Spectrophotometric study on the supramolecular interactions of nile blue sulphate with nucleic acids

Talanta 49 (1999) 495 – 503

Spectrophotometric study on the supramolecular interactions of nile blue sulphate with nucleic acids Cheng Zhi Huang *, Yuan Fang Li, Dao Jian Zhang, Xiao Ping Ao Institute of En6ironmental Chemistry, Southwest Normal Uni6ersity, Chongqing, 400715, People’s Republic of China Received 6 August 1998; received in revised form 14 December 1998; accepted 29 December 1998

Abstract The supramolecular interaction of nile blue sulphate (NBS) with nucleic acids was studied by investigating the characteristics of the interaction absorption spectra on the basis of the drug binding process in organic system in which small amount of drug interacting with large amount of biological macromolecules involves, and an accordingly binding model for organic dyes with large amount of macromolecules was established. At pH 7.40 and ionic strength 0.004, the H-aggregation of NBS occurs with increasing NBS concentration. The NBS aggregates can be bound to both calf thymus DNA and fish sperm DNA by the ratio of each nucleotide residue with a molecule of NBS if the concentration of DNAs is more than 15-fold excessive. The corresponding binding constant for the interaction of NBS with DNAs is about 103 order, with which thermodynamic parameters for the interactions, such as the change of free energy, enthalpy and entropy at 25°C, were calculated. It was found that the binding of NBS with thermally denatured DNA is similar to that with native yeast RNA, which indicates H-aggregation of NBS can be encouraged by single stranded nucleic acids. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Nucleic acids; Nile blue sulphate; Absorption spectrum

1. Introduction The study of the supramolecular recognition of organic dyes with biological macromolecules has become an increasingly interested research field for the recent decades, especially in the field to study the interactions of functional organic dyes * Corresponding author. Tel.: +86-23-6825-3822; fax: + 86-23-6886-6796. E-mail address: [email protected] (C.Z. Huang)

with biological macromolecules in order to investigate the micro-structure of biological macromolecules and to simulate the biophysical process [1,2]. The studies on the supramolecular interacting systems, because of the formation of the complex species of the interacting subunit by virtue of the intermolecular linkage, resulting in organized structures and functions which none of the interacting subunits should have, can be studied by investigating the binding characteristics of organic dyes [3]. Depending on the molecular symmetry

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and the environmental conditions, the interactions of organic dyes with nucleic acids generally involve in three mechanisms: the intercalation of the organic dyes into the base pairs of the double stranded structure of nucleic acids [4]; the groove binding in which the organic dyes locate in the major or minor groove of DNA [4]; and the long range assembly of the organic dyes on the molecular surfaces of nucleic acids [5,6]. Based on those bindings, analytical methods of biological macromolecules have been established [7 – 13]. In the drug binding process in a practical organism system, a common observed case is the interaction of small amount of drug with large amount of biological macromolecules [14]. On this account, we try to establish interaction models of organic dyes with nucleic acids on the basis of drug binding in the presence of large amounts of biological macromolecules in the present paper. It was found that each molecule of NBS is bound to one nucleotide residue of DNA and the reaction is not affected significantly by ionic strength if the concentration of nucleic acids is 15-fold larger than that of NBS. Binding constant is about 103 order, which indicates that the interaction is weak and new supramolecular complexes are formed [15].

6600 M − 1 cm − 1 and oRNA = 7800 M − 1 cm − 1, respectively [16]. 0.003 M and 7.5 × 10 − 5 M working solution of nucleic acids, including ctDNA, fsDNA and yRNA, were used according to necessity. The stock solution of nile blue sulphate (NBS) was prepared by dissolving the crystallized product (The Third Chemical Reagent Plant of Shanghai, Shanghai, China) in to water. Its working solution was 2.0× 10 − 4 M. Tris–HCl buffer solution (pH 7.40) buffer was used to control the acidity, while 0.1 and 1.0 M of NaCl solution was used to adjust the ionic strength of the aqueous solutions. All other reagents were of analytical-reagent grade without further purification. Doubly distilled water was used throughout.

