalytic activity of papain was5.5, 35 °C, 0.10 mM, 2 h, respectively. The apparent steady-state kinetic analysis suggested that papain’s catalytic reaction follow Michaelis-Menten model and ping-pong mechanism. ICP-MS data indicated that peroxidase-like catalytic activity of papain is not due to metal impurities. We also incubated papain with HRP for different time and then measured the catalytic activity of the mixture. These results showed that the peroxidase activity may be derived from an unknown impurity.(2) Detection of H2O2 has an important significance in many fields such as biology,medicine, environmental protection and food industry and so on. Due to the catalytic reaction involving hydrogen peroxide peroxidase and its mimics, the concentration of H2O2 can be directly measured according to the color change of the certain colorimetric substrates. The proposed method is based on the fact that papain in the presence of H2O2 can oxidize TMB to induce a blue color and an absorption peak centered at 652 nm. As demonstrated above, the absorbance variation of TMB oxidation catalyzed by papain was H2O2 concentration-dependent and the catalytic oxidation of glucose oxidase(GOx)produces H2O2, a method for quantitative determination of H2O2 and glucose concentration based on papain were developed.(3) Glutathione(GSH) is widely found in nature, and its thiol structure plays an important role in the maintenance of cell environment in the human body. We develop a fast and simple method for high sensitivity and high selective detection of GSH. Papain could oxidize TMB to induce a blue color in the presence of H2O2 and an absorption peak at 652 nm. The blue colored free cation radicals were generated and it could be successfully repaired with the introduction of GSH, both of which result in blue color fading and a decrease of absorbance at 652 nm. Based on this, we design a new colorimetric sensor for the detection of glutathione and the detection limit is 0.03 μM.