Interaction mechanism between berberine and the enzyme lysozyme

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 209–214

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Interaction mechanism between berberine and the enzyme lysozyme Ling-Li Cheng a, Mei Wang b, Ming-Hong Wu a, Si-De Yao b, Zheng Jiao a,⇑, Shi-Long Wang b,⇑ a b

Shanghai Applied Radiation Institute, Shanghai University, Shanghai 200444, China School of Life Science and Technology, Tongji University, Shanghai 200092, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Static quenching is the main

The possible photooxidation mechanisms of Trp and Lys caused by BBR. In the oxygen-free polar solvent, the photo-induced active radicals of BBR (BBR+/BBR(-H) and 3BBR⁄) can oxidize Trp and Lys directly via an electron transfer mechanism (Type I mechanism). In the presence of O2, the photooxidation Lys involves both Type I and II processes, and Type I mechanism is competitive with 1O2. Which reaction is major pathway is dependent on the concentrations of O2 and the polar of the solvent.

fluorescence quenching mechanism between Lys and BBR, and there is one binding site in Lys for BBR. " BBR neutral radical react with Trp (K = 3.4  109 M1 s1) via electron transfer to give the radical cation (Trp/NH+) and neutral radical (TrpN) of Trp. " BBR can selectively oxidize the Trp residues of Lys with the rate constant 4.2  108 M1 s1. " Through thermodynamic calculation, the reaction mechanisms between 3BBR⁄ and Trp or Lys were determined to be electron transfer process.

a r t i c l e

i n f o

Article history: Received 16 January 2012 Received in revised form 5 April 2012 Accepted 1 May 2012 Available online 2 June 2012 Keywords: Berberine Fluorescence spectroscopy Electron transfer Laser flash photolysis

a b s t r a c t In the present paper, the interaction between model protein lysozyme (Lys) and antitumorigenic berberine (BBR) was investigated by spectroscopic methods, for finding an efficient and safe photosensitizer with highly active transient products using in photodynamic therapy study. The fluorescence data shows that the binding of BBR could change the environment of the tryptophan (Trp) residues of Lys, and form a new complex. Static quenching is the main fluorescence quenching mechanism between Lys and BBR, and there is one binding site in Lys for BBR and the type of binding force between them was determined to be hydrophobic interaction. Furthermore, the possible interaction mechanism between BBR and Lys under the photoexcitation was studied by laser flash photolysis method, the results demonstrated that BBR neutral radicals (BBR(-H)) react with Trp (K = 3.4  109 M1 s1) via electron transfer to give the radical cation (Trp/NH+) and neutral radical of Trp (TrpN). Additionally BBR selectively oxidize the Trp residues of Lys was also observed by comparing the transient absorption spectra of their reaction products. Through thermodynamic calculation, the reaction mechanisms between 3BBR⁄ and Trp or Lys were determined to be electron transfer process. Ó 2012 Elsevier B.V. All rights reserved.

Introduction Berberine (BBR, Scheme 1) is a natural isoquinoline alkaloid and possesses a wide range of pharmacological and biochemical

activities including antiinflammatory [1], antimalarial [2], antiviral [3], antidepressantlike [4], antimicrobial [5,6], hypolipidermic [7], and anxiolytic effects [8]. Recently, BBR has been proved to possess antitumor activity in vitro and in vivo [9]. The wide ranging

⇑ Corresponding authors. Tel./fax: +86 21 66137803 (Z. Jiao), tel.: +86 21 65982595; fax: +86 21 65982286 (S.L. Wang). E-mail addresses: [email protected] (Z. Jiao), [email protected] (S.-L. Wang). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.05.035

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prepared in 2 mM sodium phosphate buffer (pH 7.0) or in its mixture with acetonitrile, and purged with nitrogen (N2, P99.99%) or oxygen (O2, P99.5%) for 20 min in different experimental situation. All experiments were carried out at room temperature. Ground-state absorption properties were studied using a UV–visible spectrometer (VARIAN CARY 50 Probe). Fluorescence measurements

Scheme 1. Molecular structures of berberine.

