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Microcalorimetric studies of the effects of MgCl2 concentrations and pH on the adsorption of DNA on montmorillonite, kaolinite and goethite
Applied Clay Science 32 (2006) 147 – 152 www.elsevier.com/locate/clay
Microcalorimetric studies of the effects of MgCl2 concentrations and pH on the adsorption of DNA on montmorillonite, kaolinite and goethite Peng Cai, Qiaoyun Huang ⁎, Xuewen Zhang State Key Laboratory of Agricultural Microbiology, College of Resources and Environment, Huazhong Agriculture University, Wuhan 430070, China Received 1 July 2005; received in revised form 15 November 2005; accepted 16 November 2005 Available online 20 December 2005
Abstract This study attempted to comprehend the interaction mechanisms between DNA and the common minerals in soil such as montmorillonite, kaolinite and goethite at various environmental conditions. The effects of MgCl2 concentrations (0, 1, 10 and 60 mM) and pH (3.0, 5.0, 7.0 and 9.0) on the adsorption of DNA on the examined minerals were investigated by the equilibrium adsorption and direct measurement of adsorption enthalpies. The adsorption isotherms fitted by the Langmuirian model revealed that the maximum capacities and affinities of DNA adsorption on minerals increased with MgCl2 concentrations and decrease of pH. No DNA was adsorbed on montmorillonite at pH 9.0. The values of DNA adsorption enthalpies (ΔHads) ranged from −0.3 to 4.9 kJ g− 1 at different systems. The ΔHads values decreased with increasing MgCl2 concentrations and decreasing pH. The adsorption of DNA on montmorillonite, kaolinite and goethite was an exothermic reaction (−0.3 b ΔHads b − 0.1 kJ g− 1) at 60 mM MgCl2 and pH 3.0, suggesting a more significant electrostatic attraction in the adsorption process. In contrast, DNA adsorption on minerals became endothermic (0.1 b ΔHads b 4.9 kJ g− 1) at 0–10 mM MgCl2 and pH 5.0–9.0 and dehydration effects were considered as the dominant driving forces for DNA adsorption on minerals. The thermodynamic parameters presented in this study have important implications for clarifying the binding mechanisms between DNA and mineral particles in soil and associated environments. © 2005 Elsevier B.V. All rights reserved. Keywords: Microcalorimetry; Thermodynamic parameters; Adsorption; DNA; Minerals
1. Introduction DNA could be released in large quantities into the soil environment from dead or moribund cells or from viable cells in specific growth phases (Ogram et al., 1988). These extracellular DNA molecules can be adsorbed by soil particles such as clay minerals, sand ⁎ Corresponding author. Tel.: +86 27 87671033; fax: +86 27 87280670. E-mail address: [email protected] (Q. Huang). 0169-1317/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2005.11.004
and humic acids and are partially protected against degradation by nucleases (Khanna and Stotzky, 1992; Crecchio and Stotzky, 1998). The persistence of DNA is one of the most important factors in horizontal transfer of genes among bacteria in soil ecosystem (Stotzky, 2000; Cai et al., 2005). Therefore, determining how DNA molecules adsorb on soil particles is of fundamental significance for the understanding of the behavior and fate of DNA in soils. The adsorption of DNA on surface-active particles in soil can be affected significantly by pH and salt
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8.0. However, no thermodynamic data directly supported the assumption. Montmorillonite, kaolinite and goethite are common and important clay minerals and oxide in soil and sediments. The main aim of our research was to obtain the adsorption enthalpies of DNA on montmorillonite, kaolinite and goethite as affected by salt concentration and pH. 2. Materials and methods 2.1. DNA Salmon sperm DNA was bought from Sigma Chemical Co., St. Louis, MO.
Fig. 1. Equilibrium adsorption of DNA (25 to 350 μg) on 10 mg of minerals at various MgCl2 concentrations. Vertical bars represent the standard deviation.
concentrations. For instance, decreasing the pH generally increased the amount of DNA adsorbed by sand, humic acids and clay minerals including montmorillonite and kaolinte (Lorenz and Wackernagel, 1987; Khanna and Stotzky, 1992; Crecchio and Stotzky, 1998). Addition of divalent cations such as Mg2+ and Ca2+ could markedly accelerate DNA adsorption and these ions probably formed bridges between the phosphate groups of DNA and the negatively charged sites of clays and sand (Romanowski et al., 1991; Franchi et al., 2003). Based on the adsorption experiment of supercoiled PUC18 DNA on silica in 6 M perchlorate at 23 °C and 37 °C, Melzak et al. (1996) proposed that DNA adsorption reactions were slightly exothermic at pH b 7.0 and endothermic at pH
Fig. 2. Equilibrium adsorption of DNA (25 to 350 μg) on 10 mg of minerals at different pH values. Vertical bars represent the standard deviation.
