Analytica Chimica Acta 459 (2002) 8391

Studies on catalytic spectrophotometry using -cyclodextrin polymerSchiff base metal complex as mimetic enzyme
Bo Tang , Guo-Ying Zhang, Yang Liu, Fang Han
Department of Chemistry, Shandong Normal University, Jinan 250014, China Received 10 July 2001; received in revised form 5 February 2002; accepted 5 February 2002

Abstract A novel catalytic spectrophotometric method for the determination of superoxide anion radical (O2 ) and superoxide dismutase (SOD) activity is proposed. The method is based on the coupling reaction of 2,4-dichlorophenol (2,4-DCP) and 4-amino antipyrine in the presence of H2 O2 , catalyzed by a Schiff base metal complex included by -cyclodextrin cross-linked polymer ( -CDP) as a mimetic enzyme. The contribution describes the design and synthesis of a novel Schiff base, salicylidene-2-amino-4-phenylthiazole (SAPTS) as well as a series of its metal complexes: M(II)(SAPTS)2 [M: Cu(II), Co(II), Zn(II), Ni(II)], among which Cu(II)(SAPTS)2 exhibits efcient horseradish peroxidase-like catalytic activity. Investigations of the optimum reaction conditions, the mechanism, and effects of coexisting substances are presented. The linear calibration range and the detect limit (3 ) of the developed method for superoxide is from 0.0 to 1.0103 and 7.54107 M, respectively. The determinations of SOD activity in human/rat blood, garlic, onion and scallion serve as models for the proposed method. A comparison of the results with established classical analysis is satisfactory. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Mimetic enzyme; -Cyclodextrin cross-linked polymer; Catalytic spectrophotometry; Superoxide anion radical; Superoxide dismutase

1. Introduction An enzyme is characterized by its high effectivity and specicity. Undoubtedly, the enzyme function relies heavily on its prosthetic group; but the immobilization from proteins three-dimensional structure is also important [1]. A natural enzyme is often expensive, affected by the reaction conditions and prone to denaturization. So studies on mimetic enzymes and their immobilization are signicant in both theory and application. Analogues for peroxidase have been widely employed in analytical chemistry [25], but their catalytic
Corresponding author. Fax: +86-5312962643. E-mail address: tangb@sdnu.edu.cn (B. Tang).

activity and specicity are all much lower than that of horseradish peroxise (HRP), as a result of the prosthetic groups freedom from immobilization. Saito et al. [6] bound a mimetic enzyme on anionexchange resin. But the recommended method does not work in a single-phase solution analysis or especially for immunoanalysis with labeled enzyme. Cyclodextrin (CD)s inner hydrophobic and outer hydrophilic characteristics [7] are extremely similar to the exact molecular recognition of an enzyme to a substrate [8], which makes it a good enzyme or enzyme immobilizer. Several reviews [911] and articles [1214] about CD mimetic enzymes have been reported. Modied CD possesses the function only of single inclusion and double recognition [15], in contrast to the multiple inclusions and multiple recognitions of

