estimation of binding sites from the adsorption profile of complexed dna

14
Estimation of Binding Sites from the Adsorption Profile of Complexed DNA SUBHENDU GHOSH AND ANJALI MOOKERJEE School of Environmental Sciences, Jawaharlal Nehru University, New Delhi- 110 067, India Abstract The adsorption profiles of free and Daunomycin bound DNA at an alumina-HzO interface have been studied. Proper experimental conditions were maintained to considerably reduce intercalation and to make electrostatic binding predominant in the DNA-Daunomycin complex. For both free and complexed DNA, Langmuir plots gave straight lines. Adsorption of drug bound DNA was less than that of free DNA. This has been explained from the viewpoint of electrostatic interactions between adsorbate and adsorbent. A Langmuir-type model for adsorption of polymers combined with Scatchard equation have been used to estimate the average value of percent of phosphates in DNA stacked with Daunomycin. Introduction The surface activity of biopolymers like nucleic acids, proteins and enzymes has been a subject of much investigation [ 1-61. An increasing number of bio- logical phenomena involve adsorption of surface-active molecules and ions at interfaces. For instance, intake of foreign molecules into cells takes place via cell membrane which offers a polar surface (lipid layer) to the foreign molecules for adsorption. Specific to the biopolymers, the phenomenon of adsorption, which is the attachment of molecules onto interface, involves mainly the electrostatic interactions between the polymers in solution and the adsorbing surface. Adsorption phenomenon at a solid-liquid interface is controlled, in most cases, by the electrical double layer. The double layer through proper mechanism imparts electrical charge over the whole surface and this charge, to a great extent, is responsible for the adsorption of the counter ions by means of electrostatic interaction. The physical state of dissolved DNA, e.g., conformation, length of segments, the tertiary structure, is very important as the surface adsorption demands specific stereo-chemical structural requirements. The interaction of DNA molecule with the solvent, e.g., water, determines the state of DNA in solution and this is also reflected in some way in the adsorbed condition. Measurements of intrinsic viscosity and sedimentation coefficient of the DNA solution confirm a structure intermediate between the rodlike and random-coil one. Further, refined analysis of hydrodynamic data and electron micrographs support the wormlike nature of DNA molecule in solution [7]. Small organic molecules, denoted as ligands, are used as “probes” to study the finer changes and distortions in the structure of the macromolecule [8-111. International Journal of Quantum Chemistry, Val. XX, 185-198 (1981) 0 1981 by John Wiley & Sons, Inc. CCC 01 61-3642/8 1 /070185- 14$01.40

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Page 1: Estimation of binding sites from the adsorption profile of complexed DNA

Estimation of Binding Sites from the Adsorption Profile of Complexed DNA

SUBHENDU GHOSH AND ANJALI MOOKERJEE School of Environmental Sciences, Jawaharlal Nehru University, New Delhi-

110 067, India

Abstract

The adsorption profiles of free and Daunomycin bound DNA at an alumina-HzO interface have been studied. Proper experimental conditions were maintained to considerably reduce intercalation and to make electrostatic binding predominant in the DNA-Daunomycin complex. For both free and complexed DNA, Langmuir plots gave straight lines. Adsorption of drug bound DNA was less than that of free DNA. This has been explained from the viewpoint of electrostatic interactions between adsorbate and adsorbent. A Langmuir-type model for adsorption of polymers combined with Scatchard equation have been used to estimate the average value of percent of phosphates in DNA stacked with Daunomycin.

Introduction

The surface activity of biopolymers like nucleic acids, proteins and enzymes has been a subject of much investigation [ 1-61. An increasing number of bio- logical phenomena involve adsorption of surface-active molecules and ions at interfaces. For instance, intake of foreign molecules into cells takes place via cell membrane which offers a polar surface (lipid layer) to the foreign molecules for adsorption. Specific to the biopolymers, the phenomenon of adsorption, which is the attachment of molecules onto interface, involves mainly the electrostatic interactions between the polymers in solution and the adsorbing surface.

