Tài liệu Báo cáo khoa học: A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity ppt

Another way to imitate the SeÆÆÆN interaction in GPX is if the selenium is not bound directly to the heteroatom N or O, but is located in close proximity to it, this Keywords cyclodextri

glutathione peroxidase activity

Shao-Wu Lv1, Xiao-Guang Wang1, Ying Mu1, Tian-Zhu Zang1, Yue-Tong Ji1, Jun-Qiu Liu2,

Jia-Cong Shen2and Gui-Min Luo1

1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, China

2 Key Laboratory for Supramolecular Structure and Materials of the Ministry of Education, Jilin University, Changchun, China

Glutathione peroxidase (GPX; EC 1.11.1.9) is a

well-known selenoenzyme that catalyzes the reduction

of harmful hydroperoxides by glutathione (GSH)

(Scheme 1) and protects lipid membranes and other

cellular components against oxidative damage [1–4] It

is related to many diseases and is regarded as one of

the most important antioxidant enzymes in living

organisms GPX enzyme activity is sometimes

increased in disease, possibly as a compensatory

mech-anism to try to counteract the oxidative stress

associ-ated with the pathology, although it is also decreased

in other diseases [5–8] Therefore, modulation of GPX

may be involved in many pathological conditions

Because natural GPX has some shortcomings, such as

instability, antigenicity and poor availability, much attention has been paid to its artificial imitation [9,10]

In synthetic approaches, an initial attempt is made

to synthesize organoselenium compounds in which the interaction of Se–N in GPX catalysis is imitated by inducing N or O in close proximity to selenium One way in which this is achieved is by binding the selen-ium atom directly to a heteroatom such as nitrogen 2-Phenyl-l,2-benzisoselenazol-3(2H)-one (Ebselen), the first biologically active organoselenium compound, represents an excellent example of a GPX mimic [9] Another way to imitate the SeÆÆÆN interaction in GPX is

if the selenium is not bound directly to the heteroatom (N or O), but is located in close proximity to it, this

Keywords

cyclodextrins; enzyme mimics; glutathione

peroxidase; selenium; substrate binding

Correspondence

G.-M Luo, Key Laboratory for Molecular

Enzymology and Engineering of the Ministry

of Education, Jilin University, 2519 Jiefang

Road, Changchun 130021, China

Fax: +86 431 8898 0440

Tel: +86 431 8849 8974

E-mail: luogm@jlu.edn.cn

(Received 12 February 2007, revised

27 May 2007, accepted 31 May 2007)

doi:10.1111/j.1742-4658.2007.05913.x

A 6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-bis-b-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX) In this novel GPX model, b-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate 6-CySeCD exhibits better GPX activity than 6,6¢-di-selenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H2O2, tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reac-tions The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics First, the substrate-bind-ing site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate⁄ ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application

Abbreviations

BHT, 2,6-di-tert-butyl-4-methylphenol; b-CD, b-cyclodextrin; 6-CySeCD, 6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-bis-b-cyclodextrin; CumOOH, cumenyl hydroperoxide; Ebselen, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one; GPX, glutathione peroxidase; GSH, glutathione; 6-SeCD, 6,6¢-diselenide-bis-cyclodextrin; TBARS, thiobarbituric acid reactive substances; t-BuOOH, tert-butyl hydroperoxide.

approach seems to help stabilize the selenolate and

enhance the GPX-like activity of diselenides [11]

