Bull Environ Contam Toxicol DOI 10.1007/s00128-014-1265-y

Quantitative Study of As (V) and As (III) Interaction with Mangrove DNA by Molecular Fluorescence Spectroscopy N. Majumder • C. Chowdhury • R. Ray T. K. Jana



Received: 10 December 2013 / Accepted: 20 March 2014 Ó Springer Science+Business Media New York 2014

Abstract This study describes the in vitro study of (1:1) one step nucleophilic displacement (S1N ) of phosphate by heavier anion arsenate and arsenite in the DNA of arsenic ridden Sundarban mangroves. Mangrove DNA was found to give rise to a broad fluorescence and its integrated fluorescence intensity was enhanced on addition of As (V) and As (III), respectively. Analyses of the fluorescence parameter showed adequacy of 1:1 model to describe substitution of phosphate of mangrove DNA chain exiplex by arsenate and arsenite with equilibrium constant (log Kc) ranging between 4.19 and 4.32 for As (V), and between 3.77 and 3.89 for As (III) at pH 7 and 25°C. In the cases, the melting temperature (Tm) and reassociation rate constant of mangrove DNA was increased on treatment with As (V) and As (III). It is suggested that heavier ion arsenate and arsenite may substitute phosphate in natural DNA. Keywords Mangrove DNA  Fluorescence enhancement  Substitution reaction  Arsenic The effect of arsenic on human health is an issue of global concern and arsenic is known to be carcinogen and mutagen posing serious health effect (Frankenberger 2002). In spite of their toxicity, arsenic salts have been used for centuries to treat a variety of medical conditions (Vernhet et al. 2001). Given the worldwide acknowledged importance of mangrove forests for protection from storm surges, as well as in serving as nurseries for marine life, Mandal

N. Majumder  C. Chowdhury  R. Ray  T. K. Jana (&) Department of Marine Science, Calcutta University, 35, B. C. Road, Kolkata 700019, India e-mail: [email protected]

et al.(2013), showed the value of mangrove forests in forming humic substances for the binding of As and their subsequent deposition into sediment. Occurrence of elevated concentrations of arsenic in the Sundarban mangrove wetland has posed an environmental risk. Acharyya et al. 2000 suggested that the ultimate source of arsenic could be from Himalayan supply. Mandal et al. (2009), showed that Arsenic concentrations varied from 336 to 2,741 ng L-1 in the Sundarban mangrove water, 310–19,900 ng L-1 in pore water, 0.6–1.53 mg kg-1 in sediment and showed several fold enrichment of arsenic in the polycheate (Nemaly castis faauveli, 4.32 lg g-1, enrichment factor, 895), and in mangrove leaves (0.17 lg g-1, enrichment factor, 35) with respect to pore water. Wolfe-Simon et al. 2010 argued that arsenate could be a viable substitute for phosphate in the DNA of the Halomonadaceae (a family halophilic protobacteria) GFAJ-1 strain which could grow in the presence of arsenate, and possibly in the absence of phosphate. This has raised much interest, but at the same time also fueled an active debate. The objective of this study was to determine whether mangrove DNA could interact with As (V) and As (III) at 25°C and pH 7.

Materials and Methods The study site is located in a natural mangrove forest, Lotthian island (two sites: 21°45.220 N, 88°20.450 E and 21°45.230 N, 88°18.480 E), Sundarbans at the confluence of the Saptamukhi River and the Bay of Bengal, and Avicennia alba, Avicennia marina, Bruguiera gymnorrhiza are the dominant species of mangroves in the island. Samples of A. alba, A. marina, B. gymnorrhiza leaves from 10 m height and A. alba roots were collected and washed with sterilized Mili Q water. For DNA analysis,

