Thermodynamic Characterization of Proflavine Binding to DNA

14 Thermodynamic Characterization of Proflavine Binding to DNA

Downloaded by UNIV OF MINNESOTA on August 15, 2013 | http://pubs.acs.org Publication Date: May 12, 1982 | doi: 10.1021/bk-1982-0186.ch014

YOSHIHIRO BABA1 and CHARLES L . B E A T T Y University of Florida, Materials Science and Engineering, Gainesville, F L 32611 AKIHIRO KAGEMOTO Osaka Institute of Technology, Asahi-Ku, Osaka 535, Japan

The interaction between DNA and proflavine was studied by means of the heating of mixing. From the results, the thermodynamic quantities were estimated for the DNA-proflavine system by an intercalation process. The thermodynamic results show that the intercalation process of DNA-proflavine system would be expected to be dominated by the enthalpy rather than the entropy. In addition, the heat of interaction depends on GC base composition of DNA; the heat of interaction between GC base pairs and proflavine i s greater than that between AT base pairs and proflavine.

It i s well known that aminoacridine dyes are bound to DNA i n two different ways depending on the concentration of dye: the first type corresponds to strong binding1) and/or intercalation process2) i n which dye molecules are bound to the DNA base at dilute concentration of dye. The other type corresponds to the weak binding1) and/or stacking process2) i n which dye molecules are bound to the outside of DNA molecules without base s p e c i f i c i t y as the concentration of dye increases.

1

Permanent address: Osaka Institute of Technology, Asahi-Ku, Osaka 535, Japan.

0097-6156/82/0186-0177$5.00/0 © 1982 American Chemical Society In Biological Activities of Polymers; Carraher, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

BIOLOGICAL ACTIVITIES OF POLYMERS


178

The interaction for the intercalation process seems to be based on an interaction of f l a t aromatic dye molecules between the base pairs of DNA as reported by many investigators"tl—I . However, the exact steric location of the intercalated dye and the heat of interaction between DNA and dye have not been established yet. In this paper, we attempt to elucidate the thermodynamic quantities of the interaction i n the dye binding by intercalation process and also explore the effect of GC base composition of DNA on dye binding by measuring the heat of mixing of proflavine (PF) and CI. perfringens DNA (DNA I ) , E. c o l i DNA (DNA II) and M. lysodeikticus DNA (DNA I I I ) , respectively because the interaction for intercalation process i s based on the interaction between dye and base pairs of DNA. EXPERIMENTAL Materials: The DNA's employed were CI. perfringens DNA (31 % GC, Sigma type XII), E. c o l i DNA (50 % GC, Sigma type VIII) and M. lysodeikticus DNA (72 % GC, Sigma type XI) and used without further purification. The dye used was proflavine (PF) which was purchased from Aldrich Chemical Co., Inc. A l l measurements were i n 0.1 mol/1 tris-HCl buffer solution (pH 7.60). Apparatus and Procedure: The calorimeter which was used for the measurement of the heats of mixing of DNA and PF was a LKB batch type microcalorimeter (LKB-10700). For calorimetric measurement, the DNA sample was dissolved into the buffer solution, and equal volumes (about 1.2 cm^) of DNA and dye solutions were mixed. In this case, the concentration of DNA was kept at a definite value of 4X10"^ mol/1 of phosphate units of DNA while for the dye solution various amounts of dye were used. The DNA concentration was determined from molar extinction coefficient at 260 nmSi E =7400, 6500 and 7000 for CI. perfringens DNA (DNA I ) , E. c o l i DNA (DNA II) and M. lysodeikticus DNA (DNA III), respectively. In order to obtain information about the binding parameter for the intercalation process, the absorption spectra of solutions containing a constant concentration of PF and varing amounts of DNA were measured by using a spectrophotometer (Perkin-Elmer, Model 552). p

RESULTS AND DISCUSSION Spectral measurement Typical absorption spectra of solutions containing a definite concentration of PF and various amounts of DNA are shown i n Figures 1(a), (b) and (c). As seen i n Figures 1, the wavelength of maximum absorbance for each system shifts to red as the

In Biological Activities of Polymers; Carraher, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In Biological Activities of Polymers; Carraher, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982. 5

X(nm)

5

s

4

Typical absorption spectra of PF solution (1.6 X 10' mol/L) containing various amounts of DNA.

X(nm)

4

4

s

4

4

s

(a) DNA II-PF system shows DNA concentrations: A, 0; B, 5.36 X IO' ; C , 1.36 X W ; and D, 5.30 X 10~ mol/L. (b) DNA l-PF system shows DNA concentrations: A, 0; B, 3.92 X 10 ; C, 9.79 X 10 ; and £>, 3.92 X 10~ mol/L. (c) DNA III-PF system shows DNA concentrations: A, 0; B, 4.01 X 10 ; C, 1.15 X IO ; and D, 4.95 X 10~ mol/L.