P. Cai et al. / Applied Clay Science 32 (2006) 147–152 Table 1 Main characteristics of minerals used in this study Mineral
PZC
CEC (cmol kg− 1)
SESA (m2 g− 1)
Montmorillonite Kaolinite Goethite
2.5 3.6 8.3
90.2 7.1 0.2
73.6 23.9 101.6
149
The microreaction system is a titration mode with a 20-ml ampoule. Continuous heat leakage measurements are taken in an isothermal system. The heat of adsorption flows through high-sensitivity thermopiles surrounded by a heat sink, which is stabilized at ±2 × 10− 4 °C. The magnitude of heat exchange of thermopile with a heat sink is proportional to the time interval of the voltage signal. In this investigation, experiment was conducted at various pH values and MgCl2 concentrations. Twenty milligrams of mineral and 4.2 ml of Tris buffer were placed in the ampoule stirring at 80 rpm. After the thermal equilibrium was reached, 0.8 ml (0.5 mg ml− 1) of DNA solution was titrated into the dispersed mineral suspension through a Hamilton syringe. The output signal was collected as power, P, versus time, t, and was integrated and quantified by the amount of adsorbed DNA in order to give the enthalpy change of adsorption. Before calculation of the adsorption enthalpy, the dilution heat of DNA and adsorbents should be subtracted to obtain the net heat of interaction between DNA and minerals at different systems. The adsorption enthalpy change (ΔHads) thus was calculated by the following equation: Qads = mqΔHads, where Qads (J) is the net heat attributed to the interaction between DNA and minerals, m (mg) represents the amount of minerals added into the suspension, q (μg mg− 1) is the amount of adsorbed DNA and it can be obtained from the isotherms. The experiment was performed at a temperature of 298 K.
Point of zero charge (PZC), cation exchange capacity (CEC) and specific external surface area (SESA) were analyzed by Mehlich and NH4AcO method (Xiong, 1985) and N2 adsorption method (Beijing Analytical Instrument Company), respectively.
2.2. Preparation of minerals Kaolin and montmorillonite (CP grade) were obtained from Shanghai Wusi Chemical Reagent Company and Henan Xinyang Montmorillonite Company, respectively. The reagent was suspended in deionized distilled water (ddH2O) and adjusted to pH 10.0 with 0.01 M NaOH and dispersed by sonication. The b 2 μm fraction was separated by sedimentation. The clays were washed with ddH2O and ethanol until free of Cl− ions and air-dried. Goethite was synthesized as described previously by Huang et al. (2003a). All minerals prepared were ground to pass a 100-mesh sieve. 2.3. Equilibrium adsorption isotherms Equilibrium adsorption isotherms were performed at various MgCl2 concentrations and pH values at 298 K. The pH of Tris buffer was adjusted from 3.0 to 9.0 with 0.01 M HCl or NaOH. Ten milligram of each mineral was mixed with 2.5 ml of 0.01 M Tris buffer containing 25–350 μg of DNA. The mixture was gently shaken at 298 K for 2 h and centrifuged at 20,000×g for 20 min. DNA in the supernatant was determined by UV at 260 nm. The amount of DNA adsorbed was calculated by the difference between the amount of DNA added and that remaining in the supernatant.
3. Results and discussion 3.1. Equilibrium adsorption analysis The equilibrium adsorption isotherms for DNA adsorption on montmorillonite, kaolinite and goethite at various MgCl2 concentrations and pH values are shown in Figs. 1 and 2. DNA adsorbed by minerals fitted a Langmuir equation which can be described as X = XmKC/(1 + KC), where X is the amount of DNA adsorbed per unit mass of mineral, Xm is the maximum capacity that DNA can be adsorbed, K is a constant related to the adsorption energy and C is the equilibrium
2.4. Heat measurement The LKB 3114/3236 TAM air isothermal calorimeter used was a thermal activity monitor controlled by Picolog software.
Table 2 Langmuir parameters for adsorption of DNA on minerals at various MgCl2 concentrations and pH values Mineral
Montmorillonite
Kaolinite
Goethite
Parameters
MgCl2 (mM) −1
Xm (μg mg ) K r Xm (μg mg− 1) K r Xm (μg mg− 1) K r
pH
0
1
10
60
3.0
5.0
7.0
9.0
6.7 0.09 0.99 6.6 0.05 0.97 3.5 0.07 0.99
16.0 0.12 0.98 8.7 0.05 0.99 4.2 0.07 0.99
21.5 0.16 0.99 17.9 0.07 0.99 5.4 0.08 0.97
31.6 0.19 0.96 23.1 0.09 0.99 7.7 0.10 0.98
38.2 0.33 0.96 19.6 0.55 0.96 42.0 0.32 0.98
8.6 0.11 0.99 11.3 0.29 0.99 4.5 0.10 0.99
6.7 0.09 0.99 6.6 0.05 0.97 3.5 0.07 0.99
0 – – 3.3 0.04 0.95 2.0 0.04 0.95
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Fig. 3. The power–time curves of DNA at various MgCl2 concentrations and pH values.
