Adsorption of Biopolymer at Solid− Liquid Interfaces. 1. Affinities of

The extent of adsorption (Γ21) of DNA from aqueous solution on different hydrophobic and hydrophilic solid surfaces has been compared as a function o...
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Articles Adsorption of Biopolymer at Solid-Liquid Interfaces. 1. Affinities of DNA to Hydrophobic and Hydrophilic Solid Surfaces S. A. Gani, D. C. Mukherjee,† and D. K. Chattoraj* Department of Food Technology and Biochemical Engineering, Jadavpur University, Calcutta 700032, India, and University College of Science, Calcutta University, 92 A. P. C. Road, Calcutta 700009, India Received June 26, 1997. In Final Form: May 17, 1999 The extent of adsorption (Γ21) of DNA from aqueous solution on different hydrophobic and hydrophilic solid surfaces has been compared as a function of pH, temperature, ionic strength of the medium, and denaturants. Γ21 at a given surface (except sephadex) increases with an increase of the nucleotide concentration (X2) of DNA in mole fraction units, but it attains a monolayer saturation value Γ2m when X2 attains a value X2m. In most cases Γ21 increases further when X2 . X2m. Various neutral electrolytes like LiCl, KCl, CsCl, KBr, CaCl2, and Na2SO4, surfactants like SDS and Triton-X 100, the denaturing action of heat, and the addition of acid and alkali have been observed to play an important role during the study of the adsorption of DNA on charcoal powder. Further, a comparative study has been performed to examine the relative affinity of DNA toward different types of solid surfaces. The significant role of different types of surfaces in controlling the adsorption process has been explained in terms of Gibbs’ surface excess quantities. The experimental results have been interpreted in terms of maximum work due to the free energy change of DNA adsorption on various solid surfaces and more quantitatively in terms of the standard free energy change (∆G°) for the saturation of the surface by DNA as a result of the change in the nucleotide concentration in the bulk from zero to unity in mole fraction units.

Introduction Because of the biological importance of cell membrane proteins, physicochemical investigations of this type of biopolymer at solid-liquid and liquid-liquid interfaces have been extensively studied using many surfacechemical techniques.1-6 Compared to this, the behavior of DNA, the most important natural polyelectrolyte controlling genetic aspects of living systems at liquid and solid surfaces, has not been investigated in detail. However, in the last few decades, it has been noted that nuclear membranes of eukaryote cells are in localized interactions with DNA present in chromosomes in an invaginated state.7 It has also been speculated that DNA is in considerable interactions with membrane materials of cells during the cell division process. Positively charged liposomes of cationic lipids are now used to entrap negatively charged DNA, and this led to the possibility of gene transfer in vivo.8,9 Two possible applications of cationic lipids are the aerosol delivery of DNA to lungs * To whom all correspondence regarding this paper should be sent at Jadavpur University. † Calcutta University. (1) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 257. (2) Hjerten, S. Adv. Chromatogr. 1980, 19, 111-123. (3) Porath, J. Pure Appl. Chem. 1979, 51, 1549-1559. (4) Greig Russell, G.; Brooks, D. E. J. Colloid Interface Sci. 1981, 83, 661. (5) Hajra, S.; Chattoraj, D. K. Ind. J. Biochem. Biophys. 1991, 28, 184. (6) Lee, S. H.; Runkenstein, E. J. Colloid Interface Sci. 1988, 125, 385. (7) Lehninger, A. L. Biochemistry, 2nd ed.; First Indian Edition, Kalyani Publishers: 1978; p 139. (8) Zhu, N.; Liggitt, D.; Liu, Y.; Debs, R. Science 1993, 261, 209-211.

and the development of cancer vaccines.10,11 Recently, an interesting paper12 has been published reporting the formation of a lipid-DNA complex in organic media which may precipitate in an aqueous surrounding. Direct interaction of plasmid DNA encoding foreign antigens into the skin surface has been shown to evoke a protective immune response.13 The method of delivery of DNA into different types of human tissues using a “gene gun” developed14-17 originally for transformation of plants involves gene-coated gold particles. In all these cases it appears that DNA-surface interaction may play a major role, and the nature of such interaction in general needs detailed investigation. Earlier, in 1968 and onward, some interest has been shown for investigating the behavior of DNA adsorbed on (9) Tsukamoto, M.; Ochiya, T.; Yoshida, S.; Sugimura, T.; Tesada, M. Nat. Genet. 1995, 9, 243-248. (10) Sarscher, E. J.; Logan, J. J. Hum. Gene Ther. 1994, 5, 10891094. (11) Nabel, G. J.; Nabel, E. G.; Yang, Z. F.; Fox, B. A.; Plautz, G. E.; Gao, X.; Huang, L.; Shu, S.; Gordon, D.; Chang, A. E. Proc. Natl. Acad. Sci. USA 1993, 90, 11306-11311. (12) Kuniharu, L.; Yoshio, O. J. Chem. Soc., Chem. Commun. 1992, 1339. (13) Ulmer, J. B.; Donnelly, J. J.; Parker, S. E.; Rhodes, G. H.; Felgner, P. L.; Dwarki, V. J.; Gromkowski., S. A.; et al. Science 1993, 259, 17451749. (14) Whalen, R. G.; Davis, H. L. Clin. Immunol. Immunopathol. 1995, 75, 1-12. (15) Vile, R.; Russel, J. J. Gene Ther. 1994, 1, 88-98. (16) Spooner, R. A.; Deonarain, M. P.; Epenetos, A. A. Gene Ther. 1995, 2, 173-180. (17) Cheng, L.; Ziegelhoffer, P. R.; Yang, N. S. Proc. Natl. Acad. Sci. USA 1993, 90, 4455-4459.