0003-2670/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 0 8 7 - 9

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natural enzymes. However, the binding constants of CD polymer and bridged CD (briCD) with prosthetic group sited on the bridge or entrapped in the cavity, and thus closer to natural enzymes in their functions, have values up to 108 M1 or more, into the region of many antigenantibodies binding constants [16]. Until now, either briCD or CD polymer has primarily been employed to x metalloporphyrins incorporating Fe and Mn to mimic peroxidase [1719]. Schiff bases, as common ligands, have such advantages over metalloporphyrins as easy synthesis, cheap chemicals used, easy dissolution and stable solutions formation. They easily form metal complexes and were widely used in metal analysis [20,21]. Recently, they have been found to have similar catalytic activity to metalloporphyrins or HRP have even though they have not the same structure [5,22]. According to our initial experiments of the two traditional metal ions, Mn(II) and Fe(III), respectively, Schiff base complex of the former exhibits very unsatisfactorily catalytic activity; as for the latter, the best is Fe(III) 2-hydroxy-1-naphthaldehyde thiosemicarbazone (HNT) [5], having 75% of HRP in relative activity. We are trying to develop new metal complexes having similar or even better catalytic functions. In this contribution, based on the stereoselectivity of -CDP to molecules with two benzene rings [23], the Schiff base salicylidene-2-amino-4-phenylthiazole (SAPTS) and a series of its metal complexes M(II)(SAPTS)2 [M: Cu(II), Co(II), Zn(II), Ni(II)] as mimetic enzymes of HRP were synthesized, with -CDP playing a role of inclusion. Owing to the enhanced hydrophobic cavity, the cooperative action among -CD molecules and the orientation of mimetic enzyme to the substrates, the catalytic activity, stability, and analytical sensitivity are all remarkably improved. The relative activity may be as high as 86% of that of HRP. According to this, a novel catalytic spectrophotometric procedure was developed for the determination of superoxide (O2 ) and superoxdase activity. A comparison between the proposed method and established methods shows that the former has satisfactory accuracy and better precision. In addition, compared with conventional techniques such as electron spin resonance (ESR) spectrometry [24], liquid chromatography (LC) [25] and chemiluminescence (CL) [26], the method is easy to follow without expensive apparatus.

2. Experimental 2.1. Apparatus A UV-265 recording spectrophotometer (Shimadzu) equipped with 1.0 cm quartz cells was used for the spectrum scanning and determination experiments. All pH measurements were made with a pHS-3C digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glasscalomel electrode. The IR spectra were recorded on a PE-983 IR spectrometer (KBr discs cm1 , Perkin-Elmer, Norwalk, CT, USA). All elementary analysis measurements were performed with a PE-240 CHN elementary analytical meter (Perkin-Elmer, Norwalk, CT, USA). A centrifuge with a speed of 8000 rpm was employed to obtain the superoxide dismutase (SOD) extract. A high-pressure mercury lamp with a wavelength range from 237.8 to 2325 nm (Nanjing Engineering Academy Radio Tube Works) was used to illuminate Vitamin B2 (VB2 ) for superoxide anion radical, combined with lters of 365, 405, 546 and 577 nm. 2.2. Reagents All chemicals used were of analytical reagent grade. Deionized water was employed throughout. VB2 (4 104 M) was prepared every other day by dissolving 15.05 mg of the reagent in 100 ml of 0.1 M NaOH and stored away from light. 4-Aminoantipyrine (4-AAP) solution (2.0 102 M) was prepared by dissolving 0.4065 g of the reagent in 100 ml of water and stored in a brown volumetric ask. 2,4-DCP solution (2.0 102 M), -CDP solution (5.0 103 M), NH2 OH HCl solution (1.0 102 M), pyrogallic acid solution (6.0 103 M) and 4-aminobenzenesulfonic acid solution (1.9 102 M) were all prepared by dissolving a suitable amount of the reagent in 100 ml of water. Cu(II)(SAPTS)2 solution (5.0 104 M) (synthesized in the laboratory) and 1-naphythylamine solution (5.5 102 M) were prepared in 100 ml of dimethyl formamide (DMF) and absolute ethanol, respectively. H2 O2 solution (5.0 103 M) was prepared by diluting 0.051 ml of 30% H2 O2 (v/v) (titration with KMnO4 ) solution to 100 ml with water. A TrisHCl buffer (0.10 M, pH = 8.20) and a phosphate buffer (0.05 M, pH = 7.80) were used.