2. Experimental

2.3. Method

2.1. Reagents

Into a 10-ml volumetric flask were added appropriate working solution of nucleic acids and 1.0 ml of buffer solution, vortexed and then added NBS solution. The mixture at last was diluted to 10.00 ml by using doubly distilled water and mixed thoroughly. All absorption measurements were obtained against the blank treated in the same way without nucleic acids.

The stock solutions of DNAs and RNA were prepared by dissolving commercially purchased calf thymus DNA (ctDNA, Beitai Biochemical Co., Chinese Academy of Sciences, Beijing, China), fish sperm DNA and yeast RNA (fsDNA and yRNA, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai, China) in doubly distilled water. For DNAs, 24 h or more were needed at 4°C, with gentle shaking occasionally. The concentrations of nucleic acids were determined according to the absorbances at 260 nm after establishing that the absorbance ratio A260/A280 was in the range 1.80 –1.90 for DNAs and 1.90 – 2.00 for yRNA. The molarities of nucleic acids were calculated by using oDNA =

2.2. Apparatus All absorption determination was made by using a Hitachi U-3400 Spectrophotometer (Tokyo, Japan). A pH S-10A digital pH meter (Xiaoshan Scientific Instruments Plant, Zhejiang, China) was used to measure the pH values of the solutions, and an MVS-1 vortex mixer (Beide Scientific Instrumental Ltd., Beijing, China) was used to blend the solutions in the volumetric flasks.

3. Results and discussion

3.1. Absorption characteristics Fig. 1 shows the absorption characteristics of NBS and those for the interactions of NBS with ctDNA. At pH 7.40, NBS has its maximum ab-

C.Z. Huang et al. / Talanta 49 (1999) 495–503

sorption in the region from 640.2 to 637.6 nm. By increasing its concentration, NBS has its a maximum absorption band characterized a few nanometer’s hypsochromic shift, with the appearance of an increasingly apparent absorption band at 603.4 nm. It was found that the ratio of Amax/A603.4 decreases with increasing NBS concentration (Table 1), which indicates some aggregation of NBS occur with increasing NBS concentration and the band located at 603.4 nm should be ascribed to H-aggregation species of NBS [17]. The change of Amax/AH can come to 0.338 with increasing NBS concentration from 0.4×10 − 5 to 2.0× 10 − 5 M. In addition, the aggregation mechanism of NBS in the aqueous medium at pH 7.40 can be supported by the absorption in the region of 640.2 – 637.6 nm which

Fig. 1. Absorption spectra of NBS and NBS–ctDNA mixtures. ctDNA concentration constant at 3.0 ×10 − 4 mol l − 1; pH, 7.40; Ionic strength, 0.004. Solid line: NBS; dashed line: NBS – ctDNA.

497

does not follow Beer’s law if the concentration of NBS is higher than 1.2× 10 − 5 M. In contrast to the H-aggregation mechanism of NBS, a new absorption band at 657.8 nm appears without any shift of the absorption band with increasing NBS concentration if the concentration of ctDNA or fsDNA is 15-fold more than that of NBS in the interacting system. One important phenomenon is that the new band at 657.8 nm shows significant hyperchromic effect compared to the band in the region from 640.2 to 637.6 nm, and no significant shoulder peak can be observed at the corresponding H-aggregation band of NBS. These absorption spectra indicate that the interaction of NBS with ctDNA or fsDNA results in new complex. It is worth noting that the absorption band should be ascribed to the absorption of the new complex because nucleic acids have no absorption band in the region under consideration, and their concentrations, 15-fold more than that of NBS, keep the concentration of free NBS very small, and to be neglected. Fig. 1 shows the newly formed complexes have larger absorptivity than that of NBS at the their maximum absorption wavelength. It was found that the aborsbance ratio at their maximum wavelength, ADNA – NBS/ ANBS, decreases with increasing NBS concentration until 1.2 ×10 − 5 M (Table 1), and then keeps constant. This phenomenon possibly indicates that the aggregation of NBS occurs with increasing NBS concentration. From Fig. 1, it is obvious that the absorption increases with increasing NBS concentration in the presence of 3.0×10 − 4 M ctDNA, and two equal absorption points can be obtained at 637.6 and 669.7 nm for each absorption spectrum of different concentration of NBS. The two equal absorption points, which can obtained arbitrarily to some degree, should be carefully determined by measuring the absorbances at different concentration levels of NBS. In the same way, we can obtain the equal absorption points at 637.6 and 670.8 nm for the interaction of NBS with fsDNA. However, we can not find the equal absorption points for the interactions of NBS with native yRNA, which is similar to the interaction of NBS with thermally denatured ctDNA (Fig. 2). So we can conclude that the interaction of NBS with