10.1021/la970686h CCC: $18.00 © 1999 American Chemical Society Published on Web 09/25/1999

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solid-liquid,18 liquid-liquid,19 and air-water20 interfaces. The techniques used for such studies were differential capacity measurement,21 microelectrophoresis,22 electron microscopy,23 and radiotracer20 methods. Chattoraj et al.18 have already investigated the adsorption of DNA at an alumina-water interface. Chari and Mookerjee24 studied the effect of pH on the adsorption of DNA on a glass surface. To understand the complex features of the interactions of DNA with heterogeneous biointerfaces, it is necessary to study the adsorption of DNA at many other model solid surfaces. To serve this purpose, we present in this paper our analytical results obtained for the adsorption of DNA on different solid surfaces such as powdered charcoal, Cl- resin, powdered silica, powdered metallic chromium, and sephadex as a function of various solution parameters. Our interest also goes to examine the competitive role of water in controlling the process of adsorption.25,26 Further, we have made an attempt to examine certain experimental as well as thermodynamic parameters which may be used to understand the DNA-solid interactions in a more quantitative manner. Experimental Section Materials. In our present investigations, calf-thymus DNA (lot No. 67F-9725) of Sigma Chemicals Company was used. DNA is an anionic polymer formed by the linear polymerization of monomer units called nucleotides (vide Figure 1A). A nucleotide residue of DNA consists of a deoxygenated ribose unit, a phosphate anion, and one of the four organic bases such as adenine, guanine, cytosine, and thymine. The bases which are purine and pyrimidine derivatives are relatively hydrophobic in nature (vide Figure 1B). The backbone of the DNA chain consists of deoxyriboses covalently linked with phosphate groups so that each nucleotide contains one monovalent phosphate anion.27 The DNA macromolecule thus behaves as a polyelectrolyte containing polyphosphate anion and corresponding sodium counterions. Each β-D-2-deoxyribose in the DNA polyanion is also covalently linked with one type of organic base by elimination of a water molecule formed by an OH group of the sugar in the 1′ position and a hydrogen atom attached to the NH group of A and G at the 9 position or that of C and T in position 1. Since the deoxyribose group in the DNA chain contains two CH2 groups without any free hydroxyl group, it is relatively hydrophobic in nature. From a comparison of the optical densities of a solution of DNA in 0.1 M NaCl at 260 and 230 nm as well as from a negative Folin reagent test, the absence of protein in the sample was confirmed.25,26 Powdered charcoal, silica, and amberlite resin in chloride form (IRA-402 of E. Merck, Germany), powdered chromium of BDH, England, and sephadex (G-100) of Sigma Chemicals Company of standard grade were used. All inorganic salts like LiCl, NaCl, KCl, KBr, KI, CsCl, CaCl2, and Na2SO4 and surfactants like SDS and Triton-X 100 were of analytical grade and hence used directly without further purification. Double-distilled water was used throughout the experiments. Powdered silica, charcoal, and chromium were washed thoroughly with distilled water and then dried for 4-5 h at 250-300 °C in a hot chamber. Cl- resin was washed with distilled water and (18) Upadhyay, S. N.; Chattoraj, D. K. Biochim. Biophys. Acta 1968, 161, 561. (19) Fromer, M. A.; Miller, I. R. J. Colloid Interface Sci. 1966, 21, 245. (20) Fromer, M. A.; Miller, I. R. J. Phys. Chem. 1968, 72, 2862. (21) Miller, I. R. Biochim. Biophys. Acta 1965, 103, 219. (22) Chattoraj, D. K.; Chaurashi, P.; Chakravarti, K. Biopolymers 1968, 6, 97. (23) Gordon, C. N.; Kleinschmidt, A. K. J. Colloid Interface Sci. 1970, 34 (1), 131. (24) Chari, R. V. J.; Mookerjee, A. Ind. J. Biochem. Biophys. 1975, 12, 219. (25) Chattoraj, D. K.; Bull, H. B. Arch. Biochem. Biophys. 1971, 142, 363. (26) Chattoraj, D. K.; Bull, H. B. J. Colloid Interface Sci. 1971, 35, 220. (27) Stryer, L. In Biochemistry, 4th ed.; W. H. Freeman & Co.: New York, 1995.

Figure 1. Structure of nucleotide unit in DNA chain. Table 1. Characteristics of Solid Adsorbents Used

adsorbent

surface area (m2/g)

charcoal 50.0 ( 1.0 silica 100 ( 1.0 chromium 80.6 ( 1.5 Cl- resin 29.0 ( 1.0 sephadex 57.3 ( 0.3 alumina 69.5 ( 3.3

mol of water/kg of solid avg particle (P/P0 ) 0.95) size (µm) 0.221 33.9 0.0185 38.3 30.1 48.7

7(2 295 ( 5 9(2 big big 125 ( 8

charge negative positive neutral positive

dried in hot air till attainment of constant weight. Sephadex was dried in a vacuum desiccator containing concentrated H2SO4 for 3 days to a constant weight. The surface areas of powdered silica, chromium, Cl- resin, sephadex, and alumina were measured by the method of palmitic acid adsorption from benzene solutions at 28 °C, developed by Russel et al.28 The surface area of charcoal was determined by the method28 of acetic acid adsorption from an aqueous medium. Average values of the specific surface area of different solid adsorbents are presented in Table 1. Using the isopiestic method of vapor pressure measurements29 at 0.95 water activity, the number of moles of water vapor bound per kilogram of different solids has also been presented in Table 1. The powdered particles of different solids were suspended in water, and the size of one hundred particles was examined microscopically. The values of average particle size of different solids are also given in Table 1. In the case of alumina and silica, a dye (disulphine blue) was used to make particles opaque for size measurement. A relatively concentrated stock solution of DNA was prepared by dissolving a definite weight of DNA in 1 L of NaCl solution of concentration 0.002 M, and this was preserved in a refrigerator. For a particular set of experiments, the solution of required concentration was prepared by proper dilution of this stock (28) Russel, A. S.; Cochran, C. N. Ind. Eng. Chem. 1950, 42, 1332. (29) Chattoraj, D. K.; Birdi, K. S. In Adsorption and Gibbs’ Surface Excess; Plenum Press: New York, 1984.