Although some GPX mimics show some increased

activity, most show only limited catalytic enhancement

Based on an understanding of the structure of GPX,

its mode of molecular recognition and catalysis, as well

as previous studies [12–14], we believe that generation

of specific binding ability for the thiol substrate and

correct incorporation of the functional selenium group

should be critical in the construction of an effective

GPX model Previous studies by our group in

prepar-ing GPX models usprepar-ing a mAb technique [15,16],

bio-imprinting [17] and the chemical modification of native

enzymes [18] have supported this hypothesis Recently,

we developed some GPX mimics in which the

b-cyclo-dextrin (b-CD) cavity provided a hydrophobic

environ-ment for substrate binding [19–23] For example,

6,6’-diseleno-bis-cyclodextrin (6-SeCD) activity for the

reduction of hydrogen peroxide (H2O2) by GSH is 4.3

times that of Ebselen because of the role of the

hydro-phobic cavity of b-CD in binding substrate

In this study we designed and synthesized a new GPX

mimic,

6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-bis-b-cyclodextrin (6-CySeCD), in which the

cyclo-hexylamine group was incorporated in the proximity of

the selenium atom and the b-CD cavity provided a

hydrophobic environment for substrate binding

6-CySeCD showed higher GPX activity than 6-SeCD for the reduction of H2O2, tert-butyl hydroperoxide (t-BuOOH) and cumenyl hydroperoxide (CumOOH)

by GSH, indicating that incorporation of the imido-group in the proximity of the selenium atom may increase the stability of the nucleophilic intermediate selenolate and enhance GPX-like activity in selenium-containing GPX mimics We also studied the catalytic mechanism using steady-state kinetics of 6-CySeCD catalysis and investigated the antioxidant ability of 6-CySeCD using a mitochondria injury system

Results and Discussion

Synthesis and characterization of 6-CySeCD The synthetic routes of 6-CySeCD are shown in Scheme 2 6-CySeCD was analyzed using elemental analysis, found (calculated for C96H160O66N2Se2Æ6H2O)

%: C, 43.67 (43.28); H, 6.49 (6.31); N, 1.06 (1.05) IR (KBr): 3376(-OH), 2926(CH,CH2), 1644(-OH), 1568, 1467(-NH-), 1158, 1079, 1031(-O-), 948, 840, 755, 709,

579 cm)1 13C NMR (400 MHz, D2O) d(p.p.m.): 102.5(C1), 81.9(C4), 73.3(C3), 72.7(C5), 72.0(C2), 60.3(C6), 59.2(C6¢), 52.3(C7), 35.1(C8), 28.5(C10), 25.7(C9)

The content and valence of selenium in 6-CySeCD were measured by X-ray photoelectron spectroscopy The Se3d electronic-binding energy of 6-CySeCD

is 54.9 eV, which approaches the binding energy of SeCys (55.1 eV), indicating that the selenium in 6-CySeCD is present in the form of)1 valence

(diseleni-um bridge, -Se-Se-) The experiment also gave the C⁄ Se ratio, which is 48.3 : 1 (calculated 48 : 1), indicating

2 mol of selenium per mol of mimic Thus, the structure

of 6-CySeCD should be as shown in Scheme 3

GPX activity of 6-CySeCD The initial reaction rate for the reduction of hydro-peroxides by GSH was determined by observing the change in NADPH absorption at 340 nm (Eqns 1,2)

Scheme 2 Synthetic route of 6-CySeCD.

Scheme 1 Catalytic cycle for GPX.

The GPX activities of 6-CySeCD and other GPX

mim-ics catalyzed the reduction of hydroperoxides by GSH

are listed in Table 1

ROOH + 2GSH!GPXROH þ GSSG þ H2O ð1Þ

GSSGþ NADPH þ Hþ!GSH reductase 2GSHþ NADPþ

ð2Þ The GPX activities of 6-CySeCD and 6-SeCD for the

reduction of H2O2 by GSH were 7.9 and 4.2 min)1,

respectively, indicating that 6-CySeCD and 6-SeCD

display higher GPX activity than Ebselen This result

is not surprising, because b-CD shows good substrate

binding compared with Ebselen [15] When the

sub-strates were H2O2, t-BuOOH and CumOOH, we found

that the GPX activities with 6-CySeCD for the

reduc-tion of H2O2, t-BuOOH and CumOOH by GSH were

higher than with 6-SeCD

In the investigation of GPX mimics, Wilson’s disele-nides are successful [11] As shown in Scheme 4, there are two processes (oxidation and reduction with thiols)

in the mechanism, and the N near the selenium moiety apparently helps stabilize the selenolate and enhance the GPX-like activity of diselenides In this study, a cyclohexylamine group was incorporated in the prox-imity of the active selenium atom in the 6-CySeCD molecule and the GPX activity for 6-CySeCD was higher than for 6-SeCD