Adsorption phenomenon at a solid-liquid interface is controlled, in most cases, by the electrical double layer. The double layer through proper mechanism imparts electrical charge over the whole surface and this charge, to a great extent, is responsible for the adsorption of the counter ions by means of electrostatic interaction.

The physical state of dissolved DNA, e.g., conformation, length of segments, the tertiary structure, is very important as the surface adsorption demands specific stereo-chemical structural requirements. The interaction of DNA molecule with the solvent, e.g., water, determines the state of DNA in solution and this is also reflected in some way in the adsorbed condition. Measurements of intrinsic viscosity and sedimentation coefficient of the DNA solution confirm a structure intermediate between the rodlike and random-coil one. Further, refined analysis of hydrodynamic data and electron micrographs support the wormlike nature of DNA molecule in solution [7].

Small organic molecules, denoted as ligands, are used as “probes” to study the finer changes and distortions in the structure of the macromolecule [8-111.

International Journal of Quantum Chemistry, Val. XX, 185-198 (1981) 0 1981 by John Wiley & Sons, Inc. CCC 01 61-3642/8 1 /070185- 14$01.40

Page 2: Estimation of binding sites from the adsorption profile of complexed DNA

186 GHOSH AND MOOKERJEE

It is already known that drug molecules can bind with DNA in two ways, weak binding or stacking which takes place at the phosphate sites and strong binding or intercalation between the bases. On the other hand, it has been indicated that the adsorption of DNA on alumina surface is mainly due to electrostatic inter- action between the negatively charged phosphate groups on the outer region of the macromolecular chain and the positively charged alumina surface. Hence, the adsorption on alumina surface is a function of the number of negatively charged sites on the DNA template.

As the adsorption profile of complexed DNA is not known, the present work has been undertaken with a view to elucidate the adsorption profile of ligand- bound DNA. The antibiotic Daunomycin has been used as a ligand for com- plexation with DNA. From the data obtained, a model has been proposed, combining the Langmuir model and Scatchard equation, for the quantitative estimation of the drug bound (stacked) phosphate sites.

Materials and Methods

DNA: Highly polymerized calf thymus DNA type I obtained from Sigma Chemicals, USA, was used for all experiments.

Daunomycin Hydrochloride: Daunomycin [Fig. I (a)] was obtained in the form of a hydrochloride from “Calbiochem” under the trade name of Daunorubicine. Since this was available in the analytical grade, it did not require further puri- fication. Concentration of Daunomycin used ranged from 3.5 to 27 pM.

For Daunomycin HCI literature from Calbiochem was available which gives the molecular weight as 527.5.

Sodium Chloride: Analar quality NaCl from BDH was used. Alumina: Alumina supplied by BDH had already been standardized for

Glassware: Corning glassware was used throughout the experiments. Preparation of Different DNA Samples: Native DNA-A stock solution of

DNA at a concentration of 200 pg/mL in 0.002M NaCl was prepared. This solution was then diluted to concentrations ranging from 10 pg/mL (30.30 pM) to 70 pg/mL (212.1 pM) and used for adsorption studies.

Procedure for Preparing DNA-Daunomycin Complex: Daunomycin was bound to DNA by mixing the two solutions in a test tube and gently shaking or rotating the test tube between the two palms for 10 min followed by an interval of 30 min for the process to attain a steady state. The concentrations (in pM) of DNA were the same as those used for adsorption of free DNA. The macromolecule to ligand ratio, written P/D, was maintained around 8 as any further addition of DNA only increases the number of free binding sites [ 121. Thus, at P/D around 8, there will be no further complexation, either intercalation or stacking, with Dau- nomycin, and free phosphate sites will be available for adsorption on to alu- mina.

Spectrophotometry: The optical densities of free and complexed DNA in the uv range at 260 and 320 nm were measured by Carl Zeiss PMQ I1 and ECIL

chromatographic adsorption analysis.