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Figure 2. Plot of concentration versus time for the adsorption of DNA on the surface of charcoal and alumina at 28 °C. pH 6.5 and ionic strength 0.05. (O) charcoal; (b) alumina. solution with a 0.002 M NaCl solution. From the known percent concentration of DNA in the prepared stock solution, the molar concentration of nucleotide in the solution can be calculated, since the average molecular weight of nucleotide30 may be taken as 330 so that 1 g of DNA contains on the average 0.003 mole of nucleotides. Heat-denatured DNA was prepared by heating a suitable volume of the stock DNA solution in a well-stoppered volumetric flask at 90-95 °C for 0.5 h. The solution was suddenly cooled in a bath of melting ice.31 For the preparation of acid- and alkalidenatured DNA, 0.1 M HCl or 0.1 M NaOH was added to a definite volume of the stock solution of DNA of known concentration respectively to bring the pH to either 2.5 or 12.5. The solution was kept at that condition for 24 h. The pH was then adjusted to the required value by the addition of requisite quantities of acid or alkali. The hyperchromic shifts of DNA solutions after denaturation32 by heat, acid, and alkali were found to be 38, 36, and 38%, respectively, at 260 nm. For a particular set of experiments, unbuffered solutions of DNA (native or denatured) were prepared by varying the amount of DNA in the presence of a constant amount of HCl and salt. Finally, the pH and ionic strength of all the solutions in a set were brought to the same value by suitable addition of dilute HCl, salt, and water. For preparing buffer solutions at pH 6.5 and 9.0, phosphate buffer was used and for pH 3.5, acetate buffer was used. The pH of the solution was measured with a digital pH meter (L-120, Elico, India) with an accuracy of (0.01 pH. Adsorption Experiment. To a definite volume V (equal to 40 mL) of solution of either native or denatured DNA taken in a 250 mL well-stoppered conical flask, a measured amount W grams of dry solid adsorbent (charcoal ∼ 1.0 g, Cl- resin 0.2 g, and chromium and sephadex each about 0.5 g) was added. The weights were taken by means of a Metler electric balance with an accuracy of (0.0002 g. The suspensions were shaken gently for 15 min and allowed to attain the adsorption equilibrium in an incubator at constant temperature maintained at 28.0 ( 0.1 °C. Each solution was shaken by means of a mechanical shaker intermittently for 4 h. The solutions were then kept without disturbance for 20 h to settle down the solid particles, although preliminary studies of the adsorption kinetics indicated that the adsorption equilibrium could be reached after 10-12 h (vide Figure 2). The almost clear solution from the top was pipetted out, and on measuring its absorbance at 340 nm, it was found to be completely free of solid particles.33 The absorbance of each solution was then measured with a Beckmann DU-6 spectrophotometer at 260 nm against a blank. The blank solution was the solvent containing all the components except DNA. Under identical conditions, the same measurements with DNA solutions of known concentrations were taken in the absence of solid adsorbent. Using the standard curve, the nucleotide concentration (30) Falk, M. Can. J. Chem. 1966, 44, 1108. (31) Marmur, J.; Doty, M. J. Mol. Biol. 1962, 5, 104. (32) Hotchkiss, R. D. In Methods of Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1957; Vol. III. (33) Bull, H. B. Biochem. Biophys. Acta 1956, 19, 464.

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Figure 3. Study of desorption followed by adsorption of native DNA at charcoal-water and alumina-water interfaces at 33 °C, pH 6.0, and ionic strength 0.15: I, adsorption (O, charcoal; b, alumina); II, desorption (4, charcoal; 2, alumina). (C2) in the solution after adsorption was determined with 2-4% standard error. The number of moles (Γ21) of nucleotide adsorbed per square meter of dry solid powder can be calculated from eq 1.

Γ21 )

(C2t - C2)Vt 1000

(1)

Here C2t is the molar concentration of the nucleotides in a solution of volume v mL before addition of the solid powder and C2 is its molar concentration in the bulk solution at adsorption equilibrium after the addition of W grams of solid powder. Vt is equal to v/WA, A being the specific surface area in square meters per gram of solid. Since the solution is dilute with respect to nucleotides, C2 may be taken as equal to 55.5X2, where X2 stands for the mole fraction of the nucleotide in the bulk solution at adsorption equilibrium. Each experiment was repeated four times, and the average value of Γ21 was presented graphically at the mean value of C2. The standard deviation in the values of Γ21 did not usually exceed 6%. The limits of experimental errors are shown in one isotherm in each figure (vide Figures 4-7). To study the desorption of DNA from the powdered solid surfaces of alumina and charcoal followed by adsorption, the experiment was set up as follows: After the attainment of adsorption equilibrium in each flask containing 20 mL of DNA solution at pH 6.0 and ionic strength 0.15 and 1.0 g of dry powdered solid surface, the extent of adsorption (Γ21) was estimated by the method described earlier. Then, for the study of desorption followed by adsorption, a 5 mL aliquot was withdrawn from each flask after complete settling of solid particles and the volume was again adjusted to 20 mL by the addition of 5 mL of buffer solution and a further 1.0 g of solid surface was added. This process of dilution and subsequent addition of excess solid powder was started from the set corresponding to the point S at the saturation level, as indicated on the adsorption curves in Figure 3. The flasks were then placed on a mechanical shaker kept in a room of constant temperature at 33.0 ( 0.1 °C for 3 days. After the attainment of further adsorption equilibrium, the concentration of DNA in each set was again estimated and the extent of adsorption (Γ21) was calculated using eq 1.

Results and Discussion Watson and Crick34 have proposed that two polynucleotide chains of DNA form a double-helix structure with complementary base pairing between A, T, and G, C, respectively. The base pairing between A and T occurs due to the formation of two hydrogen bonds from positions 3 and 4 of thymine to positions 1 and 6 of adenine, (34) Watson, J. D.; Crick, F. H. C. Nature 1953a, 171, 737.

Affinities of DNA to Solid Surfaces

Figure 4. Plot of Γ21 against X2 for the adsorption of native DNA on different solid surfaces at 28 °C, pH 6.5, and ionic strength 0.05 in the presence of NaCl: (O) silica; (4) chromium; (b) sephadex; (2) Cl- resin; (#) charcoal; (Y) alumina (25 °C).

