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Adsorption of Biopolymer at Solid-Liquid Interfaces. 2. Interaction of BSA and DNA with Casein S. A. Gani, D. C. Mukherjee,† and D. K. Chattoraj* Department of Food Technology and Biochemical Engineering, Jadavpur University, Calcutta 700 032, India, and University College of Science, Calcutta University, 92 A. P. C. Road, Calcutta 700 009, India Received June 26, 1997. In Final Form: May 18, 1999 The extent of adsorption (Γ21) of bovine serum albumin (BSA) and calf-thymus deoxyribonucleic acid (DNA) on the surface of casein has been studied as a function of pH, temperature, ionic strength of the medium, and denaturants. Γ21 in all cases increases with an increase of biopolymer concentration (X2) in the bulk, and it attains a saturation value (Γ2m) when X2 reaches X2m. In the case of DNA, Γ21 increases further with an increase of X2 beyond the saturation value when X2 reaches a critical value X2a. The influence of various inorganic electrolytes and denaturants on the extent of adsorption of BSA on casein has also been examined. Native DNA is not able to accumulate at the surface of casein in the presence of NaCl only, but with the addition of 1.0 × 10-3 M CaCl2 or 5.0 × 10-4 M AlCl3 the extent of adsorption increases to a significant amount. However, denatured DNA is able to do so in the sole presence of NaCl under identical solution conditions. The role of water in controlling the process of adsorption has been explained in terms of a Gibbs’ surface excess quantity (Γ21).
Binding of proteins with DNA in aqueous media has been extensively studied in the last few decades, and attempts have been made to relate binding processes with the control of genetic and other biological properties.1-6 Binding of the basic proteins histones and many acidic proteins present in chromosomes with DNA in aqueous media has been investigated by several workers with interesting results.7-10 Calorimetric studies11 on polypeptide-DNA systems have revealed that the formation of macromolecular complexes is an entropy-driven process. Using a gel chromatography technique,12 DNA-protein binding constants in aqueous media have been evaluated. Cross-linking between DNA and protein in aqueous media with application of UV laser and other techniques has been recently reported by several workers.13,14 Proteinprotein and protein-nucleic acid interactions occuring in λ repressor-operator systems have been recently reviewed by Roy.15 Study of protein-protein interaction in aqueous media has also been carried out recently using immunoprecipitation,16 fractionation chromatography,16,17 the
blotting electrophoresis technique,18 and size exclusion chromatographs.19 Although interactions of DNA and protein with other proteins in aqueous media have been investigated in recent years, there exists no detailed study of the adsorption interaction of these two macromolecules with the hydrophilic surface of many insoluble proteins. Such interaction between biopolymers dissolved in solution and insoluble proteins in contact with such a biofluid containing dissolved protein and nucleic acid may actually occur in living systems. Further, from a surface chemical standpoint, immobilization of enzymes and nucleic acids on the surface of insoluble and hydrophilic protein may be of considerable theoretical and technological interest. In part 1 of this paper,20 we have presented our data on adsorption of DNA on various types of rigid solid surfaces. In our present investigation, an attempt has been made to study the extents of adsorption of native and denatured BSA and DNA on powdered insoluble milk protein casein at different values of pH, ionic strength, and temperature and in the presence of different neutral salts and surfactants.
* To whom all correspondence regarding this paper should be sent at Jadavpur University. † Calcutta University.