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they were crushed in liquid nitrogen and homogenized in extraction buffer (100 mM Tris–HCl, 10 mM EDTA, and 500 mM NaCl). The mixture was treated with 20 % SDS and incubated at 65°C followed by the addition of 50 mM ammonium acetate and incubation at 0°C. The mixture was centrifuged to remove precipitate of protein (Deshmukh et al. 2007) and the centrifugate was incubated at -20°C for more than 1 h after mixing with isopropyl alcohol. The DNA precipitate was separated by centrifugation and washed with 70 % ethyl alcohol. Purified DNA was obtained through a standard phenol chloroform extraction method and was further purified from RNA contamination by treatment with LiCl overnight at 0°C. DNA concentration was measured by a spectrophotometric method (Burton 1956). The minimum detection limit for this process was 0.46 lg. DNA solutions (25 lM P) of different mangrove species were prepared in saline citrate buffer. Concentrated solutions of 2 mM As (III) and As (V) were prepared by dissolving As2O3 in 0.1 N NaOH and Na2HAsO4 salt in MiliQ water and pH of all solutions were adjusted to pH 7. A series of mixtures were prepared containing (0–25 lM) of As (III) and As (V) with a constant proportion of DNA solution (25 lM P). The fluorescence spectrum was recorded against a reference solution of saline citrate buffer using a Hitachi F-7000 FL Spectrophotometer (Serial Number: 2025-020). The instrument settings were between 330 and 560 nm emission wavelength, 10 nm excitation and emission slit width and scan rate of 240 nm/min. The fluorescence enhancement experiment was repeated using duplicate samples of mangrove DNA. Computational Details The experimental data were fitted to a 1:1 model to describe the binding between the mangrove DNA (L) and As (III and V) and is represented by the equation (Charges omitted) As þ L $ AsL:

ð1Þ

The corresponding binding constant (Kc) is given by Kc ¼ ½AsL =½As ½L;

ð2Þ

where [AsL] is the concentration of DNA bound to As, [As] and [L] are the concentration of free As and L (DNA). If a is the fraction of the total DNA bound to As and CL is total concentration of DNA, then a = [AsL]/CL. Therefore, CAs ¼ ½As þ ½AsL;

ð3Þ

and CL ¼ ½L þ ½AsL ¼ ½L þ Kc ½As½L

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ð4Þ

or, Kc ¼ CAs  ½As = ðCL  CAs þ ½AsÞ or; Kc ½As2 þ ½As f KcCL  KcCAs þ 1g  CAs ¼ 0: ð5Þ Combining Eqs. 2, 3 and 4, the following relation is obtained a = Kc [As]/1 ? Kc [As], where [As] is the real root of the Eq. (5). As the enhancement of the flurorescence signal is proportional to the concentration of the [AsL], the following relation is valid (Esteves Da Silva et al. 1996): IF  I0 = Imax  I0 ¼ a ðIFM  I0 Þ = Imax  I0 ;

ð6Þ

where the fluorescence intensities are as follows: I0, when no As is present; IF throughout the titration; IFM, due to the [AsL]; and Imax, total fluorescence intensity at the end of the titration. Analysis of the spectral data were scaled between 0 and 100, i.e., I max = 100 and I0 = 0 and the Eq. (6) is simplified to IF ¼ a IFM or; IF ¼ fKc ½As =1 þ Kc ½Asg fIFM g:

ð7Þ

Concentration of DNA (CL) was kept constant in terms of phosphorus (P) concentration at 25 lM P and the enhancement of its fluorescence intensity (IF) was obtained on gradual increase of arsenate and arsenite concentration (CAs) to 25 lM. Equations (5) and (7) were solved from the known values of CL, CAs, IF and IFM was considered in terms of Imax (100). The kinetics of reassociation of thermally denatured mangrove DNA in presence of As was considered a second order reaction. The reaction rate is expressed by the equation: da/ dt = k (C0 - a)2, where C0 is the initial concentration of single stranded DNA, and ‘a’ denotes the concentration of the reassociated reactants. The fraction of a single-stranded molecule (f) decreases with time according to the expression: f = (C0 - a)/ C0 = 1/(1 ? kC0t) or, (1 - f)/f = kC0t, in which the initial concentration of single-stranded molecules is expressed in moles and the rate constant k is expressed in mol-1 s-1.