B. Tang et al. / Analytica Chimica Acta 459 (2002) 8391 Table 1 Physical properties and analytical data of the ligand and complexes Compound mp ( C) Yield (%) Color Elemental analysis (%) (calculated) C L CuL2 ZnL2 NiL2 CoL2 119120 >300 291292 >300 >300 72.0 65.0 50.7 47.5 52.3 Yellow Brown Orange Orange Purple 68.84 61.49 61.72 2.41 62.46 (68.55) (61.80) (61.59) (62.26) (62.23) H 4.41 3.51 3.48 3.51 3.68 (4.31) (3.57) (3.56) (3.60) (3.59) N 9.89 8.87 8.75 8.84 8.92 (9.99) (9.01) (8.98) (9.08) (9.07) M 10.68 10.39 9.82 9.62

85

(10.21) (10.48) (9.51) (9.54)

2.3. Synthesis and characteristics of -CDP A 10.5 g of -CD was dissolved in 25 ml of 35% NaOH. Into this, 8.5 ml of epichlorohydrin was added dropwise, with stirring. The mixture was heated to 90 C for 5 min, then cooled down to room temperature by stirring continuously, and neutralized by 6 M HCl, to give a pale yellow, transparent solution. The solution obtained was dialyzed through a dialysis bag (molecular weight <3500), and 8.5 g of -CDP solid was obtained after cool-drying in a low pressure. The polymer was ascertained to have retained the characteristic structure of -CD by IR spectroscopy [27] and to contain 34 -CD molecules by the phenolsulfuric acid method [28]. 2.4. Synthesis and characteristics of the ligand and complexes 2.4.1. Synthesis of the ligand A 30 ml of absolute ethanol containing 1.76 g (0.01 mol) of 2-amino-4-phenylthiazone (synthesized according to [29]) was prepared. Into this, 1.22 g (0.01 mol) of salicylaldehyde was added dropwise at room temperature. The mixture was stirred for 10 min, heated to 80 C and reuxed for 3 h. After most of the ethanol had been evaporated, the product was ltered at the pump and recrystallized twice from 95% ethanol. After being dried under vacuum, 2.81 g of the ligand SAPTS was obtained with a yield of 72%. The reaction is as follows:

2.4.2. Synthesis of complexes A 1.12 g (4 mmol) of ligand was dissolved in 15 ml of absolute ethanol. Into this, 2 mmol of Zn(CH3 COO)2 in ethanol solution was added dropwise with heating and stirring. The mixture was heated to 75 C for reaction for 4 h with magnetic stirring, then ltered at the pump and washed three times with 95% ethanol. After being dried under vacuum, the complex Zn(SAPTS)2 was obtained. Complexes of Cu(II), Co(II) and Ni(II) were synthesized according to the same procedure except that Ni(II) was used as its chloride and nitrogen gas was needed to protect Co(II) from oxidation. 2.4.3. Characteristics of ligand and complexes Structural properties of the ligand and complexes are shown in Tables 1 and 2. In the IR spectra of the complexes, the disappearance of the OH stretching vibration and the violet shift of ArO vibration simultaneously demonstrate the formation of a bond between oxygen with a metal ion [30]. The intense bands at 16051610 cm1 in complexes are attributed = to the C= N stretching vibration, with a red shift of 1520 cm1 compared with that of the ligand, which = indicates the participation of the nitrogen in HC= NH in coordination; the new peak at 484492 cm1 is assigned to the N M bond [31], for cannot be the thiazole nitrogen because of steric effects. The mole ratio method and the continual variation method both proved that the ratio between metal and ligand is 1:2, which is conrmed by elemental analysis. From the characteristics earlier, the structure of the mimetic

86 Table 2 IR data of the ligand and complexes Compound L CuL2 ZnL2 NiL2 CoL2 OH

B. Tang et al. / Analytica Chimica Acta 459 (2002) 8391

= C= N 1625 1605 1610 1605 1608 (s) (s) (s) (s) (s)

= C= N (ring) 1555 1525 1530 1520 1525 (s) (s) (s) (s) (s)

ArO 1280 1330 1335 1320 1330 (s) (s) (s) (s) (s)

MN 484 492 489 491 (w) (w) (w) (w)

27503150 (bw)

enzyme is suggested to be:

2.5. Procedure 2.5.1. Determination of superoxide and SOD activity Into a 10 ml color comparison tube were added 2.0 ml of TrisHCl buffer (pH = 8.20), 2.5 ml of 5.0 103 M -CDP, 0.9 ml of 5.0 104 M Cu(II)(SAPTS)2 , 0.8 ml 2.0 102 M 4-AAP, 1.0 ml of 2.0 102 M 2,4-DCP and 0.5 ml 4.0 104 M VB2 in sequence. Water was added to mark. The solution was mixed and then illuminated under a high-pressure mercury lamp with the exception of the reference solution. The absorbance was measured at 508 nm in a UV spectrophotometer. The plot of absorbance versus illumination time (dynamic method) and the absorbance at equilibrium (static equilibrium method) were recorded. In actual sample analysis, 10 l of SOD extract was injected after VB2 , illuminated for 20 min, and the absorbance at 508 nm was recorded against a reagent blank. 2.5.2. Determination of SOD activity by the 1-naphythylamine-4-aminobenzene sulfonic acid (NABSA) method [32] Into 1 ml of 0.05 M phosphate buffer (pH = 7.80) were added SOD extract, 0.1 ml of 10 mM

NH2 OH HCl and 0.3 ml of 4.0 104 M VB2 . The solution was mixed and illuminated for 20 min. Then 2 ml of 5.5 102 M 1-naphythylamine and 2 ml of 1.9 102 M 4-aminobenzenesulfonic acid were added and mixed. The absorbance of the mixture was measured at 530 nm against a reagent blank. The inhibition [33] extent (P%) of SOD to superoxide = [(Ablank Asample )/Ablank ] 100. If the SOD amount with the P% at 50% is assumed as a unit, the calculation formula of SOD activity is as follows: SOD activity (U/ml) = PVT n/(50%V M ), where VT is the total volume of the solution to be determined (ml), VM the volume of the SOD extract and n the diluting times of the SOD tract (VT /VM ). 3. Results and discussion 3.1. Comparison of catalytic activity among different enzymes According to the method described in the procedure, catalytic activities of four metal complexes were examined in the absence of -CDP (illuminated for 20 min). The results (Fig. 1) indicate that the four mimetic enzymes can all catalyze the reaction but Cu(II)(SAPTS)2 stands out for the most remarkably catalytic activity, sensibilizing the absorbance value by 150%. So Cu(II)(SAPTS)2 was selected as the optimum mimetic enzyme. 3.2. UV absorption spectra In order to determine the optimum-working wavelength, spectral characteristics were studied at various pH and in the presence or absence of different catalytic agent and sensitizer. The corrected absorption spectra (Fig. 2) shows that the reaction is catalyzed more effectively by Cu(II)(SAPTS)2 than by Cu(II) and

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sensibilized more remarkably by -CDP than by -CD. Under these conditions, the maximum absorbance wavelength of the oxidation-coupling product is at 508 nm. 3.3. Selection of the optimum pH The pH of the medium had a great effect on the absorbance of the oxidation-coupling product. The effect of the pH range from 3.0 to 12.0 was investigated. The results show that the pH between 7.5 and 8.5 is best. To obtain the most stable and maximum absorbance (the following optimum experimental conditions were all chosen according to the same principle), a pH of 8.2 was xed with the use of TrisHCl buffer (Fig. 3). 3.4. Effect of the amount and order of reaction agents The addition order (Table 3) and the amounts of the reagents were investigated and found to have effects on the absorbance. According to the earlier mentioned principle the sequence and amounts selected as the optimum was 2.0 ml buffer, 2.5 ml of 5.0 103 M -CDP, 0.9 ml of 5.0 104 M Cu(II)(SAPTS)2 , 0.8 ml of 2.0102 M 4-AAP, 1.0 ml of 2.0102 M 2,4-DCP and 0.5 ml of 4 104 M VB2 . 3.5. Effect of illumination wavelength The O2 was obtained by illuminating VB2 with a high-pressure mercury lamp [34]. The wavelength

Fig. 1. Comparison of mimetic enzymes catalytic activity: (1) in the absence of mimetic enzyme, (2) in the presence of Cu(II)(SAPTS)2 (4.5 105 M), (3) in the presence of Co(II)(SAPTS)2 (4.5 105 M), (4) in the presence of Ni(II) (SAPTS)2 (4.5 105 M), (5) in the presence of Zn(II)(SAPTS)2 (4.5 105 M).