Page 3: Estimation of binding sites from the adsorption profile of complexed DNA

ESTIMATION OF BINDING SITES 187

0 OH

I 0 OH

H

WAVE LE NG 1 H, n m

(b) Figure 1. (a) Structure of Daunomycin. (b) uv absorption spectra of free Daunomycin in 0.002M NaCI; peaks at 235 and 255 nm.

spectrophotometers. The complete spectra were recorded by a Shimadzu MP 5000 spectrophotometer.

Particle Size: The average size of alumina particles was measured using a micrometer and microscope. The diameter of alumina particles ranged from 4 to 6 pm.

Procedure for Adsorption: Alumina surface is found to develop electrical charges depending on the p H of the solution in contact with it, which gives rise to electrostatic interactions with charged molecules or ions in liquid phase. The point of zero charge of alumina is at pH 9.0, below this p H value alumina par- ticles will be positively charged [ 131, and this was maintained throughout the experiments.

Solutions of different concentrations of free and Daunomycin bound DNA, were adjusted to pH 7.0 by HCl. Ionic strengths as determined by experimental conditions were adjusted by addition of requisite amounts of NaCl. Ten milli- liters of each solution was added to 1 g of alumina in 50 mL stoppered conical

Page 4: Estimation of binding sites from the adsorption profile of complexed DNA

188 GHOSH AND MOOKERJEE

flasks and shaken intermittently for 4 h at 20°C. The solutions were left un- disturbed for another 20 h, for attainment of equilibrium. The optical densities of the supernatant solutions were measured at 260 nm both before and after adsorption. Absorption was also measured at 320 nm to check on interference of any adsorbent particles.

Results and Discussion

On comparison of the uv absorption spectra of Daunomycin-DNA complex (at P/D = 8) and of free DNA at the same concentration as in complex (Fig. 2) it was seen that although the optical densities differed due to complexation there was little change in their contour patterns excepting a hump near 240 nm, and a blue shift of the DNA peak at 260 nm by 2 nm. Due to stacking the elec- tronic structure of DNA (the p~ and drr orbitals) remains more or less un- changed. Intercalation leads to changes in the electronic structure [8,11] changing the uv absorption spectra. Therefore, it is justifiable to consider that in the above case stacking is dominant and intercalation is reduced considerably. Under such circumstances, when both free and complexed DNA give absorption maxima at or near 260 nm, the optical density measurements at 260 nm can be taken to be proportional to the concentration of number of DNA molecules per mL. The amount of free and complexed polymer adsorbed has been calculated in terms of percentage.

Now, in case of complexation-when drug molecules are stacked to DNA-then, the number of phosphate sites remaining vacant for adsorption should be reduced. Hence the adsorption on alumina surface should be com- paratively less in the case of the complexed DNA than that of the free DNA.

,. 1.0 I \ 1

WAVELENGT H,nm

Figure 2. uv absorption spectra of free and complexed DNA in 0.002M NaCl; concentra- tions of DNA are 14.50 and 42.75 fig/mL, respectively and a P/D value of 8 as calculated. The weighed values of P ~ o and P40 are shown on the curves.

Page 5: Estimation of binding sites from the adsorption profile of complexed DNA

ESTIMATION OF BINDING SITES 189

Adsorption process is dependent on the ionic environment. When the exper- iments were carried out in a solvent of 0.05M sodium chloride [4,6], no signif- icant difference was found between the adsorption of free DNA and DNA- Daunomycin complex at P/D ratio equal to 8. Peacocke and Skerett [8] have shown that in case of proflavine-DNA complex, the number of dye molecules ( r ) bound per unit of nucleic acid (nucleotide phosphorus P) decreased with increase in ionic strength. It has also been pointed out that in general, low values of r and hence high values of P/D mainly correspond to intercalations, and stacking takes place at higher r values ( r > 0.22).