biological activities of BBR in general and its anticancer activities in particular have generated considerable interest to study its mechanism of action. Several studies show that BBR is able to produce 1 O2 and radical species if it is exposed to UVA irradiation [10–12], which means that it might be used as a new type of photodynamic therapeutic agent. Photodynamic therapy (PDT) is an approved, minimally invasive therapeutic approach for the management of a variety of tumors and certain benign diseases, which based on the combination of a photosensitizer that is selectively localized in the target tissue and illumination of the lesion with light of specific wavelength, resulting in photodamage and subsequent cell death [13–17]. The photosensitization process is classified into two major types of reaction. Type I process involves the direct interaction of the actives radicals of photosensitizer with a substrate (proteins, lipids or nucleic acids), and Type II involves reaction between sensitizer and oxygen to form reactive oxygen species (ROS) [18–21]. Both Type I and Type II reactions can occur simultaneously, and the ratio between these processes depends on the type of photosensitizer used, as well as the concentrations of substrate and oxygen [22,23]. In our previous studies [24,25], we have identified that BBR could be excited by both 355 and 266 nm laser to produce BBR excited state (3BBR⁄), BBR radical anion (BBR), BBR radical cation (BBR+) and BBR neutral radicals (BBR(-H)) through the fast deprotonation of BBR+. And the related photophysical and photochemical behaviors of them was also studied by means of time-resolved laser flash photolysis (LFP). All these results show that BBR might be a new type of photosensitizer using in PDT. In spite of the potential importance of BBR as a photosensitizer in photobiology and photomedicine, few studies of protein photodamage initiated by BBR have been carried out. To gain further insight into the important biological process and the pharmacological mechanism under physiological conditions, in the present paper, some photobiological characters of BBR were studied in details. By taking lysozyme (Lys) as a model globular protein, the mechanism of interaction between Lys and BBR was explored by fluorescence spectroscopy. Furthermore, the possible interaction mechanisms between BBR and tryptophan (Trp) or Lys under the photoexcitation were studied by LFP, and the related photodynamic parameters were also obtained.

Experimental Chemical reagent BBR, tryptophan (Trp) and lysozyme (Lys) were purchased from Sigma and used as received. Acetonitrile was spectrophotometric grade. All solvents were of the highest available commercial grade except the water, which was triply distilled. The samples were

Fluorescence spectroscopy experiments were performed on an F-4500 Fluorescence Spectrophotometer (Hitachi). Spectra were measured at an excitation wavelength of 280 nm and an emission wavelength range of 300–500 nm, using a 1  1 cm pathlength quartz cuvette (2 mL) with slit widths 5 nm for both excitation and emission channels. In order to eliminate the inner filter effects of protein and ligand, absorbance measurements were performed at excitation and emission wavelengths of the fluorescence measurements. The fluorescence intensity was corrected using the following equation [26,27]:

F cor ¼ F obs 10ðA1 þA2 Þ=2

ð1Þ

where Fcor and Fobs are the fluorescence intensities corrected and observed, respectively; A1 and A2 are the sum of the absorbance of protein and ligand at the excitation and emission wavelengths, respectively. Laser flash photolysis experiments LFP experiments were carried out using Nd: YAG laser of 355 nm light pulses with a duration of 5 ns and the energy of 60 mJ per pulse used as the pump light source. A 250 W xenon lamp was employed as detecting light source. The laser and analyzing light beam passed perpendicularly through a quartz cell with an optical path length of 10 mm. The transmitted light entered a monochromator equipped with an R955 photomultiplier. The output signal from the Agilent 54830B digital oscillograph was transferred to a personal computer for data treatment. The LFP setup has been previously described [24]. Results and discussion Characterization of the binding interaction of BBR with Lys by fluorescence measurements Fluorescence quenching BBR emits fairly feeble fluorescence in aqueous solution, for which quantum yield of UF = 4.7  104 has been reported in D2O [10]. On photoexcitation, BBR is primarily excited to singlet state, then by rapid intersystem crossing come to the non-fluorescent triplet state. On the other hand, Lys is a well-known fluorescent protein due to the intrinsic fluorescence of its Trp residues. Therefore, to study the interaction of Lys and BBR with the fluorescence techniques, we have chosen Lys as a fluorophore and BBR as a quencher. Although, the absorption wavelength of BBR (Fig. 1B) lies very close to the emission wavelength of Lys, the insignificantly small quantum yield of BBR renders no significant interference to the emission of Lys. As shown in Fig. 2, the fluorescence intensity of Lys decreased regularly with an increasing concentration of BBR. With addition of BBR of varying concentration (0.01–0.08 mM), the fluorescence intensity of Lys with wavelength maximum around 340 nm due to the presence of six native Trp residues decreased significantly with a new peak generated with a maximum around 370 nm. The fluorescence quenching of Lys around 340 nm indicated that