Experimental Section
Introduction
(1) Olins, A. L.; Olins, D. E. Science 1974, 183, 330. (2) Crick, F. H. Nature 1971, 234, 25. (3) Brayer, G. D.; Mcpherson, A. Biochemistry 1984, 23, 340. (4) Ollis, D. L. Nature 1985, 313, 762. (5) Powell, M. D.; Gray, D. M. Biochemistry 1993, 32, 12538. (6) Frontali, C. Biopolymers 1998, 27 (8), 1329. (7) Bartley, J. A.; Chalkley, R. J. Biol. Chem. 1972, 247, 3647. (8) Chattoraj, D. K.; Bull, H. B.; Chalkley, R. Arch. Biochem. Biophys. 1972, 152, 778. (9) Yu, S. S.; Li, H. J.; Shih, T. Y. Biochemistry 1976, 15, 2027. (10) Weiskopf, M. Biopolymers 1977, 15, 669. (11) Giancotti, V.; Cesaro, A.; Crescenzi, V. Biopolymers 1975, 14, 675. (12) Kalambet, Yu. A.; Burova, E. I. Symp. Biol. Hung. 1988, 37, 251. (13) Kubasek, W. L.; Hockensmith, J. W. Ber. Bunsen-Ges. Phys. Chem. 1989, 93 (3), 406. (14) Greipl, J.; Urbanke, C.; Maass, G. Top. Mol. Struct. Biol. (Protein-Nucleic Acid Interact.) 1989, 10, 61. (15) Roy, S. Curr. Sci. India 1996, 71 (2), 100. (16) Fields, S.; Song, O. K. Nature (London) 1989, 340 (6230), 245.
Materials. In the present investigation, calf-thymus DNA (lot No. 67F-9725) and BSA (lot No. 116F-9390) of Sigma Chemicals Company were used. Powdered casein of N.B. Chemicals, Cleveland, OH, of standard grade was used as an adsorbent which was dried in a desiccator containing concentrated H2SO4 for 4-5 days. Inorganic salts, surfactants, and folin reagent were of analytical grade and hence used directly without further purification. Urea was twice recrystallized from warm ethanol. Double-distilled water was used throughout the experiment. Buffered and unbuffered solutions of DNA at specific pH and ionic strength in native, heat-denatured, acid-denatured, and alkali-denatured states were prepared by the same procedure21 (17) Dickinson, E.; Rolfe, S. E. Food Hydrocolloids 1989, 3 (3), 193. (18) Carr, D. W.; Scott, J. D. Trends Biochem. Sci. 1992, 17 (7), 246. (19) Sebille, B.; Vidal-Modjar, C.; Jautmes, A. In HPLC Proteins, Pept. Polynucleotides; Hearn, M. T. W., Ed.; New York, 1991; p 395. (20) Gani, S. A.; Mukherjee, D. C.; Chattoraj, D. K. Langmuir 1999, 15, 7130.
10.1021/la970687+ CCC: $18.00 © 1999 American Chemical Society Published on Web 09/24/1999
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as that described in part 1 of this paper.20 The concentration of DNA solution in this experiment did not exceed 0.04 mass % (w/v). For the preparation of unbuffered solution of BSA, a stock solution of the protein was prepared in 0.05 M NaCl solution and a stock solution of the salt of same ionic strength was also prepared. Both these solutions were brought to the same pH by the addition of the requisite volume of dilute HCl or NaOH solution. For the preparation of denatured BSA in urea solution the required amount of urea was mixed with the stock salt solution and then the protein solution was slowly added to it. Finally, the pH was adjusted to the desired value by the addition of a suitable volume of dilute HCl or NaOH solution. A buffered solution of BSA was prepared by dissolving the required amount of protein in a phosphate buffer of fixed pH and ionic strength. Finally, the desired ionic strength was maintained by the addition of a suitable amount of solid neutral salt. The concentration of BSA solution in this experiment did not exceed 0.1 mass % (w/v). The pH values for all the solutions thus prepared were measured with a digital pH meter (L-120, Elico, India) with an accuracy of (0.01 pH. Adsorption Experiment. To 40 mL solutions of different concentrations of either native or denatured BSA in 250 mL well-stoppered conical flasks, accurately weighed amounts (0.5 g) of dry powdered casein were added. The suspensions were shaken gently for 15 min and allowed to attain the adsorption equilibrium in an incubator 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. A preliminary study of the adsorption kinetics indicated that the adsorption equilibrium could be reached after 6-8 h. The solutions from the