study on photosensitizers has received more and more attention . however , the effect of the photosensitization on dna , protein , cell , or even an organ should be given more attention , no matter whether the effect is harmful or helpful for the potential clinical application . it can be extracted from h. perforatum , a popular medicine for the treatment of depression [ 18 ] . it has been used as a conventional medication for the treatment of depression and wound healing for a long time . recently , more and more interest has been given to some other important pharmaceutical potentials of this species , such as antivirus activity , anti - hiv , antibacterial activity and antitumor activity [ 1012 ] . most of the clinic functions are proposed to be related to the phototoxicity of hy . for instance , the cytotoxic activity can be greatly enhanced after light activation [ 9 , 1316 ] . the mechanism how it works is still to be explored , however , it is proposed that after being exposed in visible light at the wavelength of 540600 nm , hy will transfer light energy to oxygen to generate reactive oxygen species ( ros ) , which may further induce the apoptosis of cells . some other studies report that hy preferentially accumulates in the cell membranes , especially the mitochondria membrane , which might be the target in the photodynamic therapy . both the type i and type ii photodynamic reactions can take place in the photoactivation of hy , resulting in the formation of radicals , such as singlet oxygen and superoxide radical . in the paper , we report our studies of the photodynamic effect of hy on the structure and function of a protein by employing uv - vis spectroscopy and electrochemical methods . hemoglobin ( hb ) , a kind of heme protein , is used as the target protein for this work . on one hand , the structure and function of hb are relatively very clear , which is easier to get to know the information of the conformational and functional changes . on the other hand , it is relatively easier to design the experimental protocols , since its electrochemistry has been somewhat largely studied in our lab [ 19 , 20 ] . besides , we have found that the reactive molecules , such as hydroxyl radical , superoxide anions , may influence the peroxidase activity of the protein , which may be ascribed to the conformational transformation of the protein . based on the data obtained in this study , we found that visible light irradiation of the protein in the presence of hy would cause the change of its conformation . and , the peroxidase activity of the protein towards hydrogen peroxide ( h2o2 ) can be obviously enhanced . since the structural and formational changes can take place after the treatment of the protein with visible light , the process of which is so common to occur , this study should be very interesting to lots of scientists in different research areas . uv - vis spectroscopy is a very helpful technique to study the conformational changes of heme proteins , since the soret band of the heme which is located at 407 nm can provide very useful information on the secondary structure of heme proteins [ 2326 ] . the position of the soret band will shift or the absorption will decrease if the structure of a heme protein is transformed . figure 1 shows that the absorption of the soret band remains almost unchanged even after 3h irradiation by visible light in the absence of hy , however , the peak will decrease evidently only after 1 hour treatment if this medicinal species is in the presence . therefore , the heme moiety of the protein can be hardly influenced only by visible light . nevertheless , irradiation treatment on hb together with hy , the structure of the protein , especially the microenvironment of the heme ring , will be changed or even be badly damaged . we propose that the ros , generated by the irradiation of hy , may react with the protein at the heme position , and change the microenvironment of the heme ring . it can be also observed from fig.1 that the decrease of the absorption is much less significant for the case of lower hy concentration . so , the photodynamic effect of hy on hb is not only a time dependent but also a concentration dependent process . we have further employed electrochemical method to check whether the dynamic activity of hy may have any effect on the peroxidase activity of the protein . as is well known , , some other activities of this protein have also been revealed . especially in recent years , the peroxidase activity of hb has been largely studied , and these findings have been employed for the development of third - generation biosensors . based on our previous work of the preparation of protein - film modified electrodes and the fabrication of hb - based h2o2 biosensors with this protein [ 1921 ] , we have first prepared an hb - hy film modified electrode to study the catalytic activity of hb towards h2o2 . as is shown in figure 2 , the reduction peak of hb will increase gradually with the addition of h2o2 in the test solution . further studies reveal that a linear relationship between