respectively. Further base pairing between G and C involves formation of three hydrogen bonds from positions 2, 3, and 4 of cytosine to positions 2, 1, and 6 of guanine, respectively (vide Figure 1B). Four ring-structured hydrogen-bonded bases occurring in the double-helix structure as well as deoxyribose residues are hydrophobic in nature. Also each phosphate group of a nucleotide contains one negatively charged hydrophilic phosphate ion, so that molecules of DNA possess surface active properties like those of proteins. Fromer and Miller20 have shown from radioactive study that a DNA molecule from bulk solution may be accumulated in excess at the air-water interface. However, these workers as well as Chattoraj, Bull, and Chalkley35 have shown that high-molecular-weight DNA on addition to water is unable to lower the surface tension γ, since the surface area of the air-water interface exposed for measurement of γ is small, so that C2t is almost equal to C2 in eq 1. On the other hand, the powdered surface of each solid exposes a large extent of the solid-liquid interface for adsorption of DNA from an aqueous solution, so that C2t is significantly different from C2 and Γ21 can be measured directly using eq 1. In Figure 4, the moles of nucleotide adsorbed per square meter of the surface of charcoal, chromium, silica, alumina, Cl- resin, and sephadex have been plotted against the mole fraction X2 (equal to C2/55.5) of nucleotide present at equilibrium in the bulk medium. The experiments were carried out at 28 °C, keeping the ionic strength and pH fixed at 0.05 and 6.5, respectively. Except in the case of sephadex, Γ21 of DNA for each surface is observed to increase with an increase of X2 at low DNA concentrations, but it reaches a steady value (Γ2m) when the concentration of DNA reaches a critical value X2m. Apparently, Γ2m may be regarded as the moles of nucleotides required to cover one square meter of the solid-liquid interface, thus effectively forming a saturated monolayer. The values of Γ2m and X2m for different adsorption isotherms are included in Tables 2 and 3. The initial slopes (dΓ21/dX2) at X2 f 0 represent the energy of the direct interaction of DNA with the solidliquid interface.36 Values of Γ2m for various solids representing packing of nucleotides per square meter of the surface are also widely different from each other, since (35) Chattoraj, D. K.; Bull, H. B.; Chalkley, R. Arch. Biochem. Biophys. 1972, 52, 778. (36) Norde, W. Proteins at Interfaces. Doctoral Thesis, Agricultural University, Wageningen, The Netherlands, 1976.

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the orientations and interactions of adsorbed nucleotides with each other are different and also because the natures of different surfaces and their interactions with solvent are different. Γ2m values for different surfaces stand in the order chromium > charcoal > silica > Cl- resin > alumina > sephadex. However, when the value of X2 far exceeds X2m beyond a certain limiting concentration X2a, defined as the critical aggregation concentration, for the surface of charcoal, Clresin, and silica, Γ21 again increases sharply. This may be due to the fact that DNA molecules packed in a monolayer start to accumulate further, resulting in molecular aggregation or interfacial coagulation at the solid surface. The values of X2a are included in Tables 2 and 3. The ionic strength and pH values in all these experiments were the same, and the temperature of measurement in all cases was 28 °C except for alumina, where the measurement was taken at 25 °C. From Table 1, one finds that chromium powder adsorbs negligible amount of water vapor at 0.95 relative humidity, so that a major fraction of its surface is highly hydrophobic in nature. Besides, there exists a small fraction of surface active spots containing positively charged chromium ions where negatively charged phosphate groups can bind to a maximum extent if the orientation of the DNA molecule is horizontal in nature, so that interaction between DNA and the hydrophobic region of the surface becomes maximum. The surface of charcoal must be hydrophobic in nature, and its water absorption capacity at 0.95 relative humidity is also very small (vide Table 1), as expected. If the adsorbed DNA molecules in contact with the bulk phase orient horizontally on the hydrophobic surface, there will be maximum hydrophobic interaction between adsorbate and adsorbent. We also note from Table 1 that, even at 0.95 relative humidity, silica, Cl- resin, alumina, and sephadex have a strong affinity for water vapor adsorption.37 DNA has to compete with water for adsorption at these hydrophilic solid surfaces. At relative humidity nearly unity, water intake by these solids is quite high. Since solutions of DNA containing nucleotides were dilute, the molar concentrations C2t and C2 in eq 1 can be replaced by the molal concentrations m2t and m2, respectively, so that 1

Γ2

(m2t - m2)W1t ) 1000

(2)

Here W1t replacing Vt is the total weight of the solvent prior to adsorption in the system (per unit square meter of the solid powder). Replacing m2t, m2, and W1t by (1000n2t/ M1nlt), (1000n2/M1n1), and M1n1t, respectively, eq 2 assumes the form

()

Γ21 ) n2t - n1t

n2 n1

(3)

Here n1t and n2t are the moles of solvent and nucleotides, respectively, before adsorption per square meter of solid powder and n1 and n2 are their values at adsorption equilibrium. Ml stands for the molecular weight of water. We now assume that the adsorbed layer is made up of ∆n1 and ∆n2 moles of solvent and solute bound per square meter of solid forming the inhomogeneous surface phase. This is in contact with the bulk phase of uniform (37) Nag, A.; Sadhukhan, B.; Chattoraj, D. K. Colloids Surf. 1987, 23, 83.

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Table 2. Evaluated Parameters for the Adsorption of DNA on the Surface of Different Solid Adsorbents at 28 °C, pH 6.5, and Ionic Strength 0.05

surface charcoal

silica chromium sephadex alumina a

additive

106X2m

107X2a

LiCl NaCl KCl CsCl KBr KI CaCl + NaCl Na2SO4 NaCl + SDS NaCl + Triton-X100 NaCl(1)a NaCl(2)a NaCl(3)a NaCl(4)a NaCl(5)a NaCl(6)a NaCl NaCl NaCl NaCl NaCl NaCl NaCl(7)a

0.54 0.81 0.54 1.08 0.59 0.81 1.35 2.70 1.35 0.96 0.60 2.96 2.32 2.16 3.78 3.24 0.27 0.60 0.60 10.3 15.2 6.4 10.2

5.12 10.2 8.64 8.10 8.90 8.94

nature of DNA native

heat-denatured acid-denatured alkali-denatured native native native native

initial slope (dΓ21/dX2)x2f0

107Γ2m (mol of nucleotide/m2 of surface)

106-∆G° (kJ/m2 of surface)

0.78 2.68 0.93 2.58 0.76 0.68 1.46 0.69 0.10 2.43 3.12 2.96 2.18 2.98 0.32 3.39 0.60 3.21 3.18 0.08 0.02 0.03 0.01

1.66 1.74 1.29 1.99 1.20 0.96 3.81 0.75 0.99 1.75 1.92 1.94. 1.62 2.10 1.59 2.05 2.27 1.93 1.93 1.27 1.99 -0.27 0.15

6.44 6.62 5.31 7.70 4.51 3.73 14.4 2.92 3.54 6.82 7.50 7.76 5.99 7.77 6.80 6.38 8.67 8.00 7.43 5.06 7.76 -1.09 5.52