top were pipetted out and centrifuged at 6000 rpm. The same treatment was carried out taking an identical set of blank solutions. The Lowry test22 with the folin reagent indicated the presence of a negligibly small extent of casein in the blank solution. BSA concentrations in the supernatant after adsorption were also determined using the Lowry test. For this, 1 mL of supernatant was taken in a test tube and 4 mL of a solution containing 2% Na2CO3 in 0.1 N NaOH (50 parts) and 0.1% CuSO4 in 2% Na-K tartrate (1 part) were added to it. The solution was mixed gently and kept for 10 min. Then after the addition of 0.5 mL of folin reagent with constant shaking, it was kept in the dark for 45 min. The absorbance of the blue color developed in the protein solution was measured with a Beckmann DU-6 spectrophotometer at 750 nm against the blank. Using the blank solution as solvent, different solutions of BSA of known protein concentrations were prepared. All these solutions were treated with folin reagent in an exactly similar manner to that described earlier. The absorbances of these solutions measured at 750 nm against that of the blank solution were plotted against known protein concentration. Using this linear plot, the BSA concentrations of the bulk solutions at adsorption equilibrium were evaluated with 2-3% standard error. For the determination of nucleotide concentration (of DNA) in the bulk supernatant solution at adsorption equilibrium, the absorbances of different solutions were measured at 260 nm in the same manner as that stated in part 1 of this paper20 against the blank solution prepared in a similar manner to that stated earlier. Using the supernatant of the blank solution as solvent, several DNA solutions of known nucleotide concentrations were prepared and their absorbances at 260 nm were measured. From the standard linear plot of absorbance versus nucleotide concentration, the concentrations (C2) of DNA in the bulk supernatant at adsorption equilibrium were estimated with the standard error not exceeding 3%. The solid-liquid systems used in these adsorption experiments were composed of an accurately weighed amount w (equal to 0.5 g) of powdered dry solid casein in contact with the bulk phase containing water, a neutral solute forming buffer, and the biopolymer (BSA or DNA) at the state of adsorption equilibrium. (21) Upadhyay, S. N.; Chattoraj, D. K. Biochim. Biophys. Acta 1968, 161, 561. (22) Lowry, O. H.; Rosenbrough, N. J.; Farr, A. C.; Randall, R. J. J. Biol. Chem. 1951, 193, 265.
Gani et al.
Figure 1. Plot of Γ21 against X2 for the adsorption of BSA on the surface of casein at 28 °C in the presence of NaCl: (O) pH 5.0, µ ) 0.05; (∆) pH 3.0, µ ) 0.05; (Y) pH 8.0, µ ) 0.05; (b) pH 5.0, µ ) 0.01; (2) pH 5.0, µ ) 0.105.
Figure 2. Plot of Γ21 against X2 for the adsorption of BSA on the surface of casein at 28 °C, pH 5.0, and µ ) 0.05 in the presence of different salts: (- - -) NaCl; (O) CaCl2; (4) AlCl3; (Y) Na2SO4; (2) Na3PO4. The extent of adsorption in moles of nucleotide or moles of BSA, respectively, per kilogram of dry casein at a fixed pH, ionic strength, and temperature may be calculated using eq 1.
Γ21 )
(C2t - C2)Vt 1000
(1)
Here C2t and C2 stand for the molar concentrations of BSA (mol. wt 68 000) or nucleotides of DNA, respectively, before and after adsorption by casein powder and Vt is the volume of the biopolymer solution in milliliters associated per kilogram of dry casein. Vt is equal to v/w, where v is the volume of the solution in milliliters (equal to 40 mL) and w is the weight of dry casein in kilograms present in the system prior to adsorption. Since the solution is dilute with respect to BSA or DNA, C2 may be taken to be equal to 55.5X2, where X2 stands for the bulk mole fraction of BSA or nucleotide. Each experiment was repeated four times, and the average Γ21 was presented at a mean value of C2. The standard error in the values of Γ21 did not exceed 6%. The errors are shown in one isotherm in each figure (vide Figures 1-5).