Kinetics of 6-CySeCD Steady-state kinetics was observed for substrates H2O2 and GSH The initial velocities for reduction of H2O2

by GSH were determined as a function of the substrate

Table 1 Comparison between GPX activities of the 6-CySeCD-cata-lyzed reduction of hydroperoxides by GSH and other species One unit of enzyme activity is defined as amount of mimic that utilizes

of 1 lmol of NADPH per minute All data are presented as means ± SD.

a Reactions were carried out in 50 m M potassium phosphate buffer,

pH 7.0, at 37 C, 1 m M GSH, 0.5 m M hydroperoxide b Obtained from Liu et al [19].

Scheme 3 Structures of 6-CySeCD.

Scheme 4 Catalytic mechanism proposed

by Wilson et al [11].

concentration at 37C and pH 7.0, by varying one

substrate concentration while another was fixed The

relevant steady-state equation (Eqn 3) for the mimic

reaction is

m0=½E0¼ kmax½GSH  ½H2O2

ðKH2O2½GSH þ KGSH ½H2O2 þ ½GSH  ½H2O2Þ

ð3Þ Where v0 is the initial reaction rate, [E]0 is the initial

enzyme mimic concentration, kmax is a

pseudo-first-order rate constant KH

2 O2and KGSH are the Michaelis–

Menten constants (Km) for H2O2and GSH, respectively

Double reciprocal plots of the initial velocity versus the

concentration of substrates gave a family of parallel

lines (Fig 1), indicating that the reaction mechanism is

a ping-pong mechanism This result demonstrated that

the GPX mimic, 6-CySeCD, has the same catalytic

mechanism as native GPX From the steady-state

equa-tion, the kinetic parameters were obtained (Table 2)

During investigation of the reduction of peroxides, it

is natural to consider the possibility of free radical

reactions Bell and Hilvert [24] used a radical trap,

2,6-di-tert-butyl-1-4-methylphenol (BHT), to inhibit the

reduction of t-BuOOH by a thioyl compound in the

presence of a GPX mimic, selenosubtilisin, and showed

that the enzyme-catalyzed reduction of hydroperoxide

proceeds via a nonradical mechanism, although the

spontaneous reduction of hydroperoxide by GSH

involves the production of free radicals The same

results were found for the 6-CySeCD-catalyzed

reduc-tion of H2O2by GSH BHT inhibited the spontaneous

reaction, but not the 6-CySeCD-catalyzed reduction

(Fig 2) This suggested that 6-CySeCD also catalyzes

the reduction of hydroperoxide by GSH via a

nonradi-cal mechanism

Protection of mitochondria against oxidative

damage by 6-CySeCD

The swelling and shrinking of mitochondria are

nor-mal physiological phenomena during respiration

How-ever, abnormal swelling disrupts the mitochondrial

membrane resulting in cell death Mitochondrial

swell-ing therefore characterizes its integrity Figure 3A

shows that the mitochondrial swelling is greatly

increased by ferrous sulfate⁄ ascorbate-induced

mito-chondrial damage and the swelling is decreased by addition of 6-CySeCD

The absorbance at 520 nm for the control group was basically constant, whereas that for the damage group decreasede considerably over time, indicating that mitochondrial swelling was increased However, the swelling in the protection group, which contained a certain concentration of 6-CySeCD, was apparently decreased compared with the damage group, and the mitochondrial swelling decreased with increasing

