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Differential pulse voltammetric studies on the effects of Al(III) on the lactate dehydrogenase activity
Chinese Chemical Letters 18 (2007) 857–860 www.elsevier.com/locate/cclet
Differential pulse voltammetric studies on the effects of Al(III) on the lactate dehydrogenase activity Kai An Yao, Na Wang, Jing Yue Zhuang, Zheng Biao Yang, Hai Yan Ni, Quan Xu, Cheng Sun, Shu Ping Bi * State Key Laboratory of Pollution Control and Resource Reuse & Key Laboratory of MOE for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China Received 4 January 2007
Abstract In this paper, differential pulse voltammetry (DPV) was applied to study the effects of aluminum Al(III) on the lactate dehydrogenase (LDH) activity. Michaelis–Menten constant (KmNADH ) and maximum velocity (vmax ) in the enzyme promoting LDH
catalytic reaction of ‘‘pyruvateðPyrÞ þ NADH þ Hþ Ð lactate þ NADþ ’’ under different conditions by monitoring DPV reduction current of NAD+ were reported. # 2007 Shu Ping Bi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Al(III); DPV; Lactate dehydrogenase; Biomarker
Aluminum (Al) has been proposed as an important factor that may contribute to several diseases [1]. It is recognized that Al occurs as many different species with varying toxicity towards organisms in natural waters [2]. Therefore, it is important to study the impact of various environmental factors on the toxicity of Al(III). Biomarkers reflect molecular and cellular alterations when organism is exposed to xenobiotics. The use of biomarkers presents the advantage of a sensitive and integrated evaluation in time and in space of pollutants, not only in terms of presence, but also in relation to the effects in the past or further [3]. LDH is one of the most important enzymes in the biological systems. The activity of LDH has been considered as a biomarker and it is used in clinical analysis, pollution monitoring and impairment assessing [4–6]. In recent years, the effects of the metal ions on LDH reaction processes have received much attention [7]. Compared to the other analytical method such as fluorescence and spectrophotometry [8], the electrochemical technique offers a number of remarkable advantages and satisfies many requires to analyze different biological systems [9,10]. In this article, we reported the effects of Al(III) on the LDH activities under different conditions. 1. Experimental A three-electrode system was used, which consisted of a hanging mercury drop electrode, a platinum foil counter electrode, and a saturated calomel reference electrode. All electrochemical experiments were performed with a * Corresponding author. E-mail address: [email protected] (S.P. Bi). 1001-8417/$ – see front matter # 2007 Shu Ping Bi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.05.011
858
K.A. Yao et al. / Chinese Chemical Letters 18 (2007) 857–860
CHI660B electrochemical system (CH Instruments Inc., Shanghai, China). The DPV parameters were scan rate 20 mV/s, pulse amplitude = 50 mV, pulse width = 50 ms. All chemicals were of analytical reagent grades. Bovine heart lactate dehydrogenase was obtained from Sigma Co. (St. Louis, MO, USA), which was diluted 100 times when used. b-NAD+ and NADH (nicotinamide adenine dinucleotide, purity 90%) were purchased from Shanghai Bio Life Science & Technology Co., Ltd. (China). 2. Results and discussion In the lactate dehydrogenase (LDH) catalytic system, before the addition of nicotinamide adenine dinucleotide (NADH), only the substrate Pyr produces the electrochemical response peak whose potential is 1.38 V (versus SCE, pH 7.5). When NADH is added, the peak current of NAD+ whose potential (to 0.89 V versus SCE, pH 7.5) increased continually while the peak current of Pyr decreased constantly along with the time (Fig. 1D). Within the first 3 min, there were positive linear relationships between the peak currents ip;NADþ and the time. So we could calculate the initial velocity (v0 ) that can be used to describe the enzymatic activity. The results showed that the effects of Al on the LDH activity exhibited two kinds of behaviors under different conditions, i.e. inhibitory effects or slightly increased LDH activity at low concentrations and inhibited at high concentrations. The inhibition effect of LDH activity increases with increasing the concentration of LDH, decreasing pH value and elevating temperature (Fig. 1). To analyze the values of KmNADH and vmax of LDH reaction system in the absence and presence of 0.04 mmol/L Al(III), it was found that the types of the inhibition of Al(III) varied with experimental conditions. It is noted that the inhibitory actions of Al(III) are all of a competitive–noncompetitive mixed type [11] with different concentrations of
Fig. 1. (A) The effects of Al(III) on the LDH activity with different concentrations of LDH. (B) The effects of Al(III) on the LDH activity at different pH. (C) The effects of Al(III) on the LDH activity at different temperatures. CAl(III) = 0, 0.10, 0.40, 0.80, 1.2, 2.0, 4.0, 8.0, 12, 20 105 mol/L. (D) The DPV responses of LDH reaction system changed with time, a ! j: t = 0, 1, 3, 5, 7, 9, 11, 13, 18, 23 min. 0.10 mol/L Tris–HCl buffer solution + 0.15 mol/L KCl, 8.0 104 mol/L Pyr, 2.0 104 mol/L NADH.