Fig. 2. Absorption spectra of reaction product, catalyzed by Cu(II)(SAPTS)2 or by Cu(II), sensibilized by -CDP or -CD. Conditions are same as described in Section 2.5. (1) TrisHCl + 4-AAP + 2, 4-DCP + VB2 ; 2 TrisHCl+ -CDP + Cu(II)(SAPTS)2 + 4-AAP + 2, 4-DCP + VB2 ; (3) TrisHCl+ -CD + Cu(II)(SAPTS)2 + 4-AAP + 2, 4-DCP + VB2 ; (4) TrisHCl + Cu(II)(SAPTS)2 + 4-AAP + 2, 4-DCP + VB2 ; (5) TrisHCl + Cu(II) + 4-AAP + 2, 4-DCP + VB2 , 4-AAP (1.6 103 M), 2,4-DCP (2.0 103 M), VB2 (2.0 105 M), Cu(II)(SAPTS)2 (4.5 105 M), -CD (6.0 103 M), -CDP (1.5 103 M).

Fig. 3. Effect of pH on absorbance of oxidation coupling product. Conditions are same as described in Section 2.5.

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Table 3 Effect of order of addition of the reagents (lighted for 20 min) Group 1 2 3 4 5 6 Order of addition TrisHCl TrisHCl TrisHCl TrisHCl TrisHCl TrisHCl + Cu(II)(SAPTS)2 + 4-AAP + 2,4-DCP + VB2 + 4-AAP + 2,4-DCP + Cu(II)(SAPTS)2 + -CDP+VB2 + -CDP + 4-AAP + 2,4-DCP + Cu(II)(SAPTS)2 + VB2 + Cu(II)(SAPTS)2 + 4-AAP + 2,4-DCP + -CDP + VB2 + -CDP + Cu(II)(SAPTS)2 + 4-AAP + 2,4-DCP + VB2 + Cu(II)(SAPTS)2 + 4-AAP + 2,4-DCP + VB2 + lighting + -CDP Absorbance (A508 nm ) 0.503 0.613 0.614 0.611 0.648 0.564

The reaction system was lighted for 20 min rst before -CDP was added in.

of light inuences the yield of O2 , so various lters combined to the lamp were examined. The experimental results (Table 4) shows that at different wavelength the relative yield was different by illuminating the same concentration of VB2 for the same long time. And the highest yield was obtained at mixture wavelength. So the work was carried out by illuminating VB2 without any lter. 3.6. Effect of illumination time Fig. 4 shows that there is a linear relationship between the absorbance and the illuminating time within 020 min. Its regression equation is A = 0.0385t + 0.0127 with a correlation coefcient (r) of 0.9992 and apparent rate constant of 6.41 104 s1 . During this time, it can be regarded as a zero-order reaction. But in the range of 2535 min the graph becomes curving with a dynamical equation of ln(A At ) = 0.0051t + 1.6448, the characteristic of one order reaction. The reaction rate in the latter period cannot reect the enzyme activity precisely. So 20 min was selected as the optimum illuminating time in the work.