the increase of the reduction peak and the h2o2 concentration can be obtained from 110mol / l to 510mol / l , and the linear regression equation is : y = 2.15459 + 0.27599x , r = 0.999 . in order to obtain an obvious contrast , and to more clearly show the change of the peroxidase activity of the protein , the value of the reduction peak with the addition of 110mol / l h2o2 is chosen as the baseline , and is set to be 0 . as is shown in figure 3 , the slope of the catalytic linearity is enhanced obviously after the irradiation of visible light on hb together with hy . , the slope increases with the irradiation time , which suggests that a longer time treatment on the samples will result in a higher catalytic ability of the protein . meanwhile , concentration of hy is found to be another key factor . as is shown in figure 4 , although the slopes will also increase for the case of low hy concentration , the changes are indistinct in the first two hours . and the increase is much smaller than that for the case with high hy concentration . these experimental results obtained with electrochemical method coincide very well with the results obtained by uv - vis spectroscopy , which further reveals that the photodynamic effect of hy is not only a time - dependent , but also a concentration - dependent process . from our further studies , this effect has also been known to be an oxygen - dependent process , although o2 is not required for the antivirus activity of hy . as is shown in fig . 5 , if the samples are prepared and irradiated in an anaerobic situation , the catalytic slope will keep unchanged even if a 3h treatment has been made . this is reasonable , since o2 is required in a photodynamic process to generate ros , which is crucial in photoactivating hy . so , no influence of this medicinal species on the peroxidase activity of hb can be obtained without o2 being involved . these results have also confirmed that the phtosensitization of hy would be achieved only in the presence of oxygen . after absorbing the light energy , hy will be transformed to be an excited state , which may further transfer the energy to oxygen directly or indirectly , both of which induce the formation of the ros , especially the singlet oxygen . ros then react with hb , which may induce the change of the microenvironment of the heme position . as a result , the catalytic ability of the protein towards h2o2 is enhanced . in summary , with uv - vis spectroscopic and electrochemical techniques , we have found that irradiation on the mixture of hb and hy by the very common visible light can activate hy to generate ros , which will make the change of the structure of hb and enhance the catalytic activity of the protein towards the h2o2 reduction . this process depends not only on the irradiation time but also on the concentration of hy . meanwhile , o2 , which is required for the generation of ros , is also found to be a vital element in the photoactive effect . this work has not only confirmed the photosensitization of hy and revealed the enhanced peroxidase activity of hb , but also be helpful to the development of more sensitive h2o2 biosensor and the usage of this medicinal herb molecule . stock solutions were prepared by dissolving known amounts of hy in 1 ml of dimethyl sulphoxide ( dmso ) . stock solutions were prepared by dissolving known amounts of hb in 1 ml of double - distilled water . dmso and h2o2 ( 30% ( w / v ) solution ) were provided from shanghai jinshan tingxin chemical reagent co. and nanjing chemical reagent co. , respectively . double - distilled water , which was purified with a milli - q purification system ( branstead , boston , ma , u.s.a . ) to a specific resistance of > 18 m  cm , was used in all the experiments , and all the solutions were stored in the refrigerator at 4 c . sample i preparation : 1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample ii preparation : 0.5l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample iii preparation:1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . this sample was thoroughly deoxygenated by blowing high - purity nitrogen for at least 1 min , and then sealed by parafilm . the samples were exposed in the visible light emitted by a 200w filament lamp , which was filtered by an orange filer to get a wide band illumination above 580 nm , for 1h , 2h , 3h , respectively , at a distance of 20 cm . uv - vis spectroscopy was performed using a uv-2550 spectrophotometer ( shimadzu , japan ) . after the treatment with visible light irradiation , the samples were then twentyfold diluted and analyzed immediately with the spectrophotometer . the substrate pyrolytic graphite ( pg ) electrode ( a = 6.28 mm ) was prepared by inserting a pg rod in a glass tube and fixing it with epoxy resin . electrical contact was made by attaching a copper wire to the rod with the help of wood 's alloy ( a fusible bismuth - based alloy ) . before the modification of the substrate electrode with hy and the protein , its surface was then polished to mirror smoothness with alumina ( particle size of approx . finally , the electrode was thoroughly washed by ultrasonicating in both double - distilled water and ethanol for approx . 