K.A. Yao et al. / Chinese Chemical Letters 18 (2007) 857–860
859
Table 1 The effects of Al(III) on KmNADH (mmol/L) and vmax (mmol/(L min)) of LDH enzyme promoting system with different concentrations of LDH (A), at different pH (B) and different temperatures (C) CAl(III) = 4 105 mol/L
CAl(III) = 0 mol/L vmax
KmNADH
vmax
KmNADH
(A) LDH amount (mL) 20 30 40
29.2 38.8 57.5
87.7 85.7 86.8
25.4 28.8 31.5
161 107 99.1
(B) pH value 6.7 7.5 8.5
37 38.8 37.7
129 85.7 94.3
12.9 28.8 30.1
86.6 107 77.1
33.8 38.8 221
75.6 85.7 500
22.1 28.8 64.5
70.5 107 207
(C) Temperature (8C) 15 25 37
LDH (Table 1). However, it is a competitive–noncompetitive mixed type at pH 7.5 and a noncompetitive– uncompetitive mixed type [11] at pH 6.7 and 8.5, respectively (Table 1). The types of the inhibition of Al(III) vary with temperature: a competitive–noncompetitive mixed type [11] at 25 8C and a noncompetitive–uncompetitive mixed type [11] at 15 8C and at 37 8C (Table 1). The effect of pH is probably due to the fact that the dominant species of Al vary with pH [1,12]. When pH descends, the cation species of Al increase and become dominant. These cation species have more effective interaction with the ionization enzyme at lower pH. The reason for the effects of temperature may due to that the high temperature not only makes the reaction rate of enzyme reaction increase but also enhances the interaction between Al(III) and LDH. Moreover, it is widely accepted that enzyme is unstable at high temperature, which may promote Al(III) to affect LDH. 3. Conclusion The activity of enzyme is one of useful biomarker on molecule level, which can reflect exposure, effect and susceptibility and widely used in environmental quality assessment [3]. Our research shows that the DPV detection of LDH promoting catalytic system could be a newly sensitive and effective method and would have potential application prospect in the future. Acknowledgments This project supported by the Natural Science Foundation of Jiangsu Province (BK 2005083), Grant of Analytical Measurements of Nanjing University, The National Science Foundation of China (No. 20575025) and Research Funding for the Doctoral Program of Higher Education (No. 20050284030). References [1] D. Orihuela, V. Meichtry, M. Pizarro, J. Inorg. Biochem. 99 (2005) 1879. [2] M.J. Gardner, E. Dixon, I. Sims, P. Whitehouse, Bull. Environ. Contam. Toxicol. 68 (2002) 195. [3] L. Lagadic, T. Caquet, J.C. Amiard, F. Ramade, Use of Biomarkers for Environmental Quality Assessment, A.A. Balkema, Rotterdam, 2000, p. 2. [4] Z.H. Gan, Q. Zhao, Z.N. Gu, Q.K. Zhuang, Anal. Chim. Acta 511 (2004) 239. [5] M. Monteiro, C. Quintaneiro, M. Pastorinho, M.L. Pereira, F. Morgado, L. Guilhermino, A.M.V.M. Soares, Chemosphere 62 (2006) 1333. [6] J.A. Almeida, Y.S. Diniz, S.F.G. Marques, L.A. Faine, B.O. Ribas, R.C. Burneiko, E.L.B. Novelli, Environ. Int. 27 (2002) 673. [7] X.X. Gao, X.Q. Wang, Chin. J. Anal. Chem. 26 (1998) 757.
860 [8] [9] [10] [11] [12]
K.A. Yao et al. / Chinese Chemical Letters 18 (2007) 857–860 M. Gulotta, H. Deng, H. Deng, R.B. Dyer, R.H. Callender, Biochemistry 41 (2002) 3353. K.A. Yao, N. Wang, Y.Q. Shi, H.Y. Ni, Z.B. Yang, C. Sun, Y.Z. Tang, S.P. Bi, Chin. J. Inorg. Chem. 22 (2006) 2202. J.W. Di, M. Zhang, K.A. Yao, S.P. Bi, Biosens. Bioelectr. 22 (2006) 247. Q.S. Yuan, Modern Enzymology, East China University of Science and Technology Press, Shanghai, 2001, pp. 31–44. G. Guibaud, C. Gauthier, J. Inorg. Biochem. 99 (2005) 1817.