3.7. Characteristic analysis of mimetic enzyme With different concentrations of 4-AAP (the optimum substrate), curves of A508 nm t were drawn to calculate initial velocity according to v = A/(tb). There was a linear relationship between 1/v and 1/[4-AAP] [35]. Regressions of different mimetic enzymes and bioenzyme are respectively as follows: (1) Cu(II)(SAPTS)2 : 1/v = 35.41/[4-AAP] + 2.083 105 (r = 0.9981). (2) -CDCu(II)(SAPTS)2 : 1/v = 14.91/[4-AAP] + 1.006 105 (r = 0.9954). (3) -CDPCu(II)(SAPTS)2 : 1/v = 3.24/[4-AAP] + 0.270 105 (r = 0.9974). (4) HRP: 1/v = 2.33/[4-AAP] + 0.233 105 (r = 0.9968). Kinetic constants are calculated from the previous LineweaverBurk equations. The results (Table 5)

Table 4 Effect of light wavelength Light lter wavelength 365 nm 405 nm 436 nm A508 nm 0.363 0.354 0.312 Light lter wavelength 546 nm 577 nm wavelength a A508 nm 0.270 0.247 0.654 Fig. 4. Effect of lighting time on the reaction system. Conditions are same as described in Section 2.5.

a O was obtained by lighting VB under high-pressure 2 2 mercury lamp without any light lters.

B. Tang et al. / Analytica Chimica Acta 459 (2002) 8391 Table 5 Comparison of the catalytic characteristics Ma 1 2 3 4
a

89

Substrate 4-AAP 4-AAP 4-AAP 4-AAP

pH 8.2 8.2 8.2 8.2

Km (M) 1.7 1.5 1.2 1.0 104 104 104 104

Vmax (M s1 ) 4.8 1.0 3.7 4.3 106 105 105 105

CM (M) 1.25 1.25 1.25 1.25 105 105 105 105

Kcat (s1 ) 0.384 0.80 2.96 3.44

Kcat /Km (M1 s1 ) 2.26 5.33 2.47 3.44 103 103 104 104

Relative activity 11 23 86 100

1: Cu(II)(SAPTS)2 ; 2: -CD-Cu(II)(SAPTS)2 ; 3: -CDPCu(II)(SAPTS)2 ; 4: HRP.

indicate that -CDP immobilizes Cu(II)(SAPTS)2 far more effectively than -CD does. 3.8. Reaction mechanism (1) O2 is obtained
.

by

illuminating

VB2 :

VB2 O2 (2) 2O2 + 2H+ = H2 O2 + O2 . (3) The coupling reaction between 2,4-DCP and 4-AAP in the presence of H2 O2 is catalyzed by Cu(II)(SAPTS)2 [34,36]

illuminating

sorbance difference, the absorption coefcient difference. Table 6 shows that -CDP includes primarily Cu(II)(SAPTS)2 rather than reaction product, which can also be explained from Table 3: the absorbance value in the order 5 increases by 28.2% in contrast to 12.1% of that in the order 6. In the Fig. 2, the sensibilization of -CD with a concentration four times as much as that of -CDP (a -CDP molecule is ascertained to consist of threefour -CD molecules [28]) is still obviously weaker, which proves the special cooperative action among -CD molecules. Obviously, the improvement of catalytic activity is attributed to the inclusion of mimetic enzyme from -CDP with enhanced hydrophobic cavity and cooperative action among -CD molecules. 3.10. Effect of foreign ions Twenty-ve ions and organic compounds were studied individually to investigate their effects on the determination of O2 obtained from 2.0105 M VB2 by the procedure. An error of 5% in the absorbance values was considered tolerable. No interference was encountered from (tolerance ratio in mass): SO4 2 , AC (2000); NO3 , Br and Cl (1500); Ca2+ , Mg2+ (1000); Ni2+ (500); Sr2+ , sucrose and glucose (400); I (300); Be2+ , Al3+ , Cr3+ (120); Zn2+ (60); glutamate and l-phenylalanine (30); Hg2+ (20); tryptophan, d-l-tyrosine (10); Co2+ , Mn2+ , V5+ , SO3 2 (1). The experimental results show that there
Table 6 Inclusion constant of -CDP to guest molecule Guest Ka (M1 ) VB2 2,4-DCP 4-AAP Cu(II) (SAPTS)2 1.75 104 Reaction product 3.02 103