L.-L. Cheng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 209–214

the environmental change of the Trp residue of Lys [28]. And the formation of a new complex with fluorescence maximum at 370 nm could be ascribed to the interaction of BBR and Lys on photoexcitation [29]. However, the absorption spectra of Lys with different concentrations of BBR (Fig. 1C) show that when BBR was added to the solution of Lys, the mixture absorption spectra had neither red or blue shifts, which means there is not chemical bond between Lys and BBR. Furthermore, the insignificant quantum yield of BBR also implied that it can be ignored the possibility of generation of any new species due to fluorescence resonance energy transfer at this longer wavelength [29]. Quenching mechanisms are usually classified into dynamic and static quenching [30]. Since higher temperature results in larger diffusion coefficients, the dynamic quenching constants will increase with raising temperature. In contrast, increased temperature is likely to result in decreased stability of complexes, and thus lower the value of the static quenching constants [31]. To confirm the quenching mechanism of the interaction of BBR and Lys, we analyzed the fluorescence spectra at different temperatures according to the Stern–Volmer equation [32]:

F0 ¼ 1 þ K SV ½Q  ¼ 1 þ kq s0 ½Q  F

ð2Þ

where F0 and F is the fluorescence intensity at 340 nm in the absence and presence of the quencher, respectively. [Q] is the concentration of the quencher, s0 is the fluorescence lifetime in the absence of quencher, kq is the quenching rate constant of the biological macromolecule and KSV is the Stern–Volmer quenching constant. The Stern–Volmer plots before and after correction were presented in Fig. 3A. It is appeared in Fig. 3A that the plot before correction was not linear. After being corrected with Eq. (1) to remove the inner filter effect, the plot showed results that agree with the Stern–Volmer Eq. (2). The corrected fluorescence data were analyzed according to F0/F versus [Q] at 298, 308 and 318 K (Fig. 3B). Eq. (2) was applied to determine KSV (Table 1) by a linear regression plot of F0/F against [Q]. The value of kq was also obtained (the fluorescence lifetime of the biopolymer (s0) is 1.0  108 s [33]). The maximum scatter collision quenching constant of various quencher with biopolymer is 2.0  1010 M1 s1 [34]. The KSV values decreased with increasing temperature and Kq was greater than 2.0  1010 M1 s1. Thus, the results indicated that the overall quenching was dominated by a static quenching mechanism in the interaction between BBR and Lys.

Fig. 2. Influence of BBR on Lys fluorescence. Conditions: Lys 0.01 mM; BBR (1–9) 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 mM; pH 7.0; T = 298 K.

Association constants and number of binding sites For the static quenching interaction, when small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Ka) and the number of binding sites (n) can be obtained from the equation [35]:

log

ðF 0  FÞ ¼ log K a þ n log½Q  F

ð3Þ

where F0, F and [Q] are the same as in Eq. (2), Ka is the binding constant and n is the number of binding sites. The values of n and Ka were calculated in Table 2. The number of binding sites n approximately equals to 1, indicating that there was one binding site in Lys for BBR during their interaction under different temperatures.

Determination of the binding forces There are four types of action forces between small molecule and biological macromolecules: hydrophobic forces, hydrogen bonds, van der Waals’ interactions and electrostatic forces. Thermodynamic parameters are important for confirming the noncovalent acting forces. Ross and Subramanian [36] have summed up the thermodynamic laws to determine the types of binding with various interactions. If DH0 < 0, DS0 < 0, the main forces are van der Waals and hydrogen bond interactions; if DH0 < 0, DS0 > 0, electrostatic effect is dominant; if DH0 > 0, DS0 > 0, hydrophobic interactions play the main roles in the binding reaction. When the change of temperature is small, DH0 can be considered as a constant. The enthalpy change (DH0), free-energy change (DG0) and the entropy change (DS0) for the interaction between BBR and Lys (Table 2) were calculated based on the van’t Hoff equation (Eq. (4)) and thermodynamic equations (Eqs. (5) and (6)):

!     K2 1 1 DH 0 ¼ ln  T1 T2 K1 R

DG0 ¼ RT ln K a 0

0

DG ¼ DH  T DS

Fig. 1. The absorption spectra of Lys (0.01 mM) (A), BBR (0.05 mM) (B), and Lys (0.01 mM) with different concentrations of BBR (from 0 to 0.04 mM) (C), respectively.