Figure 1.

X(nm)


VO


180


concentration of DNA increases and well-defined isosbestic points for each system are located at about 454 nm which i s compatible, with the previous resultS) and results reported by Lee et a l . — . . The percentage of dye bound to DNA was calculated as described by Peacocke and Skerrettli). The plots of r/Cf versus r for each DNA-PF system are shown i n Figures 2(a), (b) and (c). In this case, r i s the amount of PF bound per nucleic acid phosphate, and Cf i s the concentration of free dye. From those plots the binding parameters were estimated and are given i n Table I. It is apparent that the binding parameters for each system are i n f a i r agreement, showing that there i s no base composition of DNA selectively i n binding between DNA and PF.

Heat of Mixing The heats of mixing of DNA and PF measured at 298.15 K are found to be exothermic, indicating the attractive interaction between DNA and PF molecules. The results are shown i n Figure 3, where the heat of mixing oer mole of dye, AH i s plotted against the mole ratio of PF to DNA, [dye]/[DNA]. AH , for each system shows the sigmoidal curve such as AH increases (takes a smaller negative value) as [dye]/[DNA], that i s , PF concentration increases. Those results are compared with the successive stages of the intercalation process as described by Fredricq and Houssieri£). When [dye]/[DNA] i s less than about 0.05, the intercalated dye molecules do not interact with one another, i n the range from 0.05 to 0.13 of [dye]/[DNA] the intercalated dye molecules do interact, and then dye molecules begin to bind to the side of DNA molecules when [dye]/[DNA] i s more than 0.13. To obtain the heat of interaction between DNA and PF from calorimetric results, the following analysis i s used. M

M

M

Thermodynamic Quantities Assuming that the DNA-dye complex i s formed by following reaction process between DNA and dye, DNA + dye

(DNA - dye)

the binding constant, K, i s expressed as


(1)

ET AL.

Proflavine

Binding

to

DNA


14. BAB A


181


182


Table I The binding parameters for the intercalation process of the DNA-PF complexes estimated from spectrophotometry

DNA

K X10~

5a)

DNA I (GC 31 %)

5.6

0.20

DNA II (GC 50 %)

4.1

0.20

DNA III (GC 72 %)

6.0

0.21

Binding constant Number of binding sites per DNA phosphate


14.

BABA ET AL.

Proflavine

Binding

to DNA

183


ι. ^ 5&

·—·

SO •s δ .!£

%:

|ï

* i

I .00



184

where n i s the number of binding sites per DNA phosphate, r the amount of bound dye per DNA phosphate and Cf the concentration of free dye. From the experimental results of the heat of mixing, the heat of interaction of DNA-dye complex per mole of dye, AH, can be expressed as


*

- cr?

«>

Where AQ i s the observed heat, C5 the concentration of bound dye which i s correlated with r and the concentration of DNA phosphate P, such as C|j=rP, and V the total volume of the mixture of DNA and dye solutions. The total concentration of dye, C, i s the sum of C^ and Cf; C = C

fe

+ C

(4)

f

From Eqs. (2) to (4), AQ w i l l be represented by following complicated function of C, i n which the negative sign of the right side must be adopted because correct zero value of AQ/V i s given at zero of the concentration of dye, C. |^ = | S [ p + | + C n

v/(Pn + I + C )

2

- 4PnC

]

(5)

According to Eq. (5) we are able to calculate the value of AQ/V for a given value of C by using both the values of K and n estimated from spectrophotometry. The AH value which gives the best f i t between the calculated value and the experimental one of AQ/V i s adopted. The AH values adopted are about -70, -85 and -90 kJ/mol for DNA I, DNA II and DNA III, respectively. But the calculated value of AQ/V deviates from the experimental value in the concentration region of dye more than about 3X10"^ mol/1 as shown i n the broken lines i n Figure 4. Those deviations may be a result of K and n values estimated from spectrophotometry i n which the concentration of DNA i s different from that in calorimetry. To improve those discrepancies, K and n can be treated as variable parameters and adjusted i n such a way that the best f i t between experimental and calculated values of AQ/V i s obtained. These quantities are plotted against C as a solid l i n e in Figure 4 and the values of AH, K and n estimated are l i s t e d i n Table I I . The K values for each system estimated from calorimetry are i n agreement with those from spectrophotometry, but the n values estimated from calorimetry are a l i t t l e lower than those from spectrophotometry. The decrease i n n values may be ascribed to the intermolecular interaction of DNA which increases with the concentration of DNA and brings about the decrease of effective binding sites of DNA.



Figure 4. Dependence of the observed heat of mixing, A Q / V on the dye concentration, C, for DNA-PF system. Key: (%) DNA / - P F system; (O) DNA II-PF system; and A , DNA III-PF system; theoretical A Q / V curve calculated with Equation 5 using K and n values in Table I ( ) and in Table II ( ).