Differential pulse voltammetric studies on the effects of Al(III) on the lactate dehydrogenase activity Kai An Yao, Na Wang, Jing Yue Zhuang, Zheng Biao Yang, Hai Yan Ni, Quan Xu, Cheng Sun, Shu Ping Bi * State Key Laboratory of Pollution Control and Resource Reuse & Key Laboratory of MOE for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China Received 4 January 2007
Abstract In this paper, differential pulse voltammetry (DPV) was applied to study the effects of aluminum Al(III) on the lactate dehydrogenase (LDH) activity. Michaelis–Menten constant (KmNADH ) and maximum velocity (vmax ) in the enzyme promoting LDH
catalytic reaction of ‘‘pyruvateðPyrÞ þ NADH þ Hþ Ð lactate þ NADþ ’’ under different conditions by monitoring DPV reduction current of NAD+ were reported. # 2007 Shu Ping Bi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Al(III); DPV; Lactate dehydrogenase; Biomarker
Aluminum (Al) has been proposed as an important factor that may contribute to several diseases [1]. It is recognized that Al occurs as many different species with varying toxicity towards organisms in natural waters [2]. Therefore, it is important to study the impact of various environmental factors on the toxicity of Al(III). Biomarkers reflect molecular and cellular alterations when organism is exposed to xenobiotics. The use of biomarkers presents the advantage of a sensitive and integrated evaluation in time and in space of pollutants, not only in terms of presence, but also in relation to the effects in the past or further [3]. LDH is one of the most important enzymes in the biological systems. The activity of LDH has been considered as a biomarker and it is used in clinical analysis, pollution monitoring and impairment assessing [4–6]. In recent years, the effects of the metal ions on LDH reaction processes have received much attention [7]. Compared to the other analytical method such as fluorescence and spectrophotometry [8], the electrochemical technique offers a number of remarkable advantages and satisfies many requires to analyze different biological systems [9,10]. In this article, we reported the effects of Al(III) on the LDH activities under different conditions. 1. Experimental A three-electrode system was used, which consisted of a hanging mercury drop electrode, a platinum foil counter electrode, and a saturated calomel reference electrode. All electrochemical experiments were performed with a * Corresponding author. E-mail address: [email protected] (S.P. Bi). 1001-8417/$ – see front matter # 2007 Shu Ping Bi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.05.011
858
K.A. Yao et al. / Chinese Chemical Letters 18 (2007) 857–860
CHI660B electrochemical system (CH Instruments Inc., Shanghai, China). The DPV parameters were scan rate 20 mV/s, pulse amplitude = 50 mV, pulse width = 50 ms. All chemicals were of analytical reagent grades. Bovine heart lactate dehydrogenase was obtained from Sigma Co. (St. Louis, MO, USA), which was diluted 100 times when used. b-NAD+ and NADH (nicotinamide adenine dinucleotide, purity 90%) were purchased from Shanghai Bio Life Science & Technology Co., Ltd. (China). 2. Results and discussion In the lactate dehydrogenase (LDH) catalytic system, before the addition of nicotinamide adenine dinucleotide (NADH), only the substrate Pyr produces the electrochemical response peak whose potential is 1.38 V (versus SCE, pH 7.5). When NADH is added, the peak current of NAD+ whose potential (to 0.89 V versus SCE, pH 7.5) increased continually while the peak current of Pyr decreased constantly along with the time (Fig. 1D). Within the first 3 min, there were positive linear relationships between the peak currents ip;NADþ and the time. So we could calculate the initial velocity (v0 ) that can be used to describe the enzymatic activity. The results showed that the effects of Al on the LDH activity exhibited two kinds of behaviors under different conditions, i.e. inhibitory effects or slightly increased LDH activity at low concentrations and inhibited at high concentrations. The inhibition effect of LDH activity increases with increasing the concentration of LDH, decreasing pH value and elevating temperature (Fig. 1). To analyze the values of KmNADH and vmax of LDH reaction system in the absence and presence of 0.04 mmol/L Al(III), it was found that the types of the inhibition of Al(III) varied with experimental conditions. It is noted that the inhibitory actions of Al(III) are all of a competitive–noncompetitive mixed type [11] with different concentrations of
Fig. 1. (A) The effects of Al(III) on the LDH activity with different concentrations of LDH. (B) The effects of Al(III) on the LDH activity at different pH. (C) The effects of Al(III) on the LDH activity at different temperatures. CAl(III) = 0, 0.10, 0.40, 0.80, 1.2, 2.0, 4.0, 8.0, 12, 20 105 mol/L. (D) The DPV responses of LDH reaction system changed with time, a ! j: t = 0, 1, 3, 5, 7, 9, 11, 13, 18, 23 min. 0.10 mol/L Tris–HCl buffer solution + 0.15 mol/L KCl, 8.0 104 mol/L Pyr, 2.0 104 mol/L NADH.