211

ð4Þ ð5Þ

0

ð6Þ

where T is the reaction temperature, K1 and K2 are the binding constants (analogous to Ka in Eq. (3)) at T1 and T2, and R is the universal gas constant. Negative DG0 means that the interaction process was spontaneous. The positive DH0 and positive DS0 indicate that hydrophobic interaction played major roles in the interaction between BBR and Lys.

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The transient absorption spectra of BBR after 355 nm LFP in O2saturated acetonitrile/ phosphate buffer (4/1, v/v) solution in the presence of Trp was shown in Fig. 4. On the basis of the molar absorption coefficient of Trp at 355 nm is much less than that of BBR, the photon directly absorbed by Trp can be neglected, which is further verified by performing a blank experiment. So the laser energy was mainly absorbed by BBR. Under our experimental condition, BBR+ was initially generated by direct 355 nm photoionization, then through rapid deprotonation to produce BBR(-H) [24,25]. 3BBR⁄ (if it existed) would be quenched by oxygen and the electron should be trapped by acetonitrile to give a dimmer radical anion that did not absorb appreciably below 750 nm [41]. The absorption peak at 460 nm observed at 100 ns after 355 nm laser flash excitation should be assigned to the typical absorption of BBR(-H) (Fig. 4), which was also in good agreement with the absorption of BBR(-H) in our previous experiment [25]. As shown in Fig. 4, accompanying the decay of BBR(-H), the transient spectra showed two absorption peaks around 350 and 520 nm, which were in good agreement with the absorption of the radical cation (Trp/NH+) and the neutral radical (TrpN) of Trp reported previously [29,42–44]. Therefore, it could be confirmed that Trp was oxidized by BBR(-H) via electron transfer from Trp to BBR(-H) to produce Trp/NH+ and TrpN. In our work, for the bleaching peak caused by ground state of BBR was overlapped with the absorption peak of Trp/NH+ (350 nm), the kinetic trace of TrpN at 520 nm was chosen to be investigated as shown in the inset of Fig. 4. The growth of the trace at 520 nm occurs exactly in the same time interval as the trace at 460 nm decay (the inset of Fig. 4), which further indicates that BBR(-H) is the precursor of TrpN. In addition, enhancement of the BBR(-H) decay was observed in the presence of Trp (Fig. 5). The bimolecular rate constant for the reaction of BBR(-H) with Trp was determined to be 3.4  109 M1 s1 (the Inset of Fig. 5), and the related reaction mechanism could be shown as bellows: Fig. 3. (A) Stern–Volmer plots for the quenching of Lys by BBR at 298 K before and after correction and (B) Stern–Volmer plots for the quenching of Lys by BBR under different temperatures (corrected).

Table 1 Stern–Volmer quenching constants for the interaction of BBR with Lys at 298, 308 and 318 K. T (K)

KSV (103 M1)

kq (1011 M1 s1)

R

298 308 318

10.7 8.57 5.24

10.7 8.57 5.24

0.9980 0.9988 0.9986

Interaction of BBR(-H) with Trp and Lys Lys consists of 129 amino acid residues and contains six Trp and three tyrosine (Tyr) residues [37]. Three of Trp residues are located at the substrate binding sites, two in the hydrophobic matrix box, while one is separated from the others [37–40]. So Trp residues related to the reactive activity of Lys closely. To gain insight into the mechanism of BBR-mediated photoreaction with Lys, the interaction of BBR(-H) with Trp and Lys was investigated by LFP, respectively.