Fig 1 Double-reciprocal plots for the reduction of H2O2by GSH catalyzed by 5 l M 6-CySeCD (A) [E] 0 ⁄ v 0 versus 1 ⁄ [H 2 O 2 ] (m M )1) at

[GSH] 0.5 (j), 1 (d) and 3 m M (.) (B) [E] 0 ⁄ v 0 versus 1 ⁄ [GSH] (m M )1) at [H

2 O2] 0.5 (d), 1 (m) and 2 m M (.).

Table 2 Kinetic parameters of the 6-CySeCD Reactions were carried out in 50 m M potassium phosphate buffer, pH 7.0, at 37 C, 0.5–3.0 m M GSH, 0.5–2.0 m M H 2 O 2 All data are presented as means ± SD.

GPX mimic kmax(min -1 ) KGSH(m M ) kmax⁄ K GSH ( M )1Æmin-1 ) KH2O2(l M ) kmax⁄ K H2O2 ( M )1Æmin)1)

6-CySeCD concentration The ability of GPX mimics

(6-CySeCD, 6-SeCD and Ebselen) to protect

mito-chondria differed, as shown in Fig 3B, and 6-CySeCD

was the best among them This is in agreement with

ability of these GPX mimics to remove H2O2

In this study, we used thiobarbituric acid reactive

substances (TBARS) as a marker for lipid

per-oxidation, and 6-CySeCD, 6-SeCD and Ebselen as

antioxidants in ferrous sulfate⁄ ascorbate-induced

mito-chondrial damage to determine the levels of lipid

per-oxidation Figure 4A shows the extent of protection

afforded by 6-CySeCD The amount of TBARS

seen during mitochondrial damage was reduced

considerably in the presence of 6-CySeCD, and the

amount of TBARS decreased with increasing 6-Cy-SeCD concentration When the 6-Cy6-Cy-SeCD concentra-tion was 20 lm and mitochondria were damaged for

50 min, the TBARS content was only 24% of that in the damage group without 6-CySeCD, indicating that 76% of TBARS production was inhibited In order to gauge the ability of the three GPX mimics (6-CySeCD, 6-SeCD, and Ebselen) to inhibit TBARS production, their antioxidant activities were compared under iden-tical conditions As shown in Fig 4B, the ability of 6-CySeCD to decrease the accumulation of TBARS was greater than that of 6-SeCD and Ebselen In addi-tion, we also tested the effect of 20 lm 6-CySeCD in the absence of damage (data no shown) and the result shows that 20 lm 6-CySeCD did not have any effect

Fig 2 Plots of v0versus [H2O2] for 1 m M GSH in 50 m M

potas-sium phosphate buffer, pH 7.4, and 37 C, at [BHT] ¼ 0 l M (a) and

50 l M (b) (A) [6-CySeCD], 0 l M ; (B) [6-CySeCD], 5 l M

Fig 3 (A) Effect of concentration of 6-CySeCD on the swelling of mitochondria (a) Control; (b) damage + 20 l M 6-CySeCD; (c) dam-age + 10 l M 6-CySeCD; (d) damage + 4 l M of 6-CySeCD; (e) dam-age (B) Effect of different GPX mimics on mitochondrial swelling (a) Control; (b) damage + 10 l M 6-CySeCD; (c) damage + 10 l M 6-SeCD; (d) damage + 10 l M of Ebselen; (e) damage.

on mitochondrial swelling and lipid peroxidaton in the

absence of damage

Exposing mitochondria in vitro to redox active

xeno-biotics may simulate oxidative damage of mitochondria

in vivo The reactions for ferrous sulfate⁄

ascorbate-inducing mitochondrial damage can be proposed as

follows:

Ascorbic acid + O2! dehydroascorbic acid þ H2O2 ð4Þ

Fe2þþ H2O2! Fe3þþ OHþOH ð5Þ

Ascorbic acidþ 2Fe3þ! dehydroascorbic acid þ 2Fe2þ

where H2O2 was produced by oxidation of ascorbic acid to dehydroascorbic acid (Eqn 4) [22], in addition, mitochondria can produce superoxide by Fe(II), which could be dismutated by mitochondrial superoxide dismutase to hydrogen peroxide A hydroxyl radical was produced via the Fenton reaction (Eqns 5,6) [25–27] The biological molecules in mitochondria are easily attacked by hydroxyl radicals, when changes in composition, morphology, structure, integrity, and function of the mitochondria take place GPX mimics can scavenge hydroperoxides and block hydroxyl radical production, therefore protecting mitochondria against oxidative damage

In the ferrous sulfate⁄ ascorbate-induced mitochond-rial damage model system, swelling and TBARS con-tent were chosen according to the standard, which was used to determine the injury and extent of protection

in mitochondria 6-CySeCD reduced the mitochondrial swelling during damage and decreased the maximal TBARS content Mitochondrial swelling and the amount of TBARS were decreased in a dose-dependent manner by 6-CySeCD The inhibited TBARS content and decreased mitochondrial swelling can be explained

by 6-CySeCD acting as a GPX mimic, which effect-ively scavenged hydroperoxide and protected mito-chondria against oxidative damage

Conclusion

We developed a novel GPX mimic, 6-CySeCD In this enzyme model, the cavity of b-CD supplied a hydro-phobic environment for substrate binding, and mimic activity was increased greatly by the incorporation of a cyclohexylamine group in the proximity of the active selenium atom Compared with Ebselen and 6-SeCD, 6-CySeCD is a better GPX mimic, as evidenced by its enzymatic properties and protection of mitochondria These studies show that there are two key factors for improving catalytic efficiency of GPX mimics First, the substrate-binding site should match the size and shape of the substrates, and second, incorporation of

an imido-group increases the stability of transition state selenolate in the catalytic cycle We believe that this is significant when designing mimics with high cat-alytic efficiency

Experimental procedures

Apparatus and materials

Structural characterization of 6-CySeCD was performed with a IFS-FT66V infrared spectrometer (Bruker, Bremen, Germany), a Varian Unity-400 NMR spectrometer (Varian

Fig 4 (A) Dependence of extent of TBARS accumulation on the

con-centration of 6-CySeCD (a) Control; (b) damage + 20 l M 6-CySeCD;

(c) damage + 10 l M 6-CySeCD; (d) damage + 4 l M 6-CySeCD; (e)

damage (B) Effect of different GPX mimics on TBARS accumulated

during mitochondrial damage (a) Control; (b) damage + 10 l M

6-CySeCD; (c) damage + 10 l M 6-SeCD; (d) damage + 10 l M

Ebse-len; (e) damage Relative TBARS content calculated based on

amount of TBARS for 50 min with damage group ¼ 1.

Inc, Palo Alto, CA) and a Perkin-Elmer 240 DS elemental

analyzer (Wellesley, MA) The content and valence of

selenium in the 6-CySeCD were determined by using an

(VG Scientific, Sussex, UK) Spectrometric measurements

were carried out by using a Shimadzu UV-2550

spectropho-tometer (Kyoto, Japan)

b-CD was purchased from Shanghai Sanpu Chemical

Plant (Shanghai, China), recrystallized three times from

borohydride, selenium, BHT and 1,3-benzene-disulfonyl

chloride were obtained from Sigma (St Louis, MO) GSH,

glutathione reductase, t-BuOOH, CumOOH, and NADPH

were also obtained from Sigma Sephadex G-25 was

pur-chased from Pharmacia (Uppsala, Sweden) All the other

materials were of analytical grade and obtained from

Beijing Chemical Plant (Beijing, China)