Since adsorption of DNA on charged surface like alumina takes place through electrostatic interactions, it may be assumed that at an ionic strength of 0.05M there has not been sufficient stacking to show significant difference in adsorption patterns between the free DNA and the complexed DNA. Hence all experiments were carried out at a lower ionic strength of 0.002M NaCl, in order to get sig- nificant differences in stacking between adsorption of free and complexed DNA. The results are given in Table I and Figure 3.

In case of DNA adsorption isotherms, Chattoraj and Upadhyay have shown that for native calf thymus DNA the Langmuir plot (C/X vs. C ) gives a straight line [4]. Later this was shown to hold for heat, uv and y irradiated DNA [6]. This clearly points to the fact that the adsorption of a polyelectrolyte like DNA takes place through the same process as in Langmuir model. Figure 4 shows the Langmuir plot for free and Daunomycin-bound DNA, giving straight lines.

TABLE I. Determination of percent adsorption of free and complexed DNA.

Initial OD Final OD at Difference DNA (P) and before equilibrium in AOD% of

Complexed adsorption (after adsorption) AOD% = Pi and Ci DNA (C) ( 0 ) ( b ) a - b/a X 100 samplesa

Pi0 (14.50)b Cl0 Pro (22.75) c20 Pjo (30.75) c30 Pw (42.75) c40 Pso (46.75) c50 P60 (55.00) c60 P70 (63.25) c70

0.290 0.37 0.455 0.58 0.615 0.76 0.855 0.96 0.935 1.15 1.10 1.345 1.265 1.56

0.105 0.15 0.20 0.28 0.35 0.455 0.490 0.58 0.65 0.68 0.80 1.025 0.96 1.25

4.35

4.32

2.96

3.1 1

5.26

3.48

4.24

63.79 59.46 56.04 51.72 43.09 40.13 42.69 39.58 30.48 25.22 27.27 23.79 24.1 1 19.87

a Pi stands for free DNA at a concentration i (pg/mL) (as weighed); Ci stands for complexed DNA at a concentration i (pg/mL) (as weighed).

The calculated amount of DNA in pg/mL is written in parentheses.

Page 6: Estimation of binding sites from the adsorption profile of complexed DNA

190

70

60- 2-

5

t 50- ul

0

4 2

2 L o - n

a 30- 0

GHOSH AND MOOKERJEE

-

9 0 c 80 / \\ - F R E E D N A

H C O H P L E X E O D N A

I I I I L I I

10 20 30 40 5 0 6 0 70 I N I T I A L C O N C E N T R A T I O N O F D N A (pg/ml)

Figure 3. Percent adsorption of free and complexed DNA (average of three sets). This gives an estimate of what fractions of initial amounts of DNA are getting adsorbed at different initial concentrations of DNA.

Before adsorption of the polymer the small molecules (including the ligand molecules in the present case) and electrolytes occupy the adsorption sites on the solid surface and cover it by molecular layer formation. By means of con- tinuous shaking with the polymer solution, the previous equilibrium is disturbed while the adsorption sites are more and more exposed to the polyelectrolytes. This enables the polyelectrolytes to get adsorbed till equilibrium is attained. This has already been shown by other workers through kinetic studies.

Mathematical Model

Let Y moles of solvent be displaced by 1 mol of phosphate (v < 1). If A and A, are the polymer in solution and that adsorbed on surface, respectively, and B and B, are the solvent in solution and that adsorbed on surface, respectively, then the adsorption process can be represented by the chemical equation

A + u - B, + A, + Y - B. (1)

If N1 and N2 are the respective mole fractions of solvent and polymer in the solution, and Nf and N s are the respective mole fractions of the solvent and polymer on the surface after adsorption, then for dilute solutions, the equilibrium constant for the process at the experimental temperature is given by

K = ( N ~ ) N ’ ; / ( N ~ ) ” N ~ . (2)

Page 7: Estimation of binding sites from the adsorption profile of complexed DNA

ESTIMATION OF BINDING SITES 191

0 M F R E E D N A

C O M P L E X E D D N A

0 O t /// 2 .6

0 p/: l A - W

1 8 - 0

2 1 1 -

c -

I I I I 02 O L 0 6 0 8 1 0 1 2 11

O P T I C A L D E N S I T Y A F T E R A D S O R P T I O N

Figure 4. Langmuir plot. Instead of plotting C/x vs. C directly, equilibrium OD/AOD vs. equilibrium OD were plotted, as C/x is equal to equilibrium ODIAOD and C is proportional to equilibrium OD.