minerals. The rapidly increasing trend in the adsorption capacities of DNA on minerals at pH 3.0 in comparison with other pH values likely resulted from the denaturation of DNA and the interactions between surfaces of adsorbed DNA, leading to aggregation and/or precipitation. According to Jordan (1955), the isoelectric point of DNA was about 5.0. The values of PZC are 2.5, 3.6 and 8.3 for montmorillonite, kaolinte and goethite, respectively (Table 1). A small amount of DNA molecules are still adsorbed by kaolinite and goethite which are negatively charged at pH 9.0 (Fig. 2), suggesting that dehydration effects probably played a vital role under this condition (Norde, 1994). No DNA was adsorbed by montmorillonite at pH 9.0 (Fig. 2), which was attributed to the inhibition of electrostatic repulsion on DNA adsorption. It also indicates that DNA was adsorbed on montmorillonite mainly by electrostatic force. As listed in Table 2, the estimated K values increased with increasing MgCl2 concentrations and decreasing pH. It implies that the affinities of DNA adsorption on minerals
DNA concentration. The greater the K value, the higher the affinity between DNA and mineral surface. The adsorption isotherms belonged to the H-type at pH 3.0 whereas others were the L-type (Sparks, 1995). The amount of DNA adsorbed by minerals increased with MgCl2 concentrations from 0 to 60 mM and decrease of pH from 9.0 to 3.0. It indicates that electrostatic forces played an important role in the adsorption of DNA on
Fig. 4. The power–time curves of goethite at various MgCl2 concentrations and pH values.
Fig. 5. The power–time curves of DNA (400 μg) adsorption on 20 mg of minerals at various MgCl2 concentrations.
P. Cai et al. / Applied Clay Science 32 (2006) 147–152
Fig. 6. The power–time curves of DNA (400 μg) adsorption on 20 mg of minerals at different pH values.
were stronger at higher salt concentrations and lower pH values. 3.2. Adsorption enthalpy analysis The dilution heat of DNA and minerals such as goethite at various MgCl2 concentrations and pH values obtained by TAM air isothermal calorimeter measurements was shown in Figs. 3 and 4. The dilution heat of DNA decreased with the increase of MgCl2 concentra-
151
tions and became less exothermic at 60 mM MgCl2, which was attributed to the neutralization of Mg2+ with the negative charge of sugar-phosphate backbone and the increase of electrostatic attraction between DNA molecules (Sun et al., 2002). The dilution heat of DNA was endothermic at pH 3.0–9.0 which results from the electrostatic repulsion between DNA molecules. As shown in Fig. 4, the dilution heat of goethite decreased with increasing MgCl2 concentrations and decreasing pH. There is no significant difference in the dilution heat among the examined minerals, so the power–time curves of kaolinite and montmorillonite were not shown in the study. Figs. 5 and 6 show the power–time curves produced by DNA adsorption on minerals at various MgCl2 concentrations and pH values. The adsorption enthalpies (ΔHads) of DNA on minerals were calculated from the curves and listed in Table 3. As can be seen in Table 3, the ΔHads values of DNA adsorption on minerals ranged from 0.1 to 4.9 kJ g− 1 at 0–10 mM MgCl2 and pH 5.0–9.0 whereas the values were from − 0.3 to − 0.1 kJ mol− 1 at 60 mM MgCl2 and pH 3.0. It implies that DNA adsorption on minerals is an endothermic reaction at lower MgCl2 concentrations and higher pH values while it becomes exothermic at higher MgCl2 concentrations and in strong acidic environment. At liquid/ solid interfaces, endothermic processes include dehydration, de-ionization and structural rearrangement (Lin et al., 2001; Tsai et al., 2002; Huang et al., 2003b). Haynes and Norde (1995) reported that the conformational alternation of the protein adsorbed by ion-exchange chromatography mainly contributed to the entropy gain rather than the enthalpy. Melzak et al. (1996) also suggested that for duplex DNA adsorption on silica, the contribution of structural changes of DNA molecules to the ΔHads values is not obvious. Therefore, the decrement of ΔHads values for DNA adsorption on minerals with increasing MgCl2 concentrations and decreasing pH values was probably due to the increased heat revealed by electrostatic attractive forces of DNA on minerals and the decreased heat required for Table 3 Enthalpy values of DNA adsorption on minerals at various MgCl2 concentrations and pH values Mineral
ΔHads (kJ g− 1) MgCl2 (mM)
Montmorillonite Kaolinite Goethite
pH
0
1
10
60
3.0
5.0
7.0
9.0
3.5 1.1 1.8
0.7 0.7 0.3
0.1 0.2 0.1
− 0.2 − 0.3 − 0.2
− 0.3 − 0.1 − 0.2
0.9 0.1 0.7
3.5 1.1 1.8
– 1.5 4.9