hv

ISC

BBR ! 1 BBR ! 3 BBR hv

BBR ! BBR eaq 3

þ

ðPhotoexcitationÞ

p Ka¼4:6

þ eaq ! ðBBR  HÞ ðPhotoionisationÞ

þ 2CH3 CN ! ðCH3 CNÞ 2

BBR þ O2 ! 1 O2 þ BBR

ðEnergy transferÞ

Trp þ 1 O2 ! Oxidation Product Hþ

k

Trp þ ðBBR  HÞ  ! Trp=NHþ þ BBR ! TrpN  k ¼ 3:4  109 M1 s1 ð460nmÞ ð350nmÞ ð520nmÞ In order to find out the actual mode of interaction between BBR and Lys in excited state, the photolysis of O2-saturated phosphate buffer/acetonitrile (4/1, v/v) solution containing 0.5 mM Lys and 0.05 mM BBR was studied. Under such conditions, BBR was excited to form BBR+ then through rapid deprotonation to produce BBR(H). The absorption of Lys shows that Lys has no absorption at 355 nm (Fig. 1A), so the laser energy directly absorbed by Lys can be negligible. Transient absorption spectra recorded at 100 ns and 10 ls were obtained from 355 nm LFP of BBR in O2-saturated phosphate buffer/acetonitrile (4/1, v/v) solution with Lys. As shown in Fig. 6, the absorption peak at 460 nm produced immediately after the laser pulse 100 ns could be ascribed to BBR(-H). Subsequently,

Table 2 Binding constants Ka and relative thermodynamic patameters of the BBR and Lys system. T (K)

Ka (104 M1)

n

R

DH0 (kJ mol1)

DS0 (J mol1 K1)

DG0 (kJ mol1)

298 308 318

2.40 2.82 3.16

1.08 1.13 1.19

0.9919 0.9996 0.9976

15.04

134.3 134.0 133.5

24.99 26.24 27.40

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BBR(-H) and 1O2. However in our experiment, the Kobs of BBR(H) were used to determine the bimolecular rate constants, which exclude the effect of 1O2, and only concentrate on the reactive activity of BBR(-H) to biological molecules. Reaction mechanism between BBR and Lys In our work, BBR absorbed UVA and visible light and was turned into 3BBR⁄ and BBR(-H). The energy of the excited triplet state (ET) of BBR is 243 kJ mol1 [24,46], while the ET of Trp is 589 kJ mol1 [47,48]. Consequently, energy transfer from 3BBR⁄ to Trp is unlikely to occur. However, the thermodynamic calculation shows that 3 BBR⁄ is able to oxidize Trp by electron transfer reaction. The standard free energy change (DG) in the process is calculated to be 43.4 kJ mol1, a highly exothermic process, according to the Rehm–Weller equation [49]: Fig. 4. Transient absorption spectra from 355 nm excitation of 0.05 mM BBR in O2saturated 2 mM phosphate buffer (pH 7.0)/acetonitrile (4/1) mixtures containing 0.1 mM Trp, recorded at 100 ns (j) and 8 ls (d) after laser pulse. Inset: The absorption-time profile at 460 nm (h) and 520 nm (s), respectively.

the new absorption peaks around 350 and 520 nm were observed at 10 ls after the decay of BBR(-H) (Fig. 6) should be the reaction products of BBR(-H) and Lys. On the basis of the similarity of the transient absorption spectra of the radical species produced from the interaction of BBR(-H) with Lys to that of Trp, it can be concluded that BBR could cause selective photo-oxidation of Lys at Trp residues via electron transfer process, and the ultimate products are Lys-Trp radicals (Lys-Trp/N). The dependence of Kobs of BBR(H) on concentrations of Lys was linear (the inset of Fig. 6), and the bimolecular rate constants of the electron transfer reactions was determined to be 4.2  108 M1 s1. And the related reaction mechanism could be supposed as bellow:

DG ¼ 96:48ðEox  Ered  e2 =ea Þ  DE0;0

ð7Þ

where e2/ea is the Coulombic term and can be neglected in aqueous solution, DE0,0 is the energy level of the excited triplet state, and Eox and Ered are the half-wave potentials in volts for the oxidation of electron donor and the reduction of an electron acceptor. It should be mentioned that the redox potentials of BBR/BBR and Trp/(Trp/ NH+) are 1.06 and 1.01 V (vs. NHE), respectively [50,51]. Therefore, it is obvious that photoinduced electron transfer is thermodynamically favored from Trp to 3BBR⁄. Furthermore, it is also evident that 3BBR⁄ can oxidize the Trp residues of Lys through electron transfer process. The supposed reactions are shown as belows: Hþ

3

BBR þ Trp ! BBR þ Trp=NHþ ! TrpN

3

BBR þ Lys ! BBR þ Lys  Trp=NHþ ! Lys  TrpN:

Hþ

In our previous work [24], the 3BBR⁄ was identified under 355 nm laser excitation, which is the precursor of the singlet oxygen (1O2). And several reports have detected that 1O2 could be generated by irradiation of BBR using the electron spin resonance (ESR) associated with spin-trapping techniques [9,10,45]. So the photo-damage of Lys should be caused by both BBR(-H) and active oxygen radicals, which means the set up profiles observed in Fig. 4 and Fig. 6 should be attribute to the associative reaction of