10l of hy and hb mixture with their concentration being 110mol / l and 6.210mol / l , respectively , was mixed with 10l dmso ( 20% ) and then spread evenly on the surface of the substrate pg electrode . the hy and hb mixture had been previously treated with visible light irradiation before its immobilization onto pg electrode surface . after that , this electrode was thoroughly rinsed with double - distilled water and could be ready for use . cyclic voltammetry ( cv ) was performed with a parc 263a potentiostat / galvanostat ( eg&g ; princeton , nj , usa ) , using a three - electrode configuration at 25  0.5 c . the reference electrode was a saturated calomel electrode ( sce ) and the counter electrode was a platinum electrode . all the test solutions were thoroughly deoxygenated by bubbling high - purity nitrogen through the solution for at least 10 min . a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiments . stock solutions were prepared by dissolving known amounts of hy in 1 ml of dimethyl sulphoxide ( dmso ) . stock solutions were prepared by dissolving known amounts of hb in 1 ml of double - distilled water . dmso and h2o2 ( 30% ( w / v ) solution ) were provided from shanghai jinshan tingxin chemical reagent co. and nanjing chemical reagent co. , respectively . double - distilled water , which was purified with a milli - q purification system ( branstead , boston , ma , u.s.a . ) to a specific resistance of > 18 m  cm , was used in all the experiments , and all the solutions were stored in the refrigerator at 4 c . sample i preparation : 1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample ii preparation : 0.5l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample iii preparation:1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . this sample was thoroughly deoxygenated by blowing high - purity nitrogen for at least 1 min , and then sealed by parafilm . the samples were exposed in the visible light emitted by a 200w filament lamp , which was filtered by an orange filer to get a wide band illumination above 580 nm , for 1h , 2h , 3h , respectively , at a distance of 20 cm . uv - vis spectroscopy was performed using a uv-2550 spectrophotometer ( shimadzu , japan ) . after the treatment with visible light irradiation , the samples were then twentyfold diluted and analyzed immediately with the spectrophotometer . the substrate pyrolytic graphite ( pg ) electrode ( a = 6.28 mm ) was prepared by inserting a pg rod in a glass tube and fixing it with epoxy resin . electrical contact was made by attaching a copper wire to the rod with the help of wood 's alloy ( a fusible bismuth - based alloy ) . before the modification of the substrate electrode with hy and the protein , its surface was then polished to mirror smoothness with alumina ( particle size of approx . finally , the electrode was thoroughly washed by ultrasonicating in both double - distilled water and ethanol for approx . 10l of hy and hb mixture with their concentration being 110mol / l and 6.210mol / l , respectively , was mixed with 10l dmso ( 20% ) and then spread evenly on the surface of the substrate pg electrode . the hy and hb mixture had been previously treated with visible light irradiation before its immobilization onto pg electrode surface . the modified electrode was dried overnight at room temperature in the dark . after that , this electrode was thoroughly rinsed with double - distilled water and could be ready for use . cyclic voltammetry ( cv ) was performed with a parc 263a potentiostat / galvanostat ( eg&g ; princeton , nj , usa ) , using a three - electrode configuration at 25  0.5 c . the reference electrode was a saturated calomel electrode ( sce ) and the counter electrode was a platinum electrode . all the test solutions were thoroughly deoxygenated by bubbling high - purity nitrogen through the solution for at least 10 min . a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiments .
hypericin , extracted from h. perforatum , can induce the generation of reactive oxygen species by visible light irradiation , which may consequently induce the conformational change of hemoglobin . 
 we have not only employed uv - vis spectroscopy to observe the changes of uv - vis spectra of the protein , which reveals the conformational changes of the protein , but also employed electrochemical method to obtain its enhanced peroxidase activity . 
 the photodynamic effect of hypericin on the conformation and catalytic activity of the protein has also been proven to be strongly dependent on the irradiation time , the hypericin concentration and the presence of oxygen . 
 this work is beneficial not only to the fabrication of more sensitive hydrogen peroxide biosensor , but also to the guidance of the usage of this medicinal herb molecule , since the conformational change of the protein and the enhanced peroxidase can be easily obtained only by visible light irradiation on hypericin , the process of which is so common to happen .