10.0 8.10 10.0 6.48 6.48 5.94 5.48 8.10 5.96 5.12 5.16 5.18 16.2 10.8

(1) pH 3.5; (2) pH 9.0; (3) µ ) 0.01; (4) µ ) 0.10; (5) 37 °C; (6) 18 °C; (7) 25 °C. Table 3. Evaluated Parameters for the Adsorption of DNA on the Surface of C1- Resin at 28 °C, pH 6.5, and Ionic Strength 0.05

nature of DNA

additive

106X2m

106X2a

initial slope (dΓ21/dX2)x2f0

native heat-denatured acid-denatured alkali-denatured native

NaCl NaCl NaCl NaCl NaCl(1)a NaCl(2)a NaCl(3)a NaCl(4)a NaCl + CaCl2 Na2SO4 NaCl + SDS NaCl + Triton-X 100 CsCl

5.40 5.24 4.96 5.36 5.40 5.52 5.50 5.36 5.92 5.48 5.56 4.36 4.92

10.8 10.8 7.5 10.8 12.9 8.7 8.8 10.8 7.6 10.7 10.6 7.6 7.6

0.5 1.0 2.4 0.1 1.1 1.2 1.0 0.8 1.4 1.1 1.3 0.9 1.2

a

108Γ2m (mol of nucleotide/m2 of Cl- resin)

106-∆G° (kJ/m2 of Cl- resin)

2.9 3.8 7.8 2.6 2.4 4.2 4.0 2.4 4.2 2.2 2.0 2.9 3.0

1.13 1.50 3.08 1.03 0.95 1.60 1.55 0.90 1.70 0.86 0.80 1.10 1.19

(1) µ ) 0.01; (2) µ ) 0.10; (3) 37 °C; (4) 18 °C.

compositions n1 and n2 of solvent and nucleotide per square meter of solid, respectively. We can write

n1t ) n1 + ∆n1

(4)

n2t ) n2 + ∆n2

(5)

Combining eqs 3, 4, and 5

n2 Γ21 ) ∆n2 - ∆n1‚ n1 X2 ) ∆n2 - ∆n1‚ X1 C2 = ∆n2 - ∆n1‚ 55.5

(6)

Here X1 stands for mole fraction of free water in contact with the solid surface at adsorption equilibrium. The surfaces of charcoal and chromium are hydrophobic, so that ∆n1‚(n2/n1) in eq 6 is negligibly small and Γ21 =

∆n2. Silica and alumina are significantly hydrophilic, and they have the ability to adsorb a large amount of water and swell to form gels.37 From Table 1, we also note that the affinity of silica for water vapor uptake is considerably less than that of alumina. Also values of zero-point charge indicate that, at pH 6.5, alumina and silica particles are positively and negatively charged, respectively. At relatively high concentration of nucleotides, bound water from the silica surface can be more easily displaced than that from the hydrated alumina surface so that, according to eq 6, Γ2m for silica is higher than that for alumina. But values of Γ2m for these two hydrated surfaces are significantly lower than that of charcoal because the contributions of ∆n1‚(n2/n1) for silica and alumina are high. The electrostatic repulsion effect of silica for DNA adsorption may not be significant also at ionic strength 0.05. Particles of Cl- resin are made up of a polymer containing extensive cross-links within its chains. This results in the formation of large pores. From Table 1 we also note that even at 0.95 relative humidity a considerable amount of water vapor is accumulated within the pores which are hydrophilic in nature. The polymeric chains

Affinities of DNA to Solid Surfaces

Figure 5. Plot of Γ21 against X2 for the adsorption of native DNA on the surface of charcoal at pH 6.5 in the presence of NaCl: (O) 28 °C, µ ) 0.01; (- - -) 28 °C, µ ) 0.05; (4) 28 °C, µ ) 0.10; (Y) 18 °C, µ ) 0.05; (2) 37 °C, µ ) 0.05.

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Figure 6. Plot of Γ21 against X2 for the adsorption of native DNA on the surface of charcoal at 28 °C, pH 6.5, and ionic strength 0.05 in the presence of different salts: (O) LiCl; (4) NaCl; (2) KCl; (b) CsCl; (y) KBr; (∆) KI; (#) NaCl + CaCl2; (5) Na2SO4.

surrounding the pores are positively charged whereas Clcounterions will remain in water present in the pore. Negatively charged DNA molecules will be adsorbed by a positively charged resin chain mainly by a complex ionexchange process, so that the value of ∆n2 in eq 6 is relatively high. However, with this kind of exchange process, still some amount of strongly bound water remains in the pore, so that the contribution of ∆n1 in eq 6 is also significant and values of Γ2m will be relatively small indeed. Sephadex powder is made up of cross-linked polymer containing a large number of hydrophilic glucose rings, thus forming a flexible surface in contact with the bulk phase. Such a system contains pores which are full of solvent. Entrapped DNA in this pore will have a weak interaction with the glucose residues of sephadex, so that Γ2m will be negative according to eq 6. In Figure 5, the different isotherms for adsorption of native DNA on powdered charcoal at different ionic strengths at constant temperature and pH and at different temperatures with constant ionic strength and pH have been compared. In each of these isotherms, Γ21 initially increases with an increase of X2 until, at a critical value of X2 equal to X2m, Γ21 reaches a steady value Γ2m. Values of Γ2m and X2m are presented in Table 2. When the value of X2 far exceeds X2a (vide Table 2), Γ21 again increases sharply. From this figure, comparing the isotherms at 18, 28, and 37 °C at pH 6.5 and ionic strength 0.05 (vide Figure 5), it is observed that, with an increase of temperature, Γ2m as well as Γ21 at a given value of X2 decreases (vide Table 2). One may thus conclude that the adsorption process is physical and exothermic in nature. From the results presented in Figure 5 and Table 2, one may also note with interest that Γ21 at a given value of X2 or Γ2m for the adsorption of DNA at the charcoal-water interface increases significantly with an increase of ionic strength from 0.01 to 0.1. From Table 2 it is observed that the initial slope (dΓ21/dX2)x2f0 for the isotherms at ionic strengths 0.01, 0.05, and 0.10 are 2.18, 2.68, and 2.98, respectively. The initial slope in an adsorption isotherm in general is a relative measure of the interaction energy of an adsorbate with an adsorbent. An increase of ionic strength decreases the double-layer potential of DNA, and the radius of gyration of the DNA polyelectrolyte containing elongated and stiff segments38 is expected to decrease. Thus the value of Γ2m includes effects of both DNA-surface and DNA-DNA interactions in the interfacial phase.