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dehydration. The exothermic ΔHads values of DNA adsorption on minerals at 60 mM MgCl2 and pH 3.0 was attributed mostly to the electrostatic attraction. At lower MgCl2 concentrations (0–10 mM) and pH 5.0– 9.0, the endothermic ΔHads values of DNA adsorption on minerals was ascribed mainly to the dehydration effects. In addition, under pH 5.0 and 7.0 and lower Mg2+ concentrations (0 and 1 mM), the ΔHads values of DNA on montmorillonite were higher in comparison with kaolinite and goethite. This is probably because montmorillonite has more negative charges on its surface and results in an increase in the ΔHads values which was attributed mostly to the electrostatic repulsion. No DNA adsorbed by montmorillonite at pH 9.0 also supported this implication. 4. Conclusions To our knowledge, this is the first paper providing direct thermodynamic data of DNA adsorption on minerals at various salt concentrations and pH values. Electrostatic forces played a more marked role in DNA adsorption on montmorillonite compared with kaolinite and goethite. An increment of MgCl2 concentrations or a decrement of pH values not only enhanced the heat released by the electrostatic attractive forces between DNA molecules and minerals but also reduced the heat required for dehydration. The data provided in this study are fundamental in elucidating the binding mechanisms of DNA by soil components. Acknowledgements The authors would like to thank Professor X. Li for the help in calorimetric analysis. The research was financially supported by the National Natural Science Foundation of China (project no. 40271064). References Cai, P., Huang, Q.Y., Zhang, X.W., Chen, H., 2005. Binding and transformation of extracellular DNA in soil. Pedosphere 15, 16–23. Crecchio, C., Stotzky, G., 1998. Binding of DNA on humic acids: effect on transformation of Bacillus subtilis and resistance to DNase I. Soil Biol. Biochem. 30, 1061–1067.
Franchi, M., Ferris, J.P., Gallori, E., 2003. Cations as mediators of the adsorption of nucleic acids on clay surfaces in prebiotic environments. Orig. Life Evol. Biosph. 33, 1–16. Haynes, C.A., Norde, W., 1995. Structures and stabilities of adsorbed proteins. J. Colloid Interface Sci. 169, 313–328. Huang, Q.Y., Zhao, Z.H., Chen, W.L., 2003a. Effects of several lowmolecular weight organic acids and phosphate on the adsorption of acid phosphatase by soil colloids and minerals. Chemosphere 52, 571–579. Huang, H.M., Chen, W.Y., Ruaan, R.C., 2003b. Microcalorimetric studies of the mechanism of interaction between designed peptides and hydrophobic adsorbents. J. Colloid Interface Sci. 263, 23–28. Jordan, D.O., 1955. The physical properties of nucleic acids. In: Chargaff, E., Davidson, J.N. (Eds.), The Nucleic Acids, vol. 1. Academic Press, New York, pp. 447–492. Khanna, M., Stotzky, G., 1992. Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA. Appl. Environ. Microbiol. 58, 1930–1939. Lin, F.Y., Chen, C.S., Chen, W.Y., Yamamoto, S., 2001. Microcalorimetric studies of the interaction mechanisms between proteins and Q-Sepharose at pH near the isoelectric point (pI) Effects of NaCl concentration, pH value, and temperature. J. Chromatogr. A. 912, 281–289. Lorenz, M.G., Wackernagel, W., 1987. Adsorption of DNA to sand and variable degradation rates of adsorbed DNA. Appl. Environ. Microbiol. 53, 2948–2952. Melzak, K.A., Sherwood, C.S., Turner, R.F.B., Haynes, C.A., 1996. Driving forces for DNA adsorption to silica in perchlorate solutions. J. Colloid Interface Sci. 181, 635–644. Norde, W., 1994. Protein adsorption at solid surfaces: a thermodynamic approach. Pure Appl. Chem. 66, 491–496. Ogram, A., Sayler, G.S., Gustin, D., Lewis, R.J., 1988. DNA adsorption to soils and sediments. Environ. Sci. Technol. 22, 982–984. Romanowski, G., Lorenz, M.G., Wackernagel, W., 1991. Adsorption of plasmid DNA to mineral surfaces and protection against DNase I. Appl. Environ. Microbiol. 57, 1057–1061. Sparks, D.L., 1995. Environmental Soil Chemistry. Academic Press, San Diego, p. 267. Stotzky, G., 2000. Persistence and biological activity in soil of insecticidal proteins from Bacillus thuringiensis and of bacterial DNA bound on clays and humic acids. J. Environ. Qual. 29, 691–705. Sun, X.G., Cao, E.H., Zhang, X.Y., Liu, D.G., Bai, C.L., 2002. The divalent cation-induced DNA condensation studied by atomic force microscopy and spectra analysis. Inorg. Chem. Commun. 5, 181–186. Tsai, Y.S., Lin, F.Y., Chen, W.Y., Lin, C.C., 2002. Isothermal titration microcalorimetric studies of the effect of salt concentrations in the interaction between proteins and hydrophobic adsorbents. Colloids Surf., A Physicochem. Eng. Asp. 197, 111–118. Xiong, Y., 1985. Soil Colloids, vol. 2. Science Press, Beijing, p. 620.
