Intrinsic Peroxidase-like Activity Of Ficin And Its Analytical Applications

Enzymes are indispensable biological catalysts for self-replication and for the metabolism of organisms.They are the most specific catalysts known,both from the viewpoint of the substrate and the type of reaction performed on the substrate.In most cases,a particular enzyme fits a few types of substrate and catalyzes one type of reaction.The applications of nature enzymes are still limited because of its rigorous storage requirements,poor thermal instability,high expense,sensitivity to the environment and its short storage life due to denaturation and digestion.As a consequence,there is a good deal of current research interest in artificial enzymes,such as oxidase,protease,catalase,superoxide dismutase or peroxidase.Research of artificial peroxidase is the most widespread and profound one.To date,more and more nanozymes were found to possesse mimetic activity.These mimetic enzymes promote the development of artificial peroxidase.To one's disappointment,however,some of these non-biological catalysts often need laborious preparation procedures and modification steps to suppress aggregation,which would result in low reproducibility and low catalytic activity.Biological catalysts,in comparison to non-biological catalysts,possess particularly high catalytic efficiency,high reaction rates under very mild and favorable biological reaction conditions.For this reason,it is still highly desirable to find biological peroxidase-like materials.Here,we show our discovery that ficin not only possesses protease activity but also peroxidase-like activity.As a natural plant-protease,ficin has advantages of both biological catalysts and artificial enzymes,it shows a good prospect of application.On the basis of the peroxidase-like of ficin,some analytical application were accomplished: detection of H₂O₂ releasing from living cells,detection of L-cysteine,detection of mercury ion.?1?We found sufficient proofs for the intrinsic peroxidase-like activity of ficin,which could catalytically oxidize organic substrates?such as TMB,ABTS,OPD?by H₂O₂ to colour reactions,and designed experiments to examine its effectiveness in a variety of scenarios.Similar to HRP,the peroxidase-like activity of ficin was dependent on pH,temperature,substrate concentrations,incubation time,and showed typical Michaelis-Menten kinetics mode with a Ping-Pong mechanism.In addition,the active sites of peroxidase-like activity of ficin are different from those of protease,which reveals that one enzyme may catalyze more than one kind of substrate to perform different types of reactions.These results support deeper knowledge of enzyme functions.?2?Combining the peroxidase-like activity of ficin with peroxidase substrate TMB,a simple,sensitive,practical colorimetic detection of H₂O₂ releasing from MCF-7 cells was established.As the H₂O₂ detecting probe,ficin doesn't need complex synthetic process,any modification and it is environmentally friendly.Our findings support a wider prospect of application of ficin in biochemistry and open up the possibility of utilizing ficin as enzymatic mimics in biotechnology and environmental monitoring.?3?We provided a facile,green TMB+H₂O₂+Ficin colorimetric approach for L-cysteine detection with high specificity.Ficin acted as enzymatic mimics and catalyze the chromogenic reaction of TMB and H₂O₂.However,after the introduction of L-cysteine,L-cysteine competed with TMB for common H₂O₂,resulting in fading the chromogenic reaction.This method offered several distinct advantages: the facility of ficin as enzymatic mimics without any modification and complex synthesis;easy and direct visualization of end result with color change;low cost by the use of simple instrument;chromogenic reaction could take place under very mild and favorable biological reaction conditions.Our work could promote the application of the enzymatic mimic activity of ficin in medical diagnostics and biochemistry.This method can be successfully applied to detect L-cysteine in biological fluids,indicating its potential in biological sample analysis.?4?Heavy metals pollution,represented by mercury,is a serious threat to human health and the social environment.It is nonnegligible to establish economic and efficient mercury ion detecting method.On the basis of the peroxidase-like of ficin and the high affinity between cysteine and mercury ion,we develop a “inhibition-recovery” colorimetic quantitative detection of mercury ion.Ficin could catalyze the chromogenic reaction of TMB+H₂O₂,and the introduction of cysteine resulted in fading the chromogenic reaction?cysteine competed with TMB for common H₂O₂?.However,mercury ion combined with cysteine resulting in lower cysteine concentration and then the blue color recovered.This mercury ion detecting method is lable-free,economic,green,indicating that ficin can be a peroxidase-like probe applied to environmental analysis and ficin has great potential application in environmental monitoring.