Results and Discussion Casein is the major constituent of protein which remains in aggregated form in all kinds of animal milk. There are several forms of casein in cow milk. The powdered whole casein of bovine milk used in our experiments is practically insoluble in aqueous solution in the acidic range of pH. In alkaline pH, casein begins to dissolve in aqueous medium, forming sodium caseinate. In milk insoluble casein aggregates are in contact with solvent containing β-lactoglobulin and R-lactalbumin present in soluble form. Since such powder was present in a large amount (1 kg bottle), we have conveniently used it as adsorbent mainly at lower pH for the study of adsorption of BSA and DNA,
Interaction of BSA and DNA with Casein
Figure 3. Plot of Γ21 against X2 for the adsorption of BSA on the surface of casein at 28 °C, pH 5.0, and µ ) 0.05 in the presence of NaCl + urea: (O) NaCl + 2 M urea; (4) NaCl + 4 M urea; (b) NaCl + 6 M urea.
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surfaces of glass,23 polystyrene,24 and alumina,25 respectively, at isoelectric pH. In the presence of 6 M urea, BSA molecules adsorbed from solution become extensively unfolded and large molecules of unfolded BSA are accumulated at the interface by interfacial coagulation.26 Contrary to the case of these rigid solids, the surface of insoluble casein powder is highly hydrophilic and flexible in character. Water vapor adsorption experiments27 with casein powder indicate that the insoluble milk protein is highly hydrated close to unit water activity. When the casein powder is formed by the precipitation of casein micelles in milk, the hydrated polypeptide chains of this protein remain associated by strong protein-protein attractive forces. The intermolecular Van der Waals’ forces of attraction28 between globular protein molecules in aqueous media have already been estimated from osmotic pressure measurements. When the hydrated casein powder comes in contact with aqueous solutions of BSA or DNA, adsorption of these biopolymer molecules may occur on the bare surface of hydrated casein powder. The adsorption process may or may not lead to interfacial dehydration. It has been recently shown from the isopiestic method29 that powdered BSA and gelatin interact with each other near unit water activity, leading to the dehydration of the proteins. In Figures 1 and 2, the extents of adsorption of BSA on the bare surface of casein have been plotted against X2 at adsorption equilibrium under different physicochemical conditions. Γ21 in all cases has been found to be positive. In part 1 of this paper, it has been shown that
X2 Γ21 ) ∆n2 - ∆n1‚ X1 Figure 4. Plot of Γ21 against X2 for the adsorption of DNA on the surface of casein at pH 6.0 in the presence of NaCl + CaCl2: (O) 22 °C, µ ) 0.05; (6) 28 °C, µ ) 0.05; (Y) 37 °C, µ ) 0.05; (2) 28 °C, µ ) 0.01; (4) 28 °C, µ ) 0.005.
Figure 5. Plot of Γ21 against X2 for the adsorption of DNA on the surface of casein at 28 °C, pH 6.0, and µ ) 0.05 in the presence of salts: (- - -) NaCl + CaCl2, native DNA; (O) NaCl + AlCl3, native DNA; (Y) NaCl, heat-denatured DNA; (4) NaCl + CaCl2, heat-denatured DNA.
respectively, from aqueous solution to the hydrophilic surface of casein under different physicochemical conditions. From the studies of adsorption of BSA from aqueous solution to the rigid surfaces of various inert solid powders, it has been observed that, at the state of monolayer saturation, ellipsoid-shaped BSA molecules may orient vertically, horizontally, or in an expanded state on the
C2 = ∆n2 - ∆n1‚ 55.5
(2)
∆n1 and ∆n2 in this equation represent the number of moles of solvent and biopolymer, respectively, bound to the surface of 1 kg of casein. When the solution is dilute, the mole fraction X1 of solvent is close to unity, so that the mole fraction of adsorbate in aqueous solution is equal to C2/55.5. Since Γ21 values in all cases are positive, ∆n2 is greater than ∆n1 (C2/55.5). In each isotherm in Figures 1 and 2, Γ21 at low values of BSA concentration increases sharply with an increase of X2 but it attains a maximum and steady value Γ2m when X2 reaches X2m. Γ2m may be regarded as the amount of BSA required to saturate completely the available surface of 1 kg of casein. Many isotherms in Figures 1 and 2 are S-shaped, which may indicate that BSA-BSA attractive interactions in the adsorbed surface layer of casein are quite significant. It may also be pointed out that the shapes of many of these isotherms are similar to those observed for the adsorption of BSA on hydrophobic surfaces.20 Values of Γ2m and X2m for different isotherms are included in Table 1. (23) Bull, H. B. Biochim. Biophys. Acta 1956, 19, 464. (24) Norde, W. Proteins at Interfaces. Doctoral Thesis, Agricultural University, Wageningen, The Netherlands, 1976. (25) Hajra, S.; Chattoraj, D. K. Ind. J. Biochem. Biophys. 1991, 28, 124. (26) Sarkar, D.; Chattoraj, D. K. Ind. J. Biochem. Biophys. 1994, 31, 100. (27) Sadhukhan, B. K.; Chattoraj, D. K. Ind. J. Biochem. Biophys. 1983, 20, 59. (28) Chattoraj, D. K.; Chatterjee, R. J. Colloid Interface Sci. 1976, 54, 364. (29) Datta, P.; Hajra, S.; Chattoraj, D. K. Ind. J. Biochem. Biophys. 1997, 30, 449.