Microcalorimetric studies of the effects of MgCl2 concentrations and pH on the adsorption of DNA on montmorillonite, kaolinite and goethite Peng Cai, Qiaoyun Huang ⁎, Xuewen Zhang State Key Laboratory of Agricultural Microbiology, College of Resources and Environment, Huazhong Agriculture University, Wuhan 430070, China Received 1 July 2005; received in revised form 15 November 2005; accepted 16 November 2005 Available online 20 December 2005
Abstract This study attempted to comprehend the interaction mechanisms between DNA and the common minerals in soil such as montmorillonite, kaolinite and goethite at various environmental conditions. The effects of MgCl2 concentrations (0, 1, 10 and 60 mM) and pH (3.0, 5.0, 7.0 and 9.0) on the adsorption of DNA on the examined minerals were investigated by the equilibrium adsorption and direct measurement of adsorption enthalpies. The adsorption isotherms fitted by the Langmuirian model revealed that the maximum capacities and affinities of DNA adsorption on minerals increased with MgCl2 concentrations and decrease of pH. No DNA was adsorbed on montmorillonite at pH 9.0. The values of DNA adsorption enthalpies (ΔHads) ranged from −0.3 to 4.9 kJ g− 1 at different systems. The ΔHads values decreased with increasing MgCl2 concentrations and decreasing pH. The adsorption of DNA on montmorillonite, kaolinite and goethite was an exothermic reaction (−0.3 b ΔHads b − 0.1 kJ g− 1) at 60 mM MgCl2 and pH 3.0, suggesting a more significant electrostatic attraction in the adsorption process. In contrast, DNA adsorption on minerals became endothermic (0.1 b ΔHads b 4.9 kJ g− 1) at 0–10 mM MgCl2 and pH 5.0–9.0 and dehydration effects were considered as the dominant driving forces for DNA adsorption on minerals. The thermodynamic parameters presented in this study have important implications for clarifying the binding mechanisms between DNA and mineral particles in soil and associated environments. © 2005 Elsevier B.V. All rights reserved. Keywords: Microcalorimetry; Thermodynamic parameters; Adsorption; DNA; Minerals
1. Introduction DNA could be released in large quantities into the soil environment from dead or moribund cells or from viable cells in specific growth phases (Ogram et al., 1988). These extracellular DNA molecules can be adsorbed by soil particles such as clay minerals, sand ⁎ Corresponding author. Tel.: +86 27 87671033; fax: +86 27 87280670. E-mail address: [email protected] (Q. Huang). 0169-1317/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2005.11.004
and humic acids and are partially protected against degradation by nucleases (Khanna and Stotzky, 1992; Crecchio and Stotzky, 1998). The persistence of DNA is one of the most important factors in horizontal transfer of genes among bacteria in soil ecosystem (Stotzky, 2000; Cai et al., 2005). Therefore, determining how DNA molecules adsorb on soil particles is of fundamental significance for the understanding of the behavior and fate of DNA in soils. The adsorption of DNA on surface-active particles in soil can be affected significantly by pH and salt
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8.0. However, no thermodynamic data directly supported the assumption. Montmorillonite, kaolinite and goethite are common and important clay minerals and oxide in soil and sediments. The main aim of our research was to obtain the adsorption enthalpies of DNA on montmorillonite, kaolinite and goethite as affected by salt concentration and pH. 2. Materials and methods 2.1. DNA Salmon sperm DNA was bought from Sigma Chemical Co., St. Louis, MO.
Fig. 1. Equilibrium adsorption of DNA (25 to 350 μg) on 10 mg of minerals at various MgCl2 concentrations. Vertical bars represent the standard deviation.
concentrations. For instance, decreasing the pH generally increased the amount of DNA adsorbed by sand, humic acids and clay minerals including montmorillonite and kaolinte (Lorenz and Wackernagel, 1987; Khanna and Stotzky, 1992; Crecchio and Stotzky, 1998). Addition of divalent cations such as Mg2+ and Ca2+ could markedly accelerate DNA adsorption and these ions probably formed bridges between the phosphate groups of DNA and the negatively charged sites of clays and sand (Romanowski et al., 1991; Franchi et al., 2003). Based on the adsorption experiment of supercoiled PUC18 DNA on silica in 6 M perchlorate at 23 °C and 37 °C, Melzak et al. (1996) proposed that DNA adsorption reactions were slightly exothermic at pH b 7.0 and endothermic at pH
Fig. 2. Equilibrium adsorption of DNA (25 to 350 μg) on 10 mg of minerals at different pH values. Vertical bars represent the standard deviation.