In Table 2, values of Γ2m for the adsorption of denatured DNA of various types are found to be considerably higher than that of native DNA under similar physicochemical conditions. Γ2m for heat-denatured DNA is even higher than that of acid- or alkali-denatured DNA. The initial slopes for alkali- and acid-denatured DNA are higher than that of native DNA. This may lead to the conclusion that the interaction energy between charcoal and acid- or alkalidenatured DNA is higher than that of native DNA. On the other hand, the initial slope of heat-denatured DNA is considerably lower than that of native DNA. This result indicates that intermacromolecular interaction of denatured DNA must be very high so that the value of Γ2m becomes large. Plots of Γ21 versus X2 (not shown) for the adsorption of native DNA on powdered charcoal in the presence of NaCl + SDS (8.0 × 10-5 M) and NaCl + Triton-X 100 (8.0 × 10-5 M) have been compared with that in the presence of NaCl under identical solution conditions. Values of Γ2m in the presence of NaCl and NaCl + Triton-X 100 are almost equal to each other, whereas it is considerably lower in the presence of NaCl + SDS. The observed results can be explained qualitatively in the following manner. SDS is known not to bind DNA in bulk solution,39 but it may be adsorbed on the charcoal surface, whereby DNA uptake may be blocked due to electrostatic and other effects. Triton-X 100 binds DNA in solution,39 and the active spot for adsorption of DNA is not blocked in the presence of this nonionic surfactant at the interface. The isotherms for the adsorption of native DNA on the surface of charcoal at 28 °C and ionic strength 0.05 have been compared (not shown) at three different pH values: 3.5, 6.5, and 9.0. It is well-known27 that the double-helix structure of DNA is most stable near pH 6.0, but gradual denaturation of the biopolymer occurs as pH becomes more acidic or alkaline. Γ2m for DNA is thus observed to be minimum at pH 6.5. But with gradual acid and alkali denaturation of DNA, values of Γ2m increase, since denatured DNA molecules are more flexible so that more macroions can be accommodated at the charcoal-water interface. In Figure 6, isotherms for the adsorption of native DNA on powdered charcoal in the presence of different inorganic

(38) Davidson, N. In The Nucleohistones; Bonner, J., Tso, P. O. P., Eds.; Holdender Day: San Francisco, 1964; p 134.

(39) Chatterjee, R.; Chattoraj, D. K. Ind. J. Biochem. Biophys. 1979, 16, 236.

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salts have been compared at 28 °C, pH 6.5, and ionic strength 0.05. In all cases, different isotherms have similar shapes except in the presence of NaCl + CaCl2. Initially, the extent of adsorption increases sharply with the increase of DNA concentration in all cases and reaches almost a steady value in the presence of Na2SO4, LiCl, NaCl, and CsCl. Then it further increases sharply beyond X2 equal to 5.12 × 10-6 in the case of LiCl and 8.1 × 10-6 in the case of Na2SO4, NaCl, and CsCl. But in the presence of KCl, KBr, KI, and NaCl + CaCl2, after the initial sharp increase, the extent of adsorption still increases slowly and gradually up to the limiting concentration X2a. Values of Γ2m and X2a for such systems are included in Table 2. It is interesting to note that, in the presence of NaCl + CaCl2, no such stage for attainment of X2a is observed within the experimental concentration limit but values of Γ21 are always very high indeed. The variation of Γ2m for a charcoal surface (vide Table 2) was observed to follow the order NaCl + CaCl2 > CsCl > NaCl > LiCl > KCl > KBr > KI > NaSO4. We also note from Table 2 that the values of the initial slope stand in the order NaCl > CsCl > CaCl2 + NaCl > KCl > LiCl = KBr > KI = Na2SO4. There exists partial agreement in the orders for Γ2m and initial slope. It may be concluded that the interaction of adsorbed DNA molecules with each other plus the DNAsurface interaction (represented by the initial slopes) are included jointly in the experimental values of Γ2m for various salts in a complex manner. From electrophoretic experiments, Davidson et al.40 and others have shown that binding of different cations to DNA in the bulk solution affects the electrophoretic mobilities of DNA in solution to different extents. Different metal ions attached to adsorbed DNA molecules may also affect the values of ∆n2 and ∆n1(n2/n1) to different extents (vide eq 6), so that Γ2m for different systems may become different. Isotherms (not shown) for the adsorption of native DNA on Cl- resin at three different ionic strengths (0.01, 0.05, and 0.10) at 28 °C and pH 6.5 have been compared. Values of Γ2m are found to increase with an increase of the ionic strength of the medium. An increase of ionic strength reduces the double-layer potential of negatively charged DNA molecules in the bulk, so that the polyelectrolyte may accumulate more at the interface, thus enhancing the ion-exchange process for DNA adsorption. The initial slopes for the adsorption process at ionic strengths 0.01, 0.05, and 0.01 are 0.011, 0.012, and 0.005, respectively. Thus, the interaction energy between DNA and the resin surface is minimum at µ ) 0.10, but it decreases to a steady value when µ is reduced to 0.05 or 0.01. This leads to the contribution of the interaction energy between DNA and the resin surface being minimum for the magnitude of Γ2m and the intermacromolecular interactions of adsorbed DNA molecules being a major contribution to Γ2m. In Figure 7, the values of Γ21 were plotted against X2 for the adsorption of DNA on Cl- resin at 18, 28, and 37 °C at pH 6.5 and ionic strength 0.05. From these curves it may be noted that the extent of adsorption (Γ2m) increases with an increase of temperature. This possibly indicates that the adsorbed DNA molecules within the resin pores undergo some cooperative interaction with each other whereby water molecules within the pore become more ordered around associated DNA molecules. We shall discuss this again later on. In Table 3, we find that the values of Γ2m for heatdenatured and acid-denatured DNA increase from 1.5- to