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Table 1 a Aggregation of NBS supported by Amax/AH and ADNA−NBSmax/A max NBS ratio in aqueous medium Concentration of NBS (×10−5 M)

NBS lmax Amax/AH DNA–NBS ADNA−NBSmax/A max NBS

0.4

0.8

1.2

1.6

2.0

640.2 1.443

639.2 1.279

638.7 1.200

637.9 1.158

637.4 1.105

1.343

1.248

1.186

1.201

1.206

a AH was determined at 603.4 nm, where the absorption peak of H-aggregation band located. pH: 7.40; ionic strength: 0.004; Concentration of DNAs: 3.0×10−4 M. The change of Amax/AH with was 0.338 with increasing NBS concentration from 0.4×10−5 to 2.0×10−5 M.

nucleic acids can be used to explore the helix structure of nucleic acids.

3.2. Binding number of the interaction determined by dual wa6elength Since nucleic acids have no absorption band in the region under consideration, and the concentrations of DNA are more than 15-fold excessive, so the absorption band shown in Fig. 1 should be ascribed to the absorption of the new complex. Therefore, the absorbance at any wavelength for NBS–DNA interacting system can be expressed as: A= ANBS + ANBS – DNA =o[NBS]+ o%[NBS–DNA] (1) where o and [NBS] are the molar absorptivity and the concentration of free NBS at equilibrium, while o% and [NBS – DNA] are the molar absorptivity and the concentration of bound NBS at equilibrium, respectively. So, the absorbance difference at 637.6 and 669.7 nm, DA, can be described by: DA =A637.6 −A669.7 =(o%637.6 −o%669.7)[NBS – DNA] + (o637.6 − o669.7)[NBS]

(2)

Since the equal absorption of the complex at 637.6 and 669.7 nm, namely, o%637.6 =o%669.7, Eq. (2) can be expressed as:

DA = (o637.6 − o669.7)[NBS]

(3)

namely, DA is in proportion to the free concentration of NBS at equilibrium. So, by determining the absorbance difference at 637.6 and 669.7 nm we can calculate the value of [NBS], the free concentration of NBS in NBS–ctDNA interacting system; and by determining the absorbance difference at 637.6 and 670.8 nm we can calculate the value of [NBS] in NBS–fsDNA interacting system. Define the average binding number of NBS with each nucleotide residue of DNA, n, as: n=

cNBS − [NBS] cDNA

(4)

where cNBS and cDNA are the total concentration of NBS and nucleic acids, respectively. Rearrange Eq. (4), we have, [NBS] cNBS = n+ cDNA cDNA

(5)

so cNBS/cDNA has linear relationship with [NBS]/ cDNA by the intercept of n, the binding number of NBS with each nucleotide residue. Table 2 lists the binding number for the interactions of NBS with ctDNA and fsDNA with the dual wavelength method, from which we can conclude that each NBS molecule binds with one nucleotide residue of double-stranded DNA. So it is possible in the newly formed complex of DNA that NBS exists in monomer state, not in aggregate state.