P. Cai et al. / Applied Clay Science 32 (2006) 147–152 Table 1 Main characteristics of minerals used in this study Mineral
PZC
CEC (cmol kg− 1)
SESA (m2 g− 1)
Montmorillonite Kaolinite Goethite
2.5 3.6 8.3
90.2 7.1 0.2
73.6 23.9 101.6
149
The microreaction system is a titration mode with a 20-ml ampoule. Continuous heat leakage measurements are taken in an isothermal system. The heat of adsorption flows through high-sensitivity thermopiles surrounded by a heat sink, which is stabilized at ±2 × 10− 4 °C. The magnitude of heat exchange of thermopile with a heat sink is proportional to the time interval of the voltage signal. In this investigation, experiment was conducted at various pH values and MgCl2 concentrations. Twenty milligrams of mineral and 4.2 ml of Tris buffer were placed in the ampoule stirring at 80 rpm. After the thermal equilibrium was reached, 0.8 ml (0.5 mg ml− 1) of DNA solution was titrated into the dispersed mineral suspension through a Hamilton syringe. The output signal was collected as power, P, versus time, t, and was integrated and quantified by the amount of adsorbed DNA in order to give the enthalpy change of adsorption. Before calculation of the adsorption enthalpy, the dilution heat of DNA and adsorbents should be subtracted to obtain the net heat of interaction between DNA and minerals at different systems. The adsorption enthalpy change (ΔHads) thus was calculated by the following equation: Qads = mqΔHads, where Qads (J) is the net heat attributed to the interaction between DNA and minerals, m (mg) represents the amount of minerals added into the suspension, q (μg mg− 1) is the amount of adsorbed DNA and it can be obtained from the isotherms. The experiment was performed at a temperature of 298 K.
Point of zero charge (PZC), cation exchange capacity (CEC) and specific external surface area (SESA) were analyzed by Mehlich and NH4AcO method (Xiong, 1985) and N2 adsorption method (Beijing Analytical Instrument Company), respectively.
2.2. Preparation of minerals Kaolin and montmorillonite (CP grade) were obtained from Shanghai Wusi Chemical Reagent Company and Henan Xinyang Montmorillonite Company, respectively. The reagent was suspended in deionized distilled water (ddH2O) and adjusted to pH 10.0 with 0.01 M NaOH and dispersed by sonication. The b 2 μm fraction was separated by sedimentation. The clays were washed with ddH2O and ethanol until free of Cl− ions and air-dried. Goethite was synthesized as described previously by Huang et al. (2003a). All minerals prepared were ground to pass a 100-mesh sieve. 2.3. Equilibrium adsorption isotherms Equilibrium adsorption isotherms were performed at various MgCl2 concentrations and pH values at 298 K. The pH of Tris buffer was adjusted from 3.0 to 9.0 with 0.01 M HCl or NaOH. Ten milligram of each mineral was mixed with 2.5 ml of 0.01 M Tris buffer containing 25–350 μg of DNA. The mixture was gently shaken at 298 K for 2 h and centrifuged at 20,000×g for 20 min. DNA in the supernatant was determined by UV at 260 nm. The amount of DNA adsorbed was calculated by the difference between the amount of DNA added and that remaining in the supernatant.
3. Results and discussion 3.1. Equilibrium adsorption analysis The equilibrium adsorption isotherms for DNA adsorption on montmorillonite, kaolinite and goethite at various MgCl2 concentrations and pH values are shown in Figs. 1 and 2. DNA adsorbed by minerals fitted a Langmuir equation which can be described as X = XmKC/(1 + KC), where X is the amount of DNA adsorbed per unit mass of mineral, Xm is the maximum capacity that DNA can be adsorbed, K is a constant related to the adsorption energy and C is the equilibrium
2.4. Heat measurement The LKB 3114/3236 TAM air isothermal calorimeter used was a thermal activity monitor controlled by Picolog software.
Table 2 Langmuir parameters for adsorption of DNA on minerals at various MgCl2 concentrations and pH values Mineral
Montmorillonite
Kaolinite
Goethite
Parameters
MgCl2 (mM) −1
Xm (μg mg ) K r Xm (μg mg− 1) K r Xm (μg mg− 1) K r
pH
0
1
10
60
3.0
5.0
7.0
9.0
6.7 0.09 0.99 6.6 0.05 0.97 3.5 0.07 0.99
16.0 0.12 0.98 8.7 0.05 0.99 4.2 0.07 0.99
21.5 0.16 0.99 17.9 0.07 0.99 5.4 0.08 0.97
31.6 0.19 0.96 23.1 0.09 0.99 7.7 0.10 0.98
38.2 0.33 0.96 19.6 0.55 0.96 42.0 0.32 0.98
8.6 0.11 0.99 11.3 0.29 0.99 4.5 0.10 0.99
6.7 0.09 0.99 6.6 0.05 0.97 3.5 0.07 0.99
0 – – 3.3 0.04 0.95 2.0 0.04 0.95
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Fig. 3. The power–time curves of DNA at various MgCl2 concentrations and pH values.