Under the aerobic condition, 3BBR⁄ can be efficiently quenched by O2 (ET = 94 kJ mol1) via energy transfer to generate 1O2 [52–54]. As we know, 1O2 is a strong oxidizing agent and can induce further oxidation of Lys (Type II mechanism).Under the irradiation, BBR can also be photoionized to generate BBR(-H), especially in the polar solvents [25]. In this paper, the electron transfer from Trp and the Trp residues of Lys to BBR(-H) has been observed, which means BBR(-H) can oxidize Lys directly (Type I mechanism). Therefore, in the presence of O2, the photo-oxidation Lys involves both Type I and Type II processes, and Type I mechanism is competitive with 1O2. Which reaction is major pathway is dependent on the concentrations of O2 and the polar of the solvent.

Fig. 5. Decay profiles of BBR(-H) observed at 460 nm without (j) or with 0.1 mM Trp (d). Inset: Dependence of Kobs for BBR(-H) at 460 nm on concentration of Trp.

Fig. 6. Transient absorption spectra from 355 nm excitation of 0.05 mM BBR in O2saturated 2 mM phosphate buffer (pH 7.0)/acetonitrile (4/1) mixtures containing 0.5 mM Lys at delays of 100 ns (j), 10 ls (d).

k

þ

Lys þ ðBBR  HÞ  ! Lys  Trp=NH 8

Hþ

þ BBR ! Lys

1 1

 TrpN  k ¼ 4:2  10 M s

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Scheme 2. The possible photodamage mechanisms of Trp and Lys caused by BBR.

The possible mechanism of interactions in the photosensitization of Trp and Lys by BBR was shown in Scheme 2. Conclusion The interaction between model protein Lys and antitumorigenic BBR has been investigated by spectroscopic methods. From the fluorescence data, the binding of BBR could change the environment of the Trp residues of Lys. The main fluorescence quenching mechanism between Lys and BBR was static quenching, and there was one binding site in Lys for BBR with the binding constants of 2.40  104 M1 (298 K), 2.82  104 M1 (308 K), and 3.16  104 M1 (318 K), respectively. Quenching of the fluorescence signals by titration of Lys with BBR was utilized to evaluate the thermodynamic parameters (DH0 = 15.04 kJ mol1 and DS0 = 134.0 J mol1 K1) of the interaction between Lys and BBR. The type of binding force between them was determined by thermodynamic equations to be hydrophobic interaction. Furthermore, the photo-oxidation of Trp and Lys in the presence of BBR was studied by using laser flash photolysis. The results showed that BBR could cause photo-oxidation to Lys directly, and selectively oxidize the Trp residues of Lys. Kinetic results suggested BBR(-H) could oxidize Trp and Lys via an electron transfer mechanism with rate constants 3.4  109 and 4.2  108 M1 s1, respectively. Moreover, through thermodynamic calculation, the reaction mechanisms between 3BBR⁄ and Trp or Lys were determined to be electron transfer process. Under aerobic condition, the photo-oxidation of Trp and Lys by BBR involves both Type I and Type II processes. Photoinduced reactions of BBR with Trp and Lys can occur through different pathways in different conditions (Scheme 2). All These results provide obvious implications for understanding the mode of BBR photodynamic action. In future this study can be extended to cellular level. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 11105089), and National Postdoctoral Sustentation Fund of China (No. 2011M500759). References [1] C.L. Kuo, C.W. Chi, T.Y. Liu, Cancer Lett. 203 (2004) 127–137. [2] H. Park, M.S. Kim, B.H. Jeon, T.K. Kim, Y.M. Kim, J. Ahnn, D.Y. Kwon, Y. Takaya, Y. Wataya, H.S. Kim, Biol. Pharm. Bull. 26 (2003) 1623–1624. [3] K. Hayashi, K. Minoda, Y. Nagaoka, T. Hayashi, S. Uesato, Bioorg. Med. Chem. Lett. 17 (2007) 1562–1564. [4] S.K. Kulkarni, A. Dhir, Eur. J. Pharmacol. 589 (2008) 163–172. ˇ ern ˇ áková, D. Koštˇálová, Folia Microbiol. 47 (2002) 375–378. [5] M. C

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