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Table 1. Evaluated Parameters for the Adsorption of BSA on the Surface of Casein at 28 °C, pH 5.0, and Ionic Strength 0.05 nature of BSA native
partially denatured
a
additives
109X2m
initial slope (dΓ21/dX2)X2f0
104Γ2m (mol of BSA/kg of casein)
NaCl NaCl(1)a NaCl(2)a NaCl(3)a NaCl(4)a NaCl(5)a NaCl(6)a CaCl2 AlCl3 Na2SO4 Na3PO4 NaCl + 2 M urea NaCl + 4 M urea NaCl + 6 M urea
1.30 1.20 1.40 1.30 1.15 1.35 1.10 1.00 0.50 1.28 1.10 1.95 1.45 1.45
1095 270 572 978 1320 556 2210 2130 4130 384 614 1690 1008 840
1.59 1.35 1.20 1.35 1.65 1.45 1.60 1.80 2.10 1.42 1.10 2.05 2.06 1.50
(1) pH 3.0; (2) pH 8.0; (3) µ ) 0.01; (4) µ ) 0.105; (5) 22 °C; (6) 37 °C.
In Figure 1, the isotherms for the adsorption of native BSA in the presence of 0.05 M NaCl at three different values of pH (3.0, 5.0, and 8.0) at 28 °C have been compared. Γ2m at the isoelectric pH (5.0) of BSA is observed to be higher than those at pH 3.0 and 8.0, respectively (vide Table 1). At pH 3.0 and 8.0, protein is undergoing conformational alterations in the bulk and at the interface due to the electrostatic repulsion effect, so that BSA molecules undergo significant lateral expansion at the interface whereby values of Γ2m in both cases have been reduced. Similar types of pH effect on the adsorption of BSA on the surfaces of several rigid solid powders have been observed by earlier workers.25 At pH 5.0 and at 28 °C, with an increase of ionic strength from 0.01 to 0.10, Γ2m has been found to increase significantly (vide Figure 1, Table 1). The magnitudes of the initial slopes24 in these cases also increase with an increase of ionic strength (µ) of the medium (vide Table 1). This indicates that the interaction24 of BSA with the exposed surface of casein near the isoelectric pH increases with an increase of µ. Further BSA molecules thus adsorbed at higher values of X2 undergo intermolecular attractive interaction, which in its turn also increases with the increase of ionic strength. Due to this interaction, more BSA molecules at higher ionic strength can be accommodated at the hydrophilic surface of casein. In Figure 2, the isotherms for the adsorption of native BSA on powdered casein at 28 °C, pH 5.0, and µ ) 0.05 in the presence of different neutral salts have been presented. The values of Γ2m presented in Table 1 are found to vary in the following order of neutral salts: AlCl3 > CaCl2 > NaCl > Na2SO4 > Na3PO4. We also note from this table that the initial slopes of different isotherms follow the same order as that of Γ2m. This means that both BSA-casein interactions at low values of X2 and BSA-BSA interactions in the adsorbed layer at higher values of BSA concentration increase in the same order of neutral salts presented here. Because of these interactions, more molecules of BSA can be packed at the hydrophilic interface in the presence of AlCl3, and less, in the presence of Na3PO4. Both BSA and casein can bind Na+, Ca2+, and Al3+ counterions to different extents, which may also be responsible for the different values of Γ2m in the presence of different neutral salts in bulk medium. Three typical isotherms (Figure 3) for the adsorption of BSA on powdered casein at 28 °C, pH 5.0, and ionic strength 0.05 in the presence of 2.0, 4.0, and 6.0 M urea (mixed with NaCl) have been compared. The value of Γ2m slightly increases when the urea concentration is increased from 2 to 4 M. The globular BSA molecules unfolded to