minerals. The rapidly increasing trend in the adsorption capacities of DNA on minerals at pH 3.0 in comparison with other pH values likely resulted from the denaturation of DNA and the interactions between surfaces of adsorbed DNA, leading to aggregation and/or precipitation. According to Jordan (1955), the isoelectric point of DNA was about 5.0. The values of PZC are 2.5, 3.6 and 8.3 for montmorillonite, kaolinte and goethite, respectively (Table 1). A small amount of DNA molecules are still adsorbed by kaolinite and goethite which are negatively charged at pH 9.0 (Fig. 2), suggesting that dehydration effects probably played a vital role under this condition (Norde, 1994). No DNA was adsorbed by montmorillonite at pH 9.0 (Fig. 2), which was attributed to the inhibition of electrostatic repulsion on DNA adsorption. It also indicates that DNA was adsorbed on montmorillonite mainly by electrostatic force. As listed in Table 2, the estimated K values increased with increasing MgCl2 concentrations and decreasing pH. It implies that the affinities of DNA adsorption on minerals
DNA concentration. The greater the K value, the higher the affinity between DNA and mineral surface. The adsorption isotherms belonged to the H-type at pH 3.0 whereas others were the L-type (Sparks, 1995). The amount of DNA adsorbed by minerals increased with MgCl2 concentrations from 0 to 60 mM and decrease of pH from 9.0 to 3.0. It indicates that electrostatic forces played an important role in the adsorption of DNA on
Fig. 4. The power–time curves of goethite at various MgCl2 concentrations and pH values.
Fig. 5. The power–time curves of DNA (400 μg) adsorption on 20 mg of minerals at various MgCl2 concentrations.
P. Cai et al. / Applied Clay Science 32 (2006) 147–152
Fig. 6. The power–time curves of DNA (400 μg) adsorption on 20 mg of minerals at different pH values.
were stronger at higher salt concentrations and lower pH values. 3.2. Adsorption enthalpy analysis The dilution heat of DNA and minerals such as goethite at various MgCl2 concentrations and pH values obtained by TAM air isothermal calorimeter measurements was shown in Figs. 3 and 4. The dilution heat of DNA decreased with the increase of MgCl2 concentra-
151
tions and became less exothermic at 60 mM MgCl2, which was attributed to the neutralization of Mg2+ with the negative charge of sugar-phosphate backbone and the increase of electrostatic attraction between DNA molecules (Sun et al., 2002). The dilution heat of DNA was endothermic at pH 3.0–9.0 which results from the electrostatic repulsion between DNA molecules. As shown in Fig. 4, the dilution heat of goethite decreased with increasing MgCl2 concentrations and decreasing pH. There is no significant difference in the dilution heat among the examined minerals, so the power–time curves of kaolinite and montmorillonite were not shown in the study. Figs. 5 and 6 show the power–time curves produced by DNA adsorption on minerals at various MgCl2 concentrations and pH values. The adsorption enthalpies (ΔHads) of DNA on minerals were calculated from the curves and listed in Table 3. As can be seen in Table 3, the ΔHads values of DNA adsorption on minerals ranged from 0.1 to 4.9 kJ g− 1 at 0–10 mM MgCl2 and pH 5.0–9.0 whereas the values were from − 0.3 to − 0.1 kJ mol− 1 at 60 mM MgCl2 and pH 3.0. It implies that DNA adsorption on minerals is an endothermic reaction at lower MgCl2 concentrations and higher pH values while it becomes exothermic at higher MgCl2 concentrations and in strong acidic environment. At liquid/ solid interfaces, endothermic processes include dehydration, de-ionization and structural rearrangement (Lin et al., 2001; Tsai et al., 2002; Huang et al., 2003b). Haynes and Norde (1995) reported that the conformational alternation of the protein adsorbed by ion-exchange chromatography mainly contributed to the entropy gain rather than the enthalpy. Melzak et al. (1996) also suggested that for duplex DNA adsorption on silica, the contribution of structural changes of DNA molecules to the ΔHads values is not obvious. Therefore, the decrement of ΔHads values for DNA adsorption on minerals with increasing MgCl2 concentrations and decreasing pH values was probably due to the increased heat revealed by electrostatic attractive forces of DNA on minerals and the decreased heat required for Table 3 Enthalpy values of DNA adsorption on minerals at various MgCl2 concentrations and pH values Mineral
ΔHads (kJ g− 1) MgCl2 (mM)
Montmorillonite Kaolinite Goethite
pH
0
1
10
60
3.0
5.0
7.0
9.0
3.5 1.1 1.8
0.7 0.7 0.3
0.1 0.2 0.1
− 0.2 − 0.3 − 0.2
− 0.3 − 0.1 − 0.2
0.9 0.1 0.7
3.5 1.1 1.8
– 1.5 4.9