3.0-fold, respectively, from that for native DNA. The observation is similar to that found in the case of adsorption of DNA on the surface of charcoal. An exception is the case for Γ2m of alkali-denatured DNA, whose value remains the same as that of native DNA, in all probability due to the renaturation of DNA on the resin surface in alkaline pH. For the charcoal surface this renaturation effect is absent. The isotherms (not shown) for the adsorption of native DNA on Cl- resin in the presence of NaCl + Triton-X 100 and NaCl + SDS at 28 °C, pH 6.5, and ionic strength 0.05 have been compared with that in the presence of NaCl alone. From Table 3, it appears that the value of Γ2m in the presence of NaCl + Triton-X 100 does not differ from that in the presence of NaCl whereas in the presence of NaCl + SDS the value of Γ2m is lowered from that in the presence of NaCl alone. The same observations were also noted for the charcoal surface. In Figure 7 also, the isotherms for the adsorption of native DNA on Cl- resin in the presence of NaCl (0.04 M) + CaCl2 (0.001 M) and Na2SO4, respectively, have been compared at 28 °C, pH 6.5, and ionic strength 0.05. From these isotherms and also from Table 3, it may be noted that the value of Γ2m in the presence of Na2SO4 is less compared to that in the presence of NaCl alone. On the other hand, in the presence of NaCl + CaCl2, the value of Γ2m is found to be increased by about 50% of that in the presence of NaCl. From these observations, it may appear that the value of Γ2m in the presence of Na2SO4 decreases, possibly due to the preferential adsorption of SO42- ion at the hydrophilic surface of the resin. In the presence of NaCl + CaCl2, the extent of adsorption (Γ2m) increases, possibly due to the preferential adsorption of Ca2+ ion by the surface, whereby the positive charge at the surface is further increased, resulting an increase in the value of Γ21. The average molecular weight of calf-thymus DNA estimated from ultracentrifugal analysis is of the order 1 × 107 Da.41 According to the analysis of Watson and Crick34 on X-ray data, the diameter and cross-sectional area of a rod-shaped DNA molecule are 2.0 nm and 3.14 nm2, respectively. Following Norde,36 we also assume that one molecular layer of water is strongly associated with the outside surface of the rod-shaped biopolymer molecule, so that the diameter and cross-sectional area of the hydrated DNA are 3.0 nm and 7.06 nm2, respectively. If this rod is vertically oriented on the solid by adsorption, then the number of moles of DNA per square meter of the plane surface will be 2.35 × 10-7. Theoretically this will

(40) Olivera, B. M.; Baine, P.; Davidson, N. Biopolymers 1964, 2, 245.

(41) Chattoraj, D. K.; Chowrashi, P.; Chakravarti, K. Biopolymers 1967, 5, 173.

Figure 7. Plot of Γ21 against X2 for the adsorption of native DNA on the surface of Cl- resin at pH 6.5 and ionic strength 0.05: (4) 18 °C, NaCl; (- - -) 28 °C, NaCl; (O) 37 °C, NaCl; (b) 28 °C, NaCl + CaCl2; (2) 28 °C, Na2SO4; (#) 28 °C, NaCl + SDS.

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be equivalent to 2.35 × 10-3 kg of DNA or 7.8 × 10-4 moles of nucleotide per square meter. All values of Γ2m in Tables 2 and 3 are 103 to 104 times less than this value. Watson and Crick34 have also established from X-ray data that the pitch of the DNA double-helix representing the vertical shift of 20 nucleotides of two polynucleotide units along the helix axis is 3.4 nm, so that the projection area covered by 1 mol of nucleotide for the horizontal orientation is 3.07 × 105 m2. From this we find with interest that (Γ21)theo equal to 3.25 × 10-6 mol of nucleotides or 9.79 × 10-6 kg of DNA per m2 of plane may be completely packed without leaving any uncovered region of the surface. In the case of the DNA helix, certain parts of it may assume a bent tertiary stucture containing several pieces of rods joined to each other. In this orientation also, the value of (Γ21)theo will not alter substantially. In the presence of a chromium or charcoal surface, the value of Γ21 is about ten times less than (Γ2m)theo. All these results indicate that different rodlike horizontally oriented DNA molecules are packed on the surface in random fashion, leaving vacant surface area in contact with solvent. Monolayer packing of ellipsoidal-shaped BSA molecules on glass33 and other types of surfaces36 is compact, and there exists no vacant space on the surface in contrast to that found for rod-shaped DNA molecules at an interface. Denatured DNA adsorbed in this manner may cover more of these vacant areas because of the flexible nature of the polyelectrolyte, so that Γ21 for denatured DNA is expected to be higher than that of native DNA. Values of Γ21 for silica, alumina, Cl- resin, and sephadex surfaces are considerably less than (Γ21)theo because there exists larger vacant surface areas which are strongly attached with water. Further, the surfaces of different solids are rough with microscopic peaks and valleys where rigid segments of DNA molecules cannot react, so that Γ2m , (Γ2m)theo. On the basis of the integration of the Gibbs’ adsorption equation, Bull33 has derived eq 7, representing the maximum free energy change ∆G° for the adsorption of Γ2m moles of protein (BSA, egg albumin) per square meter of the powdered glass surface for changing active concentration a, from zero to unity in the bulk phase. 1

Γ

∫0a)1 a22 da2

∆G° ) -RT

∫0

) -RT

1

a2mΓ2

a2

da2 + RT Γ2m d ln a2m

(7)

For a dilute protein solution, Bull33 has replaced a2 by the molar concentration C2. He has also indicated that the integral part of the right hand side of eq 7 actually represents the free energy for maximum adsorption at concentration a2m whereas the second part represents the free energy of dilution of protein solution in bulk, so that a2 changes from a2m to unity, the activity of protein (one molar concentration) in its standard state. From the ratio ∆G°/Γ2m, Bull33 has calculated the standard free energy change ∆GB° for the transfer of 1 mol of bovine serum albumin or egg albumin to the surface of powdered glass with the assumption that the adsorption process is reversible. Chattoraj et al.42,43 have derived eq 7 on thermodynamic grounds for the adsorption of cationic and anionic sur(42) Das, K. P.; Chattoraj, D. K. Colloids Surf. 1983, 7, 53. (43) Chattoraj, D. K.; Ghosh, L. N.; Mahapatra, P. In Surfactants in Solutions; Mittal, K. L., Shah, D. O., Eds.; Plenum Press: New York, London, 1991.