Keeping in view the model for adsorption of polymer on solid surfaces [ 14,151 DNA here has been considered as the chain of phosphate units which are neg- atively charged and adsorbed via electrostatic interaction on the positively charged alumina surface. When undergoing adsorption a part of the chain re- mains in the solution unadsorbed. This means some fraction of the total phos- phate is bound to the solid surface. However, this takes place by replacement of solvent molecules already adsorbed on the solid surface.

If 8 is the fraction of surface covered by the polymer, then from modified Langmuir model, we get

--- 8 l V JN2

(1 -8)” N ; Now.

(3)

no. of moles of polymer molecules adsorbed (n;) total no. of moles of solvent molecules adsorbed (n?) ’

8 =

Page 8: Estimation of binding sites from the adsorption profile of complexed DNA

192 GHOSH AND MOOKERJEE

For our system nj >> n;, hence we can write

( 1 - 6’) N 1 (since 6’ << 1).

Hence, K N d N ; = O / u [from Eq. (3)], or

n y n ; = K - uNZ/NI;. (4)

The solvent in the present set of experiments is 0.002M NaCI, the function of which is mainly to stabilize DNA. The role of solvent in adsorption is played by water molecules and one can neglect the role of sodium and chloride ions in adsorption. The total amount of Daunomycin present in each complex solution is much less than that of nucleotide phosphate (at p / D = 8, D = 3.5 to 27 pM). Out of this amount of Daunomycin, a part is used for binding leaving a small fraction free in solution. Now theoretically this Daunomycin may adsorb on alumina surface and thus interfere with the adsorption of DNA. Free Dau- nomycin adsorption is evidenced from the fact that an orange colour develops on the alumina surface after adsorption. However, the surface remains white after adsorption of Daunomycin-DNA complex, indicating considerable re- duction in free Daunomycin adsorption. Thus, for all practical purposes, one can neglect the interference of free Daunomycin in adsorption of DNA. Also, the experimental solutions are dilute so that mole fraction of water remains constant for all practical purposes. Therefore,

N I 1. (4’)

Again, N2 is the mole fraction of macromolecule in solution at equilibrium, equal to moles of macromolecules per moles of macromolecules plus moles of water in solution at equilibrium. For dilute solutions,

moles of macromolecules << moles of water present. (of the order of 10-4M)

Hence

moles of macromolecules in solution ( 2 2 )

moles of water in solution ( Z l ) N2 =

at equilibrium. Therefore, from Eqs. (4) and (4’),

or

The termfis equal to the ratio of moles of polymer adsorbed on solid surface to moles of polymer present in solution at equilibrium. But, the total volume of solution in contact with 1 g of solid was kept constant (10 mL). Hence moles of polymer adsorbed is proportional to change in concentration.

And, moles of polymer in solution at equilibrium corresponds to the equilib- rium concentration of the macromolecule in solution (in mol) which is given by C = (1 / d ) O D where, E is the molar extinction coefficient, 1 is the optical path

Page 9: Estimation of binding sites from the adsorption profile of complexed DNA

ESTIMATION OF BINDING SITES 193

length, and OD is the optical density of the solution. Then polymer adsorbed from each mL of solution is given by

initial concentration - final concentration at equilibrium = ( 1 /tl)OD, - ( 1 /E/)ODF.