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dehydration. The exothermic ΔHads values of DNA adsorption on minerals at 60 mM MgCl2 and pH 3.0 was attributed mostly to the electrostatic attraction. At lower MgCl2 concentrations (0–10 mM) and pH 5.0– 9.0, the endothermic ΔHads values of DNA adsorption on minerals was ascribed mainly to the dehydration effects. In addition, under pH 5.0 and 7.0 and lower Mg2+ concentrations (0 and 1 mM), the ΔHads values of DNA on montmorillonite were higher in comparison with kaolinite and goethite. This is probably because montmorillonite has more negative charges on its surface and results in an increase in the ΔHads values which was attributed mostly to the electrostatic repulsion. No DNA adsorbed by montmorillonite at pH 9.0 also supported this implication. 4. Conclusions To our knowledge, this is the first paper providing direct thermodynamic data of DNA adsorption on minerals at various salt concentrations and pH values. Electrostatic forces played a more marked role in DNA adsorption on montmorillonite compared with kaolinite and goethite. An increment of MgCl2 concentrations or a decrement of pH values not only enhanced the heat released by the electrostatic attractive forces between DNA molecules and minerals but also reduced the heat required for dehydration. The data provided in this study are fundamental in elucidating the binding mechanisms of DNA by soil components. Acknowledgements The authors would like to thank Professor X. Li for the help in calorimetric analysis. The research was financially supported by the National Natural Science Foundation of China (project no. 40271064). References Cai, P., Huang, Q.Y., Zhang, X.W., Chen, H., 2005. Binding and transformation of extracellular DNA in soil. Pedosphere 15, 16–23. Crecchio, C., Stotzky, G., 1998. Binding of DNA on humic acids: effect on transformation of Bacillus subtilis and resistance to DNase I. Soil Biol. Biochem. 30, 1061–1067.
Franchi, M., Ferris, J.P., Gallori, E., 2003. Cations as mediators of the adsorption of nucleic acids on clay surfaces in prebiotic environments. Orig. Life Evol. Biosph. 33, 1–16. Haynes, C.A., Norde, W., 1995. Structures and stabilities of adsorbed proteins. J. Colloid Interface Sci. 169, 313–328. Huang, Q.Y., Zhao, Z.H., Chen, W.L., 2003a. Effects of several lowmolecular weight organic acids and phosphate on the adsorption of acid phosphatase by soil colloids and minerals. Chemosphere 52, 571–579. Huang, H.M., Chen, W.Y., Ruaan, R.C., 2003b. Microcalorimetric studies of the mechanism of interaction between designed peptides and hydrophobic adsorbents. J. Colloid Interface Sci. 263, 23–28. Jordan, D.O., 1955. The physical properties of nucleic acids. In: Chargaff, E., Davidson, J.N. (Eds.), The Nucleic Acids, vol. 1. Academic Press, New York, pp. 447–492. Khanna, M., Stotzky, G., 1992. Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA. Appl. Environ. Microbiol. 58, 1930–1939. Lin, F.Y., Chen, C.S., Chen, W.Y., Yamamoto, S., 2001. Microcalorimetric studies of the interaction mechanisms between proteins and Q-Sepharose at pH near the isoelectric point (pI) Effects of NaCl concentration, pH value, and temperature. J. Chromatogr. A. 912, 281–289. Lorenz, M.G., Wackernagel, W., 1987. Adsorption of DNA to sand and variable degradation rates of adsorbed DNA. Appl. Environ. Microbiol. 53, 2948–2952. Melzak, K.A., Sherwood, C.S., Turner, R.F.B., Haynes, C.A., 1996. Driving forces for DNA adsorption to silica in perchlorate solutions. J. Colloid Interface Sci. 181, 635–644. Norde, W., 1994. Protein adsorption at solid surfaces: a thermodynamic approach. Pure Appl. Chem. 66, 491–496. Ogram, A., Sayler, G.S., Gustin, D., Lewis, R.J., 1988. DNA adsorption to soils and sediments. Environ. Sci. Technol. 22, 982–984. Romanowski, G., Lorenz, M.G., Wackernagel, W., 1991. Adsorption of plasmid DNA to mineral surfaces and protection against DNase I. Appl. Environ. Microbiol. 57, 1057–1061. Sparks, D.L., 1995. Environmental Soil Chemistry. Academic Press, San Diego, p. 267. Stotzky, G., 2000. Persistence and biological activity in soil of insecticidal proteins from Bacillus thuringiensis and of bacterial DNA bound on clays and humic acids. J. Environ. Qual. 29, 691–705. Sun, X.G., Cao, E.H., Zhang, X.Y., Liu, D.G., Bai, C.L., 2002. The divalent cation-induced DNA condensation studied by atomic force microscopy and spectra analysis. Inorg. Chem. Commun. 5, 181–186. Tsai, Y.S., Lin, F.Y., Chen, W.Y., Lin, C.C., 2002. Isothermal titration microcalorimetric studies of the effect of salt concentrations in the interaction between proteins and hydrophobic adsorbents. Colloids Surf., A Physicochem. Eng. Asp. 197, 111–118. Xiong, Y., 1985. Soil Colloids, vol. 2. Science Press, Beijing, p. 620.