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Fig. 2. Absorption spectra of the interactions of NBS with single-stranded nucleic acids. (a) NBS with thermally denatured ctDNA; (b) NBS with native yRNA. Concentrations of nucleic acids constant at 3.0 ×10 − 4 mol l − 1; pH, 7.40; Ionic strength, 0.004. Solid line: NBS; dashed line: NBS with nucleic acids. Concentration of NBS in order of increasing at their maximum absorption wavelength ( × 10 − 5 mol l − 1): 0.4; 0.8; 1.2; 1.6; 2.0.

[NBS−DNA] cDNA[NBS]

3.3. Binding constant of the interaction determined by a6erage molar absorti6ity

Kapp =

According to the results of Table 2, each NBS molecule binds one nucleotide residue of DNA, so we can establish following equilibrium:

Define average absorptivity, oapp, as the ratio of the total absorbance of the solution with the total concentration of NBS, cNBS, namely:

DNA +NBSUNBS – DNA

A= oappcNBS = oapp[NBS–DNA]+ oapp[NBS]

then the apparent binding constant, Kapp, can expressed as:

where the thickness of sample cell was considered as 1 cm. Since

Kapp =

[NBS− DNA] [DNA][NBS]

(6)

where [DNA] is the equilibrium concentration of DNA. When the concentration of DNA is large enough, 15-fold excessive, for instance to keep [DNA]=cDNA, we can express Kapp as:

A= o[NBS]+o%[NBS–DNA]

(7)

(8)

(9)

so Eq. (8) can be expressed as: [NBS− DNA] o− oapp = oapp − o / [NBS] Substitute Eq. (10) to Eq. (7), yielding

(10)

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500

Table 2 The binding number of NBS with DNAs determined by dual wavelength methoda Nucleic acids

Concentration of nucleic acids (×10−6 mol l−1)

Linear regression equation

Linear correlation coefficient (r)

ctDNA

3.0 6.0 3.0 6.0

y = 1.31+1.03x y =1.26+1.15x y =1.08+1.08x y = 1.16+1.10x

0.9998 0.9997 0.9902 0.9984

fsDNA

a [NBS] was determined by using DA= −0.0066+17616.4 [NBS] (r =0.9985) which was obtained by the absorbance difference at 637.6 and 669.7 nm for a series of concentration of NBS. pH: 7.40; ionic strength: 0.004.

Kapp =

o−oapp cDNA(oapp −o /)

nNBS+ DNA“ (NBS)n DNA

(12)

where n is the number of NBS bound to each nucleotide of DNA, and (NBS)n DNA stands for the NBS–DNA complex. So we can get the binding constant of the interaction, Ka,

Rearrange Eq. (11), we can obtain: (o−oapp)=

oapp −o +(o −o /) KappcDNA

Ka

(11)

[NBS]% [n(NBS)n DNA] = [NBS][nDNA] [NBS][nDNA]

Namely, by plotting (o −oapp) against (oapp − o)/ 1 cDNA, which can yield a slope of K − app, we can calculate the binding constant. Table 3 lists the values of the binding of NBS with ctDNA and fsDNA, respectively, which were calculated according to Eq. (12) with the absorption data obtained at wavelength 637.6 nm.

According to material equilibrium principle, we have

3.4. Binding equilibrium as studied by absorbance change

[DNA]=

When NBS coexists with DNA, suppose NBS can interact only to one kind of separated binding site of DNA, then, DA = (1− x)A0 +xAb

(13)

[NBS]= cNBS − [NBS]%

(15)

(16)

and ncDNA − [NBS]’ n

(17)

Substitute Eq. (15) with Eq. (16) and Eq. (17), yielding: 1 cNBS = +1 ’ [NBS] Ka(ncDNA − [NBS]’)

(18)

Combine Eq. (14) with Eq. (18), yielding

where x is the molar ratio of bound NBS with DNA to free NBS, A0 is the absorbance at the wavelength considered without addition of DNA, and Ab is the absorbance of the NBS–DNA complex at the wavelength considered. By rearranging Eq. (13), we have, [NBS]’ DA − A0 = cNBS Ab −A0

Ka =

(14)

where [NBS]% is the total concentration of bound NBS with DNA. If DNA has an n separately independent binding site for NBS, then the interaction of NBS with DNA can be expressed as:

A0 − DA A− DA 1 cNBS = ncDNA − A0 − Ab DA − Ab Ka

(19)

So by using the slope and the intercept of the chart of (A0 − DA)cNBS/(A0 − Ab) against (A0 − DA)/(DA − Ab), we can obtain the binding number, n, and the binding constant, Ka. Table 4 lists the binding number and constant for the interactions of NBS with ctDNA and fsDNA, respectively. It can be seen that the results of Table 4 are identical with those of Tables 2 and 3. Generally, the binding of organic dyes with biological macromolecules is depicted by using

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Table 3 Binding constant of NBS with DNAs determined by average molar absorptivitya Nucleic acids

Linear regression equation

Linear correlation coefficient (r)

Binding constant (mol−1 l)

ctDNA fsDNA

y=0.00006+0.00031x y=0.000015+0.00030x

0.9999 0.9999

3225.8 3333.3

a

All data were obtained at wavelength 637.6 nm at 25°C. pH: 7.40; ionic strength: 0.004; Concentrations of DNAs: 3.0×10−4 mol l−1.

Scatchard plot [17]. It is obvious that the binding study according to Eq. (19) is different from Scatchard analysis. At first, the data treatment according Eq. (19) is very simple, it does not involve complicate calculation. In addition, Scatchard analysis treats the reaction as a true combination of the ligand with specific sites on the macromolecules, but the reality is not often the case [17].

3.5. Effects of the state of nucleic acids on the equilibrium Because of the negative backbone of nucleic acids which originates from the phosphate ion, the stranded structure of nucleic acids is very sensitive to the change of ionic strength. Generally, the controller of ionic strength of the solution, Na + , for instance, acts as a counter ion to reduce the unwinding tendency due to electrostatic repulsion between the negatively charged phosphate groups on adjacent nucleotides, and lead stability of the double stranded structure of the DNAs if the ionic strength is higher than 0.01 [3–5]. In this report, however, the concentration of nucleic acids is too excessive, the sensitive effect of ionic strength seems disappear if the ionic strength in the solution is lower than 0.02. Table 4 lists the binding number and binding constant of NBS with ctDNA and fsDNA, from which it can be seen that if the ionic strength is higher than 0.02, the binding number and binding constant decrease. One important phenomenon is the similarity between the binding of NBS with single-stranded DNA and that with native RNA. Fig. 2 shows the binding of NBS with thermally denatured ctDNA and with native RNA, respectively. It can be seen

that the interactions of NBS with thermally denatured DNA and native RNA, namely, singlestranded nucleic acids, produces two absorption bands around 610 and 655 nm, showing hypochromic effect and bathochromic shift compared with the characteristic absorption bands of NBS itself. The band located at 610 nm, corresponding the H-aggregation band of NBS itself, scarcely shows wavelength shift with increasing the concentration of NBS; the band around 655 nm, however, having larger absorptivity than that of the H-aggregation band. The absorbance ratio for the two bands, Amax/AH, decreases with increasing NBS concentration from 0.4× 10 − 5 to 2.0× 10 − 5 M, with the Amax/AH change from 0.365 to 0.414 for thermally denatured ctDNA– NBS binding and yRNA–NBS binding, respectively(Table 5).The Amax/AH ratio change, which greatly depends on increasing NBS concentration indicates that the interactions of NBS with single stranded nucleic acids result in aggregation species of NBS complex[3]. As Table 5 can be seen that the change of absorbance ratio is obviously larger than that of NBS itself in Table 1, that possibly indicates the aggregation of NBS in the presence of single stranded nucleic acids occur. In other words, H-aggregation of NBS can be encouraged by single stranded nucleic acids.