some extent in the presence of 4 M urea oriented at the interface to accommodate more BSA molecules. But at 6 M urea concentration the major fraction of BSA is denatured in solution and at the hydrophilic surface of casein so that Γ2m is reduced drastically due to the lateral expansion of biopolymer at the interface. It has also been shown in Table 1 that the initial slope of the isotherms decreases with an increase of urea concentration. This indicates that the energy of interaction between BSA and the casein surface decreases with an increase of urea concentration in the bulk phase. This particular phenomenon in all probability is due to the increased degree of denaturation of BSA in the bulk medium. Further, the flexible surface of casein must have played some major role in such a variation in the value of Γ2m in the presence of urea of different concentrations. It may be pointed out here that, on a rigid alumina surface,26 Γ2m for BSA at 2, 4, and 8 M urea decreases slowly with an increase of urea concentration in the bulk phase. The isotherms (not shown) for the adsorption of native BSA on the surface of casein studied at 22, 28, and 37 °C at pH 5.0 and ionic strength 0.05 have been compared. The solutions were unbuffered, the ionic strength was controlled by the use of NaCl only, and the pH was adjusted to 5.0 by the addition of dilute HCl. From Table 1 it is observed that both the values of Γ2m and the initial slope increase with an increase of temperature. This indicates that both BSA-casein and BSA-BSA interactions increase in the adsorbed layer with an increase of temperature. In Figures 4 and 5, moles of nucleotide (Γ21) adsorbed per kilogram of powdered casein at different physicochemical conditions have been plotted against the mole fraction (X2) of nucleotide remaining free at equilibrium in the bulk medium. We have noted with interest that native DNA does not bind to the surface of casein in the presence of NaCl alone but it does so with the addition of 1.0 × 10-3 M CaCl2. It is also known from an earlier experiment20 that the bivalent cation present in CaCl2 may bind to the phosphate group of DNA, forming an ion pair, as a result of which the charge density of the polynucleotide or the associated double-layer potential is reduced to a large extent. Under this condition DNA is able to bind to the surface of casein. It may be noted that Γ21 at a low concentration range of DNA increases with an increase of X2 and it reaches a steady value Γ2m when DNA concentration reaches a critical value X2m. Γ2m may be regarded as the number of moles of nucleotide required to saturate the available surface of 1 kg of casein powder. The values of Γ2m and X2m for different isotherms are included in Table 2.
Interaction of BSA and DNA with Casein
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Table 2. Evaluated Parameters for the Adsorption of DNA on the Surface of Casein at 28 °C, pH 6.0, and Ionic Strength 0.05 nature of DNA native
partially denatured heat-denatured heat-denatured acid-denatured acid-denatured alkali-denatured alkali-denatured a
additives
106X2m
106X2a
initial slope (dΓ21/dX2)X2f0
103Γ2m (mol of nucleotide/kg of casein)
NaCl + CaCl2 NaCl + CaCl2(1)a NaCl + CaCl2(2)a NaCl + CaCl2(3)a NaCl + CaCl2(4)a NaCl + AlCl3 NaCl + CaCl2(5)a NaCl + CaCl2(6)a NaCl NaCl + CaCl2 NaCl NaCl + CaCl2 NaCl NaCl + CaCl2
2.00 0.25 1.75 2.00 0.25 1.00 4.25 3.15 3.75 3.00 3.75 2.75 2.50 2.50
4.00 3.15 4.00 5.25 4.25 4.15
702 3160 1380 1350 2020 1320 168 170 3290 5920 2510 5100 225 940
1.45 0.55 2.10 1.00 0.66 0.95 0.50 0.60 3.66 5.18 3.50 4.72 1.20 1.80
8.10 7.00 4.50 7.00 4.75 4.25 5.00
(1) 22 °C; (2) 37 °C; (3) µ ) 0.01; (4) µ ) 0.005; (5) pH 3.0; (6) pH 8.0.