Synthesis of 6-CySeCD

Two grams of 6A,6B-diiodo-6A,6B-dideoxy-b-cyclodextrin

[28] was dissolved in 30 mL of dry dimethylformamide, and

185 lL of cyclohexylamine was added The mixture was

stirred at 45C for 4 h, dimethylformamide was evaporated

under reduced pressure The residue was dissolved in

45 mL potassium phosphate buffer (50 mm, pH 7.0) and

30 mL dimethylformamide (cosolvent) Then 7 mL of 1 m

sodium hydroselenide (NaHSe), prepared according to the

procedure of Klayman and Griffin [29] was added under

the pure nitrogen The mixture was kept under nitrogen for

36 h at 60C, oxidized in air and finally purified by

chro-matography (k¼ 254 nm) with distilled water as the eluent

The resulting solution was freeze-dried and the lyophilized

powder was washed with ethyl ether three times and dried

under vacuum to obtain a light yellow, pure sample with

34% yield The structure of 6-CySeCD was analyzed by

and valence of selenium in the 6-CySeCD was determined

by X-ray photoelectron spectroscopy The energy of the

served as standard Scans were performed 12 times

Determination of GPX-like activity and kinetics

Catalytic activities were determined by the method of

Wil-son et al [11] The reaction was carried out at 37C in

700 lL of solution containing 50 mm, pH 7.0, potassium

phosphate buffer, 1 mm EDTA, 1 mm sodium azide, 1 mm

GSH, 0.25 mm NADPH, 1 unit glutathione reductase,

5 lm 6-SeCD and 6-CySeCD The reaction was initiated by

addition of 0.5 mm hydroperoxide Organic hydroperoxides

Tri-ton X-100, which was the cosolvent and did not affect the

GPX-like activity assay Activity was determined by the

6220 m)1cm)1) Background absorption of the noncatalytic reaction was run without mimic and was subtracted The activity unit of enzyme mimic was defined as the amount of enzyme mimic, which utilizes 1 lmol NADPH per min The assay of 6-CySeCD kinetics was similar to that for native GPX [30] Initial reduction rates of H2O2 by GSH were determined by observing the change in NADPH

substrate concentration while another is fixed All kinetic experiments were performed at 37C in 700 lL of reaction solution containing 0.5–3.0 mm GSH, 0.5–2.0 mm H2O2,

50 mm potassium phosphate buffer (pH 7.0), 1 mm EDTA,

6-CySeCD Background absorption of the noncatalytic reaction was run without mimic and was subtracted Kinetic data were analyzed by double-reciprocal plotting

Preparation of mitochondria

Bovine heart mitochondria were isolated from fresh bovine heart [31] and suspended in 0.25 m sucrose, 10 mm EDTA and 25 mm Hepes-NaOH buffer, pH 7.4, and maintained

on ice The concentration of the mitochondrial protein was determined by Coomassie Brilliant Blue [32] using BSA as the standard

Ferrous sulfate⁄ ascorbate-induced mitochondrial damage

Mitochondria (0.5 mg proteinÆmL)1) suspended in medium

10 mm potassium phosphate buffer, pH 7.4) were subjected,

in the absence and presence of the mimic, to oxidative stress generated by 0.5 mm ascorbate plus 12.5 lm ferrous

without mimic and known as the damage group; the experi-ment carried out without the mimic, ascorbate, and ferrous sulfate was known as the control group

Biological analysis of mimics against mitochondrial damage

Mitochondrial swelling was assayed as described by Hunter

et al [33] Mitochondrial swelling was measured by the decrease in the turbidity of the reaction mixture at 520 nm The decrease in absorbance indicated an increase in mito-chondrial swelling and a decrease in mitochondria integrity

mitochondria was analyzed by thiobarbituric acid assay [34] In this assay, thiobarbituric acid reacts with malonal-dehyde and⁄ or other carbonyl by-products of free-radical-mediated lipid peroxidation to give 2 : 1 (mol⁄ mol) colored conjugates, which have an A532value

This research was supported by Natural Science

Foun-dation of China (Project no 20572035 and 20534030)

and Jilin University, Changchun, China

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