3.6. Thermodynamic parameters of the interactions of NBS with DNAs In the binding system of supramolecular interactions, Gibbs free energy change, DG 0cpl, is a very important parameters, which reflects the binding degree and the stability of the newly formed supramolecular complex. DG 0cpl can be calculated according following equation [18]:

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502

Table 4 Binding number and constant of NBS with nucleic acids determined by the change of absorbancea Nucleic acids

Ionic strength

Linear regression equation

Linear correlation coefficient (r)

Binding number Binding constant (mol−1· l)

ctDNA

0.004

0.9967

0.97

3225.8

fsDNA

0.004

0.9991

1.02

3448.3

ctDNA

0.010

0.9971

1.00

3333.3

ctDNA

0.020

0.9981

0.93

3225.8

ctDNA

0.030

0.9979

0.83

2857.1

ctDNA

0.040

y =0.00029 +0.00031x y =0.00031 +0.00029x y =0.00030 +0.00030x y =0.00028 +0.00031x y =0.00025 +0.00035x y =0.00020 +0.00042x

0.9991

0.67

2380.1

a

All data were obtained at 25°C at the wavelength 657.8 nm. pH: 7.40; Concentrations of DNAs 3.0×10−4 mol l−1.

DG 0cpl = − RT ln K

(20)

Since the study was constructed at constant temperature and pressure, DH 0cpl approximates to zero. So we can calculate the values of DS 0cpl for the interactions of NBS with DNAs. Table 6 lists the thermodynamic parameters for the suprmolecular interactions of NBS with DNAs. From Table 6 it can be seen that the interaction is a spontaneous process characterized entropy increase.

encouraged by single stranded nucleic acids. Considering the drug binding process in organic system in which small amount of drug interacting with large amount of biological macromolecules involves, an binding model for organic dyes with large amount of macromolecules was established, by which the interaction of NBS with double stranded nucleic acids were investigated. It was found that the interaction of NBS with double stranded nucleic acids occurs by the ratio of each nucleotide residue of DNAs binding one NBS molecule with the binding constant at 10 − 3 order. Calculation of the thermodynamic parameters of the interaction, including Gibbs free energy change, DG 0cpl, Helmholz free energy, DH 0cpl, and entropy change, DS 0cpl, shows supramolecular complexes formed, in which NBS exists in monomer.

4. Conclusion

Acknowledgements

At near physiological acidity and ionic strength 0.004, the H-aggregation of NBS resulting from increasing NBS concentration was characterized, and the aggregation band locates at 603.4 nm. It was found that the H-aggregation of NBS was

This research gets the supports of the National Natural Science Foundation of China (NSFC No.: 29875019) and the Municipal Science Foundation of Chongqing for Young and Middle Scientists(No.: 97-4729).

where the R is molar gas constant with the value 8.31 J K − 1 mol − 1, T the absolute temperature, K the binding constant. The calculated DG 0cpl is related to the change of Helmholz free energy, DH 0cpl, and entropy change, DS 0cpl, of the supramolecular interactions [18]: DG 0cpl =DH 0cpl −TDS 0cpl

(21)

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503

Table 5 Aggregation of NBS in the presence of single-stranded nucleic acidsa Concentration of NBS (×10−5 M)

0.4

0.8

1.2

1.6

2.0

ctDNA–NBSb

lmax (nm) Amax/AH lmax (nm) Amax/AH

657.6 1.448 657.3 1.536

656.8 1.287 657.4 1.376

654.6 1.194 654.2 1.258

654.1 1.135 654.1 1.180

yRNA–NBSc

652.3 1.085 652.3 1.117

a

pH: 7.40; ionic strength: 0.004; Concentration of nucleic acids: 3.0×10−4 M. The change of Amax/AH was 0.365 and 0.414 for thermally denatured ctDNA–NBS binding and native yRNA–NBS binding, respectively, with increasing NBS concentration from 0.4×10−5 to 2.0×10−5 M. b Thermally denatured ctDNA. c Native yRNA.

Table 6 Thermodynamic parameters of the interactions of ST with nucleic acidsa Thermodynamic parameters

DG 0cpl (kJ mol−1)

DH 0cpl (kJ mol−1)

DS 0cpl (kJ mol−1·K)

ctDNA fsDNA

−20.006 −20.172

0 0

26.806 28.655

a

T =298 K; pH: 7.40; ionic strength: 0.004.

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