Result and Discussion The mean mangrove DNA concentrations varied between 14.8–39.52 lg g-1 and the fluorescence spectra of different species of mangrove DNA showed excitation maxima at 302, 301, 303, 303 nm and emission maxima at 397, 399, 397, 397 392 nm. Stoke’s shift varied between 94 and 98 nm. The fluorescence intensity of different mangrove DNA (25 lM P) as measured in saline citrate buffer was enhanced by addition of increasing amounts of As (Figs. 1, 2). The highest enhancement was obtained at the (1:1) molar ratio of DNA and As, at which the integrated fluorescence intensity was enhanced 1.1–1.23 fold for As (V) and 1.08–1.11 fold for

Bull Environ Contam Toxicol

1600

(a)

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Fig. 1 Stoichiometric fluorescence intensity of mangrove DNA a Bruguiera leaf. b A. marina leaf. c A. alba leaf. d A. alba root with As (V)

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Fig. 2 Stoichiometric fluorescence intensity of mangrove DNA. a Bruguiera leaf. b A. marina leaf. c A. alba leaf. d A. alba root with As (III)

430

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0-25 µM

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As (III). The fluorescence quantum yield (a) of mangrove DNA (2.38 9 10-4–2.97 9 10-4) was enhanced in both buffers containing As (V) (2.63 9 10-4–3.64 9 10-4) and As (III) (2.59 9 10-4–3.29 9 10-4) (Table 1). The variation of the IF - I0/IFM (y) versus As concentration (V and III) was found to be linear (y = 0.039 As (V) -0.012,

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R2 = 0.99, p = 0.001, y = 0.048 As (III) -0.043, R2 = 0.97, p = 0.001 for A. alba leaf DNA; y = 0.04 As(V) -0.034, R2 = 0.99, p = 0.001, y = 0.047–0.055, R2 = 0.95, p = 0.005 for A. marina leaf DNA; y = 0.039 As (V) -0.019, R2 = 0.99, p = 0.001, y = 0.039 As (III)0.001, R2 = 0.99, p = 0.001 B. gymnorrhiza leaf DNA and

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Bull Environ Contam Toxicol Table 1 Species wise variation of mangrove DNA, Stoke’s shift, quantum yield of molecular fluorescence and binding constant (log Kc) for interaction of DNA with arsenic (III and V) at pH 7 and 25°C DNA (lg g-1)

Species

Bruguiera leaf

34.27 ± 5.4

Stoke’s shift (nm)

Quantum yield (a)

95

DNA

log Kc

DNA ? As V

DNA ? As III

DNA ? As V

DNA ? As III

2.38 9 10-4

2.66 9 10-4

2.62 9 10-4

4.32

3.82

-4

-4

2.79 9 10

2.59 9 10-4

4.25

3.89

2.63 9 10-4 3.64 9 10-4

2.61 9 10-4 3.29 9 10-4

4.19 4.19

3.89 3.77

A. marina leaf

39.52 ± 5.5

98

2.41 9 10

A. alba leaf A. alba root

37.14 ± 4.9 14.80 ± 3.0

94 94

2.40 9 10-4 2.97 9 10-4

0.48

0.09

y = 6 X 10 5 x y = 4 X 10 5 x

0.08 0.07

y = 5 X 10

0.06

(1 f)/f

Absorbance

0.47

0.46

x

0.05 0.04 0.03

DNA DNA with As V DNA with As III

0.45

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DNA

0.02

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0.01

DNA with As III

0

0

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Fig. 3 Mangrove DNA melting curve with and without As (III and V) plotted as UV absorbance versus temperature

Fig. 4 The fraction of single-stranded DNA, C0 = 2.59 9 10-5 M (P) in absence and presence of As (III and V) plotted as a function of time (t)

