Analytical Applications Of Papain As Peroxidase Mimetics

Peroxidase as an important catalyst has attracted much interest for application in chemical industry,medicine,and analytical chemistry.Peroxidase widely exists in plants as well as microorganisms and it can catalyze the oxidation of some electron donor substrates,such as 2,3-dimethoxyphenol,guaiacol,luminol,2,2'-Azinobis-?3-ethylbenzthiazoline-6-sulphonate??ABTS?and 3,3?,5,5?-tetramethylbenzidine?TMB?by hydrogen peroxide.As biological catalysts,peroxidases exhibit some remarkable advantages such as good water solubility,excellent biocompatibility,high catalytic activity and high substrate specificity under mild conditions.However,they can easily be denatured by environmental change?such as pH and temperature?and digested by protease.Furthermore,they are usually expensive and time-consuming for preparation and purification.Recent years,In order to extend the applications of peroxidase,researchers have been exploring the similars which would overcome the limitations of peroxidase-artificial enzyme mimics.So far,artificial enzyme are widely used in various fields because of its high catalytic activity,good stability,tolerance in extreme environment,reuse.Compared with nature enzymes,enzyme mimetics can be designed flexibly and synthesized with low cost as well as high stability.In order to stabilize the nanoenzymes,they are generally required capping agents,which will cause the loss of the catalytic activity,a lot of nanomaterials is also very difficult to mass production,the performance of different batches of the same materials often have great differences.It is significant to explore new peroxidase mimetics.In the previous study,we have found that papain possesses peroxidase-like activity.consequently,The method of colorimetric detection of uric acid sarcosine and AA was established.At the same time,we also investigated the effect of metal ions-Mn²⁺ on the activity of papain.?1?Peroxidases have attracted much attention due to their potential applications to varies fields.However,the application of natural peroxidase is limited for it is expensive and easily influenced by the environment.In this paper,we have demonstrated that papain possessed peroxidase-like activity,which could catalyze the oxidation of the peroxidase substrate 3,3?,5,5?-tetramethylbenzidine?TMB?by H₂O₂ to a blue colored product.A low-cost colorimetric method was developed for uric acid?UA?detection using uricase and papain.As low as 2.4 ?M UA could be detected with a linear range from 20 to 200 ?M and the proposed method was successfully applied for the determination of UA in human serum sample.?2?Sarcosine is the marker of prostate cancer,Therefore,it is significant for us to detect the concentration of sarcosine in biomedicine.As mimetic peroxidase,Papain can catalyze the oxidation of 3,3?,5,5?-tetramethylbenzidine?TMB?in the presence of H₂O₂ to produce colored reaction and appear the maximum absorbance value in the A652.A rapid,simple,high selectivity and sensitivity method for quantitative determination of sarcosine has been developed.?3?Ascorbic acid is also known as vitamin C,which is one kind of essential cofactor in human body,the deficiency of ascorbic acid is usually related with some diseases,such as cardiovascular desease,cancer and so on.Thus,it is meaningful to detect ascorbic acid.Papain can efficiently catalyze the oxidation of TMB by H₂O₂.The reaction could be hindered in the presence of ascorbic acid,for which can be oxidized by H₂O₂.So we established a colorimetric method for detecting the content of ascorbic acid.?4?Mn²⁺ is one kind of important metal elements in nature,as well as a trace element needed in human body.Papain,which possesses peroxidase enzyme activity,can catalyze the oxidation of the peroxidase substrate 3,3?,5,5?-tetramethylbenzidine?TMB?to a blue colored product in the presence of H₂O₂.The peroxidase-like activity of papain can be inhibited by Mn²⁺,resulting in the blue faded and the decrease of absorbance value in 652 nm.We explored inhibition curve under the optimal conditions.Thus,we investigated the inhibition effect of metal ions Mn²⁺ on the peroxidase-like activity of papain.