factants at liquid and solid surfaces and indicated that ∆G° represents the standard free energy change in joules per square meter of the surface, so that values of ∆G° for different systems are comparable under a universal scale of thermodynamics. They have also assumed that adsorption processes for such systems are reversible and that a2 should be replaced by mole fraction X2 (instead of C2) for rigorous comparison of values of ∆G°. Values of ∆G° in kilojoules per square meter of surface for various systems undergoing adsorption of DNA are presented in Tables 2 and 3. In Figure 2 values of Γ21 for adsorption of DNA on the hydrophobic surface of charcoal and the hydrophilic surface of alumina powder have been plotted as a function of time. The results indicate that the adsorption equilibrium in each case is attained after the lapse of 10-12 h. Since we have allowed 24 h in all our measurements for adsorption to occur, values of Γ21 in each adsorption isotherm (vide Figures 4-7) are all equilibrium values, so that ∆G° calculated with the help of eq 7 represents the amount of maximum work due to DNA adsorption, as envisaged originally by Bull33 himself. We have also compared the isotherms for the adsorption of native DNA on charcoal and alumina, respectively, with the corresponding isotherm involving the DNA desorption step under the same physicochemical conditions. We note with interest that the isotherms for adsorption and for that involving the desorption step are almost identical with a hydrophobic charcoal surface. But for a hydrophilic alumina surface, values of Γ21 (measured after a lapse of 72 h) involving a desorption step in the intermediate region of X2 are higher than those of the full adsorption process (measured after 24 h) under comparable conditions. This may possibly indicate the following: (i) The process involving a desorption step needs a much larger elapsed time than 3 days for the attainment of equilibrium. (ii) Isotherm II containing a desorption step is not shifted far from adsorption isotherm I at equilibrium. This means that deviation of the value of Γ21 in curve II from that of curve I is not wide, so that the application of the Gibbs’ adsorption equation for the nonequilibrium process not too far from the equilibrium stage44 may be taken as valid. Application of eq 7 for curves II in a hypothetical approach leads to values of ∆GII° equal to 5.66 × 10-7 and 1.45 × 10-3 kJ per square meter of alumina and charcoal, respectively, which are close to the values of ∆G° (4.66 × 10-7 and 1.68 × 10-3 kJ/m2, respectively). (iii) We have seen in all our calculations of ∆G° that the free energy of dilution in eq 7 serves as nearly 90% or more of the value of ∆G°. From these observations, we accept the approach of Bull33 and consider that the maximum work for the adsorption of biopolymer at solid-liquid interfaces is identical or close to the standard free energy of adsorption. When Γ21 < Γ2m, the apparent standard free energy change ∆Gap° can be calculated using eq 8.

∆Gap° ) -RT

[∫

1

x2Γ2

0

x2

dx2 + Γ2m ln

]

1 x2

(8)

Here it is assumed that each value of Γ21 for an experimental value of x2 remains hypothetically constant up to x2 ) 1, so that ∆Gap° for each event can be estimated from the graphical integration. ∆Gap° will be less than ∆G°, since the fraction θ of the surface, equal to Γ21/Γ2m, covered by the DNA molecules is less than unity. ∆Gap°/θ representing the standard free energy change due to the (44) Defay, R.; Prigogine, I.; Bellemans, A. In Surface Tension and Adsorption; John Wiley and Sons: New York, 1966; p 374.

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Figure 8. Plot of -∆G° against Γ2m for the adsorption of native DNA on different solid surfaces: (O) charcoal; (4) Cl- resin; (Y) silica; (2) chromium; (b) alumina.

hypothetical coverage of the unit fraction of the surface by nucleotide now represents ∆G° for unsaturated states of adsorption,43 so that

Γ21 ∆Gap° ) ∆G°θ ) ∆G° m Γ2

(9)

Plots of ∆Gap° against Γ21/Γ2m (not shown) were found to be linear, and the values of ∆G° obtained from the slopes according to eq 9 in all cases were found to remain the same as that already evaluated for the state of saturation presented in Tables 2 and 3. This result confirms that the standard free energy change for unsaturated and saturated states due to adsorption of DNA on a given adsorbent surface remains unchanged. This means that even if the solid surface structure is uneven and specific surface area and composition may alter with progress of DNA adsorption, the standard free energy change in all cases remains the same. Thus ∆G° calculated from the integrated form of Gibbs’ adsorption equation includes all types of effects of interaction between DNA and different solid surfaces. In Figure 8 a plot of ∆G° versus Γ2m is found to be linear for different adsorbents such as charcoal, Cl- resin, silica,

chromium, and so forth. The slope of this linear plot representing ∆GB° is equal to -38.3 ( 2.4 kJ/mol of nucleotide. This standard free energy was first obtianed by Bull33 in the practical scale for the adsorption of albumin on glass powder in 1956, and it has been termed by us ∆GB° in his honor. The order of Γ2m and that of ∆G° are found to be the same, and so ∆G° represents the relative affinities of nucleotides for binding to different surfaces. In the case of charcoal and resin surfaces, values of ∆G° have been calculated for some systems at 291, 301, and 310 K, so that by application of the Gibbs-Helmholtz equation, the average values of the free energy change ∆Hav°, the entropy contribution Tav∆Sav°, and the enthalpy change ∆Hav° have been calculated. One finds that, at 296 K for charcoal and resin surfaces, the values of ∆Hav° are 0.604 × 10-6 and 5.79 × 10-6 kJ/m2 whereas the Tav∆Sav° values for these two surfaces are 7.10 × 10-6 and 6.80 × 10-6 kJ/m2, respectively. This indicates that, for a hydrophobic charcoal surface, the adsorption process becomes largely entropically controlled whereas on the ionic surface of a resin ∆Hav° = Tav∆Sav°, indicating that both enthalpy and entropy effects are controlling the adsorption of DNA on this surface. The same phenomenon has been observed also at the average temperature 305.5 K. From all these observations we conclude that DNA from its aqueous solution may be adsorbed to different extents to the hydrophobic and hydrophilic surfaces of various types of powdered or porous solids. The maximum adsorption (Γ2m) of DNA depends significantly on the pH, ionic strength, temperature, and nature of the neutral salts and solid surfaces. Its value also depends on the state of the DNA (native or denatured). The rod-shaped DNA molecules in all probability lie flat on the solid surface in random orientation, so that a large amount of surface remains in contact with water also. The order in the values of Γ2m representing the maximum affinity of DNA for a surface is linearly related to the order of the values of -∆G° for adsorption. Acknowledgment. Financial assistance of Indian National Science Academy, New Delhi, is acknowledged with thanks. LA970686H