Putting a = OD1 and b = ODF, and (1 /d) = J the fraction of macromolecules per unit volume of the solution adsorbed on the solid surface is then equal to J ( a - b)/Ja. Therefore, percent of macromolecules adsorbed is given by

( a - b)/a x 100 = AOD% (7)

AOD% is determined from experimental data (Table I). By using this method of calculation, evaluation of J is not necessary. Also, f = (a - b ) / b from Eq. ( 6 ) for free DNA. Similarly, let dashed quantities denote the parameters for complexed DNA. Then from Eq. ( 6 ) for complexed DNA, we get

f = K'nju'/Z,. (8) As for dilute solutions ni and Z1 remain almost the same. Dividing Eq. (8) by (61, we get

In order to evaluate K/K' a Scatchard type of plot has been used. Considering the binding between alumina surface and DNA one may express the amount of phosphates ( r ) bound per gm of alumina in a way similar to drug-DNA binding [8,11,14,16,17] as

r4 = nKCfree/(l + Kcfree), (10)

where Cf,, is the concentration of free phosphates, K is the equilibrium constant of the binding process at that temperature, n is the total number of binding sites, and 4 is a constant depending on moles of alumina present.

From Eq. ( l o ) , by simplification, we get

( 1 1 )

Hence, a plot of r/Cfree vs. r should be a straight line with a slope of - K . How- ever,

r -- - ( K / t ) ( n - t r ) . Cfree

r A O D X ~ O = 10 f values. -- -

Cfree ODfinal

In order to evaluate r , we first calculate the amount in micromoles of DNA using Beer's law from AOD values and the extinction coefficient t (Table 11). The latter is calculated from optical density versus concentration of DNA plot (Fig. 5). From Fig. 5,

for free DNA, for complexed DNA,

tP = 0.006 pM-lcm-', t, = 0.007 pM-'cm-l.

Page 10: Estimation of binding sites from the adsorption profile of complexed DNA

194 GHOSH AND MOOKERJEE

TABLE 11. Concentration of DNA adsorbed in p M / g of alumina along with the corresponding fand f' values.

a - b a' - b' (a) Free DNA - X 10 (b) - X 10 Complexed DNA

€ € '

Amount of Amount of DNA complexed

adsorbed in DNA adsorbed f values DNA pM/g of fvalues Complexed in pM/g of (a' - b')/ (Pi) AOD alumina ( a - b) /b DNA (Ci) AOD alumina b'

Pi0 0.1917 319.5 1.8603 Ci0 0.2283 326.1 1.5996 P20 0.2517 419.5 1.2385 C2o 0.2966 423.7 1.0413 P30 0.2833 472.2 0.8484 C30 0.3147 449.6 0.6928 P40 0.3633 605.5 0.7441 C40 0.3550 507.1 0.6337 Pso 0.2866 477.7 0.4410 C50 0.2900 414.3 0.3335 P60 0.3233 538.8 0.3819 C.50 0.3100 442.9 0.301 1 P70 0.3116 519.9 0.3270 C7o 0.3233 461.9 0.2618

In the Scatchard type of plot (Fig. 6) straight line graphs with negative slopes were obtained for both free and complexed DNA. The graph for complexed DNA runs below that of free DNA, and

4.0 X 10 6.0 X lo2 '

for free DNA slope -K =

3.78 X 10 5.85 X lo2

for complexed DNA slope -K' = -

Dividing (1 2a) by (1 2b), we get

1 2 t P+D

C O N C . O F DNA I N ? g / m t Figure 5. Standard plot of optical densities vs. concentrations (in pg/mL) of free and complexed DNA to evaluate amounts of DNA adsorbed/g of lumina which is proportional to AOD values. For determination of c, 330 g / L of DNA = 1 M has been used.

Page 11: Estimation of binding sites from the adsorption profile of complexed DNA

ESTIMATION OF BINDING SITES 195

Figure 6. Scatchard type plot r/Cf,, vs. r, where r is the amount (pM) of nucleotide phos- phates bound per gram of alumina site; Cfrae is the concentration of free nucleotide phos- phates.