K.A. Yao et al. / Chinese Chemical Letters 18 (2007) 857–860
859
Table 1 The effects of Al(III) on KmNADH (mmol/L) and vmax (mmol/(L min)) of LDH enzyme promoting system with different concentrations of LDH (A), at different pH (B) and different temperatures (C) CAl(III) = 4 105 mol/L
CAl(III) = 0 mol/L vmax
KmNADH
vmax
KmNADH
(A) LDH amount (mL) 20 30 40
29.2 38.8 57.5
87.7 85.7 86.8
25.4 28.8 31.5
161 107 99.1
(B) pH value 6.7 7.5 8.5
37 38.8 37.7
129 85.7 94.3
12.9 28.8 30.1
86.6 107 77.1
33.8 38.8 221
75.6 85.7 500
22.1 28.8 64.5
70.5 107 207
(C) Temperature (8C) 15 25 37
LDH (Table 1). However, it is a competitive–noncompetitive mixed type at pH 7.5 and a noncompetitive– uncompetitive mixed type [11] at pH 6.7 and 8.5, respectively (Table 1). The types of the inhibition of Al(III) vary with temperature: a competitive–noncompetitive mixed type [11] at 25 8C and a noncompetitive–uncompetitive mixed type [11] at 15 8C and at 37 8C (Table 1). The effect of pH is probably due to the fact that the dominant species of Al vary with pH [1,12]. When pH descends, the cation species of Al increase and become dominant. These cation species have more effective interaction with the ionization enzyme at lower pH. The reason for the effects of temperature may due to that the high temperature not only makes the reaction rate of enzyme reaction increase but also enhances the interaction between Al(III) and LDH. Moreover, it is widely accepted that enzyme is unstable at high temperature, which may promote Al(III) to affect LDH. 3. Conclusion The activity of enzyme is one of useful biomarker on molecule level, which can reflect exposure, effect and susceptibility and widely used in environmental quality assessment [3]. Our research shows that the DPV detection of LDH promoting catalytic system could be a newly sensitive and effective method and would have potential application prospect in the future. Acknowledgments This project supported by the Natural Science Foundation of Jiangsu Province (BK 2005083), Grant of Analytical Measurements of Nanjing University, The National Science Foundation of China (No. 20575025) and Research Funding for the Doctoral Program of Higher Education (No. 20050284030). References [1] D. Orihuela, V. Meichtry, M. Pizarro, J. Inorg. Biochem. 99 (2005) 1879. [2] M.J. Gardner, E. Dixon, I. Sims, P. Whitehouse, Bull. Environ. Contam. Toxicol. 68 (2002) 195. [3] L. Lagadic, T. Caquet, J.C. Amiard, F. Ramade, Use of Biomarkers for Environmental Quality Assessment, A.A. Balkema, Rotterdam, 2000, p. 2. [4] Z.H. Gan, Q. Zhao, Z.N. Gu, Q.K. Zhuang, Anal. Chim. Acta 511 (2004) 239. [5] M. Monteiro, C. Quintaneiro, M. Pastorinho, M.L. Pereira, F. Morgado, L. Guilhermino, A.M.V.M. Soares, Chemosphere 62 (2006) 1333. [6] J.A. Almeida, Y.S. Diniz, S.F.G. Marques, L.A. Faine, B.O. Ribas, R.C. Burneiko, E.L.B. Novelli, Environ. Int. 27 (2002) 673. [7] X.X. Gao, X.Q. Wang, Chin. J. Anal. Chem. 26 (1998) 757.
860 [8] [9] [10] [11] [12]
K.A. Yao et al. / Chinese Chemical Letters 18 (2007) 857–860 M. Gulotta, H. Deng, H. Deng, R.B. Dyer, R.H. Callender, Biochemistry 41 (2002) 3353. K.A. Yao, N. Wang, Y.Q. Shi, H.Y. Ni, Z.B. Yang, C. Sun, Y.Z. Tang, S.P. Bi, Chin. J. Inorg. Chem. 22 (2006) 2202. J.W. Di, M. Zhang, K.A. Yao, S.P. Bi, Biosens. Bioelectr. 22 (2006) 247. Q.S. Yuan, Modern Enzymology, East China University of Science and Technology Press, Shanghai, 2001, pp. 31–44. G. Guibaud, C. Gauthier, J. Inorg. Biochem. 99 (2005) 1817.