y = 0.04 As (V) -0.025, R2 = 0.99, p = 0.001, y = 0.04 As (III) -0.001, R2 = 0.99, p = 0.002 for A. alba root) and the values of the corresponding binding constant are given in Table 1. As an example of the results, Fig. 3 shows the shift of the mangrove DNA melting curve due to binding interaction with As (III and V). The linear plot of (1 - f)/f versus time (t) are given in Fig. 4. The mean melting temperature (Tm) of mangrove DNA (62.5°C) was increased on treatment with As (III) and (V) (64–67°C). The corresponding mean values of k (rate constant for reassociation) and C0t0.5 (50 % reassociation) were found to be 1.54, 0.64 for mangrove DNA without As, 1.93, 0.51 with As (III), and 2.31, 0.43 with As (V). The concentrations of mangrove DNA was found within the range reported for other plants (Permingeat et al. 1998; Ceccherini et al. 2003). Fluorescence maxima agreed well with the literature range for model duplexes (294–420 nm) (Vaya et al. 2010; Markovitsi et al. 2007; Kwok et al. 2008). There was an overlap region between excitation and emission spectra of all DNA samples and an almost mirror image relationship was obtained. This indicated that the excited state had very similar geometry with that of the ground state and that the vibration levels of excited states resembled that of the ground state. Mangrove DNA

exhibited broad fluorescence; Anders 1981 suggested that this type of characteristic band could be due to the emission from exciplexes (S–S, p–p*). The fluorescence quantum yield (a) of mangrove DNA was found similar to the value reported for calf thymus DNA (3.1 9 10-4; Vaya et al. 2010). The enhancement of a indicated binding interaction of DNA to As. The corresponding values of log Kc were greater for As (V) (4.19– 4.32) than for As (III) (3.77–3.89). Arsenate and arsenite closely resemble to phosphate reactivity and Tawfik and Viola (2011), suggested that a strict distinction between them might be rather challenging. Elements with same periodic table the order of nucleophilicity depends on the capability to polarize i.e. the nucleophilicity for halogen is in the decreasing order of mass (I-1 [ Br-1 [ Cl-1) or RS- [ RO- (Finar 2002). However, the difference in atomic weight and thermochemical radius between phosphorus (30.97, 2.38 A°) and arsenic (74.9, 2.48 A°) suggested that heavier arsenite and arsenate anion could be a better nucleophile than phosphate and could replace phosphate at the 5/ end of DNA chain exiplex by one step nucleophilic displacement (S1N ). However, the log Kc value was found to be lower than the value (log K, 6.16) observed for intercalative mode of ground state DNA binding with Cu (II) complex with sulphonamide (Bembee

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Devi et al. 2012). The values of C0t0.5 indicated that mangrove DNA without arsenic reassociated 1.25–1.49 times as slowly as DNA with As (III and V) and was within the range observed for highly repeative (1.3 9 10-3) and moderately repeative (1.9) DNA (Melcher 2000). During the measurement of fluorescence of mangrove DNA, the pH and ionic strength (I) of the saline citrate buffer was maintained at 7 (pH) and 0.24 (I), respectively at 25°C, in order to avoid the variation of fluorescence properties of mangrove DNA as a result of structural disorder and transition of conformation due to the change of p–p stacking among interacting bases at different pH other than 7 (Douki 2006) and for the variation of ionic strength (Saenger 1984). Had the all added As (25 lM) in the medium was present as free anion without any interaction with mangrove DNA, the increase of the medium I (ionic strength) would have been insignificant (0.047 %). The increase of melting temperature indicated that interaction of phosphate in mangrove DNA with As resulted helix stability, and Eichnorn and Shin 1968 suggested that the decrease of melting temperature could occur due to the destabilize the helical form for the interaction at DNA base site.

Conclusion This study indicates (1:1) replacement of phosphate by arsenate and arsenite. Combining results of such studies with the important amount of information accumulated on interaction of As with DNA will certainly help to validate the hypothesis that arsenate and arsenite in DNA could play equivalent biological role as phosphate. Acknowledgments Financial support was provided by UGC, New Delhi. Thanks are also due to the Sundarban Biosphere Reserve and Divisional Forest Office, Government of West Bengal, for giving permissions to bring required mangrove samples for the experiment. Thanks to Dr. Anett Trebitz, US EPA Mid Continent Ecological Division, 6201, Congdon Blvd, Duluth MN 5804 for her valuable comments and English language editing.

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Quantitative study of As (V) and As (III) interaction with mangrove DNA by molecular fluorescence spectroscopy.

This study describes the in vitro study of (1:1) one step nucleophilic displacement ([Formula: see text]) of phosphate by heavier anion arsenate and a...
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