. 3.9. Sensitization mechanism of -CDP At the coloration acidity of pH = 8.20, absorption spectra of guest molecules are drawn in the presence of -CDP of different concentrations. The results show that the absorbance of VB2 , 4-AAP and 2,4-DCP increases little or does not increase; while that of Cu(II)(SAPTS)2 and the reaction product increase greatly. It indicates that the cavity of -CDP is suitable to molecules with two benzene rings. The inclusion constants (Table 6) are calculated from BenesiHildebrand equation C G C CDP / A = (K a + C CDP )/ [37], where CG and CCDP are the known starting concentration of the guest molecule and CDP, respectively; A is the ab-

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B. Tang et al. / Analytica Chimica Acta 459 (2002) 8391 Table 7 Determination of SOD activity in samples of human blood, rat blood, garlic, scallion and onion Group Young rat Old rat Young man Old man Garlic Scallion Onion Number 8 8 8 8 8 8 8 Proposed method (x S.D.) 1213 568 110 152 194 79 53 49 31 11 13 7 7 7 Standard method (x S.D.) 1197 572 115 147 191 77 55 56 37 14 15 10 12 9

is no interference for most biological samples, so the proposed highly selective method can be applied to determine O2 and the activity of SOD in various biological and organic systems. 3.11. Analytical characteristics Because of the difcult measurement of O2 due to its instant existence, the relationship between its concentration and the absorbance at 508 nm may be derived from that between the concentration of H2 O2 and the absorbance at 508 nm. The O2 , for every 2 mol produces 1 mol H2 O2 [36]. Under the optimum experimental conditions, there is a linear relationship between the absorbance and the concentration of superoxide in the range of 0103 to 1.0103 M with a correlation coefcient of 0.9986. The regression equation was A = 0.00412 + 3.58 103 c (O2 , M). The S.D. of the absorbance measurements was 0.0027 obtained from a series of 10 blank solutions. The detection limit, as dened by IUPAC [38], was determined to be 7.54 107 M when the K value was taken as 3. 3.12. Determination of SOD activity 3.12.1. Sample pretreatment A 5 ml of physiological saline was added into 0.5 ml of the whole blood of a human/rat. The mixture was isolated by centrifugation at a speed of 2000 rpm. Following phase separation, the aqueous layer was removed, and 2 ml of cooling water was added and mixed. Into this, 1 ml of 95% ethanol was added and the mixture was shaken for 20 min, followed by 1 ml of trichloromethane and instant mixing. Then the mixture was centrifuged for 5 min at a speed of 4000 rpm. The obtained upper layer was just the SOD extract for future work. SOD extraction in garlic, scallion and onion were acquired in terms of [39]. 3.12.2. Determination of sample SOD activity in samples was determined according to previous procedure. The experimental results obtained by proposed method were compared with that obtained by standard method: NABSA for blood and pyrogallic autoxidation method [40] for garlic, scallion and onion (Table 7).

4. Conclusion The development of mimetic enzymes is a focus in chemistry, aiming to synthesize a sort of organic complex with not only functions of enzymes but advantages over bioenzymes excessive sensitivity to the environment. The exact recognition of -CDP to molecules with two benzene rings, along with its three-dimensional reticular structure and cooperative action among -CD molecules, makes -CDP an excellent immobilizer to the mimetic enzyme Cu(II)(SAPTS)2 , similar to the function of the protein to an bioenzyme. Owing to the favorable microenvironment created by -CDP, the catalytic activity of the mimetic enzyme is remarkably improved. So, the paper gives a new vision on mimetic enzymes. Acknowledgements This work was supported by the Natural Science Foundation of China (no. 29975016) and the Importance Natural Science Foundation of Shandong province in China (no. Z2000B03). References
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