Initially the increase of Γ21 in all cases is due to the strong affinity of DNA molecules to the bare surface of casein, and then as the number of available active spots decreases, the amount of adsorption reaches a steady value Γ2m till the whole surface is covered effectively with bound DNA molecules. Γ21 remains steady at Γ2m up to a certain limiting concentration X2a, termed the “critical aggregation concentration”. These are included in Table 2. When X2 exceeds X2a, Γ21 again increases for all systems. This may be due to the molecular aggregation or interfacial coagulation of DNA molecules on the surface of casein with the formation of multilayers. Such a phenomenon was also observed for adsorption of DNA on charcoal, resin, and alumina.20 In Figure 4, the isotherms for the adsorption of native DNA on the surface of powdered casein at three different ionic strengths (0.005, 0.01, and 0.05) at 28 °C and pH 6.0 have been compared. The ionic strength of the medium was maintained with a mixture of NaCl and 0.001 M CaCl2. With the increase of the ionic strength of the medium, Γ2m increases (vide Table 2). The initial slope signifying BSAcasein interaction is decreased with the increase of ionic strength from 0.005 to 0.010, but on a further increase of ionic strength, its value remains constant. Also in Figure 4, the plots of Γ21 against X2 for the adsorption of native DNA on casein at 22, 28, and 37 °C at constant pH 6.0 and ionic strength 0.05 in the presence of NaCl + 0.001 M CaCl2 have been presented. From these isotherms it is observed that with the increase of temperature the extent of adsorption Γ21 at a given value of X2 increases. This may be due to the increase in DNAprotein interaction at the interface with temperature increase by some kind of cooperative effect. In Figure 5, the isotherms for the adsorption of native DNA in the presence of NaCl + 0.001 M CaCl2 and NaCl + 5.0 × 10-4 M AlCl3 and for heat-denatured DNA in the presence of NaCl and NaCl + 0.001 M CaCl2 at 28 °C, pH 6.0, and ionic strength 0.05 have been presented. Unlike native DNA, denatured DNA is able to accumulate at the surface of casein powder in the sole presence of NaCl and in the complete absence of CaCl2 or AlCl3. This is only possible due to the substantial reduction of the negative charge density of DNA by extensive denaturation whereby the charged phosphate groups are widely separated from each other in the aqueous system. From these isotherms it is observed that the extent of adsorption Γ21 of heatdenatured DNA on the surface of casein is more compared to that of the native DNA at a given value of X2. This is similar to that observed for adsorption of native and denatured DNA on the surface of charcoal.20 The values of Γ2m as recorded in Table 2 are found to vary in the
following order: heat-denatured DNA (NaCl + CaCl2) > heat-denatured DNA (NaCl) > native DNA (NaCl + CaCl2) > native DNA (NaCl + AlCl3). In the presence of denatured DNA, more sites of the casein surface are available for nucleotide binding, so that ∆n2 in eq 2 will be increased. Further, the second term ∆n1 (C2/55.5) may also be affected to different extents for native and denatured DNA in the presence of different inorganic salts. These may cause variation in the values of Γ2m for different systems. The isotherms (not shown) for the adsorption of heat-, acid-, and alkali-denatured DNA in the presence of NaCl and NaCl + CaCl2 (0.001 M) at 28 °C, pH 6.0, and ionic strength 0.05 have been compared. From these isotherms it is observed that Γ2m in the presence of NaCl + CaCl2 is higher than that in the presence of NaCl alone. The variation in the values of Γ2m as recorded Table 2 is as follows: alkali-denatured DNA (NaCl) < alkali-denatured DNA (NaCl + CaCl2) < acid-denatured DNA (NaCl) < heat-denatured DNA (NaCl) < acid-denatured DNA (NaCl + CaCl2) < heat-denatured DNA (NaCl + CaCl2). The initial slopes of these isotherms follow almost the same order (vide Table 2). The variation in the extent of adsorption Γ2m is mainly due to the different degrees of denaturation