4.0


oo


-32 -33

5

4.1 XlO

6.0 XlO

DNA II(GC 50 %)

DNA III(GC 72 %)

1

-180 -190

-85 -90

1

AS J K~ mol* -120

1

-70

AH kJ mol"

mol refers to the number of moles of PF a) ' Binding constant Number of binding sites per DNA phosphate

-33

5

AG kJ mol' 5

1

5.6 XlO

1 mol"

DNA I(GC 31 %)

DNA 1

Table II The thermodynamic quantities for intercalation process of DNA - PF system estimated from calorimetry at 298 K


0.18

0.18

0.17

14.

BAB A

ET

Proflavine

AL.

Binding

to

187

DNA

The free energy change, AG, and the entropy change, As, for each system are easily calculated from the following equations,


AG = -RT In K,

AS - (AH - AG)/T

(6)

and are l i s t e d i n the third and f i f t h columns i n Table I I , respectively. As seen i n Table I I , the values of AH for each system are different and decrease with decreasing GC base composition of DNA although the values of AG are nearly equal. The AH dependence on GC base composition of DNA seems to be based on difference of the heat of interaction between GC base pairs and PF and between AT base pairs and PF. Base composition effect of heat of interaction Assuming that the three types of DNA/dye interaction ( i . e . between GC pairs, AT pairs and GC-AT base pairs) occur i n numbers given by the probability of existence of such pairs when DNA molecule corresponds to a random chain, then the heat of interaction, AH, i s represented as 2

AH = n^A^

+ n AH + n ^ A ^

(7)

2

where AH^, AH« and AH~ are the heats of interaction between GC base pairs ana dye, AT base pairs and dye and GC-AT base pairs and dye, respectively, and n and n are fractions of GC and AT base pairs i n DNA molecule, since n i s equal to one minus n^, AH i s also written as the function of n , A AH = (AH^ +AH - 2AH )n* + 2(AH - AH )n + AH^ (8) fi

A

2

3

3

£

A

The plots of AH estimated from calorimetry versus GC fraction of DNA are shown i n Figure 5, and the solid line i n Figure 5 shows the best f i t curve of Eq. (8). From this curve, we can obtain AH^-96, and AH =-50 kJ/mol. 2

Conclusion From the thermodynamic quantities estimated from calorimetry, the intercalation process of DNA - PF system would be expected to be dominated by the enthalpy rather than entropy because of negative value of the heat of interaction and positive value of entropy change for the interaction between DNA and PF. The heat of interaction between DNA and PF i s greater than that between DNA and acridine orangeil). This greater heat of interaction may be based not only on interaction between base pairs of DNA and PF but on hydrogen bonding between phosphate group with negative charge of DNA and amino group on 3 and/or 6 position of PF as reported by Peacocke et al.i-U.


188



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I ι υ § < sf ο

s I •S 'δΙ •δ·

I I


14.

BABA ET AL.

Proflavine

Binding

to

DNA

189

In addition the dependence of the heat of interaction of GC base concentration of DNA may be based on the difference of the heat of interaction between GC base pairs and PF and AT base pairs and PF because the heat of interaction of GC base pairs and PF i s greater than that between AT base pairs and PF, Aolmowledgment


The authors (Y. Baba and C. L. Beatty) would l i k e to acknowledge p a r t i a l support of this work by the Biomedical Center of Excellence, University of Florida. Literature Cited 1. A. Blake and A.R. Peacocke, Biopolymers, 6, 1225 (1968). 2. L.S. Lerman, J . Mol. B i o l . , 3, 18 (1961). 3. A. Blake and A.R. Peacocke, Biopolymers, 5, 39 (1967). 4. A Blake and A.R. Peacocke, Biopolymers, 5, 871 (1967). 5. D.M. Neviller, J r . , and D.R. Davies, J . Mol. B i o l . , 17, 57 (1966). 6. B.J. Gardner and S.F. Mason, Biopolymers, 5, 79 (1967). 7. N.F. Gersch and D.O. Jordan, J . Mol. B i o l . , 13, 138 (1965). 8. G. Felsenfeld and S.Z. Hirschman, J . Mol. B i o l . , 13, 407 (1965). 9. S. Tanaka, Y. Baba, A. Kagemoto and R. Fujishiro, Makromol. Chem., 181, 2175 (1980). 10. CH. Lee, C.T. Change and J.W. Wetmur, Biopolymers, 12, 1099 (1973). 11. A.R. Peacocke and J.N. Skerrett, Trans. Farady Soc., 52, 261 (1956). 12. E. Fredericq and C. Houssier, Biopolymers, 11, 2281 (1972). 13. Y. Baba, C.L. Beatty and A. Kagemoto, i n preparation. 14. N.J. Pritchard, A. Blake and A.R. Peacocke, Nature (London), 212, 1360 (1966). RECEIVED February 5, 1982.


Thermodynamic Characterization of Proflavine Binding to DNA

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