KIK‘ = 1.031. (13)

Therefore, from Eqs. (9) and (13),

u r / u = 1.03 f/f

or

u - Y’ f- 1.03f -- - U f

We consider the term ( u - u’) /u to be the measure of the fraction of phosphates stacked with Daunomycin.

In order to evaluate ( u - v’) /u by using Eq. (14), f andf were calculated and plotted against concentration of DNA (40 to 200 pM) as shown in Figure 7. Now taking f andf values from the graph the values of (v - v’ ) /u were computed from Table 111.

The values of ( u - d ) / u against concentration of DNA (pM, as shown in Table 111) have been plotted in Figure 8. It shows a rise in the value of percent phosphates stacked with Daunomycin with increase in concentration of DNA: the value ranges from 7% to 19% for a concentration range 40 to 200 pM. This result may be interpreted as follows:

Bradley and Wolf [ 181 showed in case of Acridine Orange that binding of drug at any site of DNA would enhance or favor the binding of drugs on the

Page 12: Estimation of binding sites from the adsorption profile of complexed DNA

196 GHOSH AND MOOKERJEE

2.2 c 1.6

;; 1.k > -- 1.2

04 -”: 0 . 2

0 2 4 6 E 10 12 1L 16 18 2 0 2 2 C O N C . O F POLYMER ( D N A ) I N y M X t 0 - l

Figure 7. f a n d f values vs. concentration of polymer (DNA) in q M (Table 111)

neighboring sites due to cooperativity factor (a probabilistic interpretation has also been given). Following similar argument one would expect that as con- centration of DNA is increased keeping the DNA to drug ratio constant, drug molecules may be expected to bind to phosphates (of DNA) via a stepwise mechanism, where each binding results in increased probability for the subse- quent binding to occur. Thus, due to cooperativity the number of drug-bound phosphate sites would increase (with multiplicity) with increase in phosphate concentration.

TABLE 111. Evaluation of percent of phosphates stacked with Daunomycin.

u - U I -X

U

f - 1.03f 100 = ___

f x 100

a - b f = a ’ - b ’ = %of phosphates stacked Concentration of f=- DNA (@MI b b’ with Daunomycin

40 60 80

100 120 140 160 180 200

2.12 1 .57 1.20 0.94 0.74 0.6 0.48 0.39 0.33

1.9 0.42 1.08 0.86 0.66 0.51 0.39 0.32 0.26

7.689 6.815 7.600 9.043 8.108

12.600 16.250 15.384 18.788

Page 13: Estimation of binding sites from the adsorption profile of complexed DNA

ESTIMATION OF BINDING SITES 197

f 19

17

v 15 Y

Q

0 2 1 -

W

-l + 11-

L k k 10 12 1L 16 18 20 I I I I I I 2( 5 L

L 6 8 10 12 1L 16 18 20

C O N C . O F DNA I N p M X lo-‘

Figure 8. Percent of phosphates stacked with Daunomycin ( u - v’)/u vs. concentration of DNA in FM.

While calculating the percent of binding sites stacked, the value of [(v - v’)/v] has been determined. This is done on the basis of the fact that even if the binding of the drug to DNA chain is not a random process, the chain may be considered as consisting of regions of adsorption sites and these regions are randomly dis- tributed over the entire DNA chain. This allows one to conclude that the number of segments of the macromolecular chain getting adsorbed on the solid surface will be distributed at random. The same also holds in case of complexed DNA with bound phosphate sites due to drug interaction.

Although the work is on Calf thymus DNA and a specific “drug” (Dau- nomycin) this model suggests a general method for estimating the number of binding sites stacked with a drug. At present the model is limited to stacking only. A modification to deal with complexes where intercalation predominates, and also a study of the dependence if any, on the type of drug and DNA, are interesting fields of work to be undertaken.

Acknowledgments

The authors are thankful to Dr. Rita Mukhopadhyay, School of Environ- mental Sciences, JNU, for her critical comments on this paper. One of us (S.G.) received financial assistance from Jawaharlal Nehru University, New Delhi.

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198 GHOSH AND MOOKERJEE

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