of DNA and counterion binding to different extents to the negatively charged phosphate group, which may lead to the reduction of the negative charge density of the biopolymer prior to adsorption. Plots of Γ21 versus X2 (not shown) for the adsorption of DNA on casein powder at 28 °C, ionic strength 0.05, and three different values of pH (3.0, 6.0, and 8.0) in the presence of NaCl + CaCl2 (0.001 M) have been compared. From these isotherms it is found that Γ2m at pH 6.0 is higher than that at pH 3.0 or 8.0. At pH 3.0, the surface of casein powder becomes positively charged whereas at pH 8.0 it becomes negatively charged, since the isoelectric pH of casein is 5.9. It appears that near the isoelectric point of casein, that is, at pH 6.0, the DNA-protein interaction is more pronounced. The values of Γ2m and initial slopes included in Table 2 are in conformity with the experimental observation. A similar result was observed during the adsorption of BSA on casein, as discussed earlier. In Figures 1-5, one finds that the number of kilograms of BSA or nucleic acid adsorbed per kilogram of casein is always a small fraction of the order 10-4 to 10-3. This means that the interaction of BSA or DNA occurs on the exposed sites of solid casein powder. One may assume that the surface area of casein available for adsorption remains unaltered due to the adsorption of biopolymers
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from solution up to X2m so long as Γ2m remains constant. Beyond this point, Γ21 sharply increases by surface aggregation, as observed also in the case of adsorption of DNA on a rigid solid surface. Alternatively, it may be assumed that the highly hydrated casein may swell significantly27 at this stage, thus exposing a large number of sites for further adsorption. For all cases of adsorption of BSA and DNA on the surface of casein, each measured value of Γ21 (or Γ2m) may be regarded as the time independent equilibrium value at a given value of X2 (or X2m), so that the apparent values of the standard free energy change ∆G° in kilojoules per kilogram of casein for the change of biopolymer concentration in the bulk from zero to unity in mole fraction units can be evaluated using the Bull23 equation (eq 7) given in part 1 of this series. The evaluated apparent values of -∆G° in kilojoules per kilogram of casein for various systems for each biopolymer (BSA or DNA) are found to increase linearly with an increase of Γ2m in moles of BSA or nucleotide (of DNA) adsorbed per kilogram of casein. The slope of this linear plot representing the standard free energy change ∆GB° for both biopolymers is 39 kJ per mole of BSA or nucleotide (of DNA) transferred from the bulk solution to the surface of casein. Strictly, however, ∆G° or ∆GB° may be regarded as the true standard free energy change if the isotherms for the adsorption and that involving desorption steps are very
Gani et al.
close to each other. This has been discussed in detail in part 1 of this series. Our experiments on the desorption step for BSA and DNA from the casein surface are found to be in gross error because of the occurrence of a large inaccuracy in the determination of adsorbate concentrations. It has also been anticipated that the desorption equilibrium needs a much longer time (maybe 1 week or more), during which putrefication of biopolymers has actually occurred. Under this circumstance the value of ∆G° or ∆GB° based solely on adsorption data according to Bull23 represents the maximum change of work for the process rather than the true standard free energy change for adsorption. We may thus conclude that BSA and DNA dissolved in aqueous solvent can be adsorbed to the highly hydrophilic surface of insoluble casein powder and that the maximum value of adsorption of these biopolymers depends on pH, ionic strength, temperature, the nature of the neutral salts, and the states (native or denatured) of the biopolymers in solution. Acknowledgment. Financial assistance of Indian National Science Academy, New Delhi, is acknowledged with thanks. LA970687+
