Adsorption of nucleic acids at the alumina-water interface From recent measurements of particle mobility b y the microelectrophoretic method ~,2, evidence was obtained b y us for the adsorption of DNA and RNA from solution onto the surface of solid alumina powder. In this communication we present our analytical results for the adsorption isotherms. Calf thymus DNA (lot No. 637) and yeast RNA (lot No. 6KB) used in the present investigation were supplied b y the Worthington Biochemical Corporation (U.S.A.). The detailed specifications of the acids were previously discussed 1,~. The surface areas of the two samples of pro-analysis grade alumina powders, as determined b y the B E T method of nitrogen gas adsorption, were, respectively, 69. 5 and lO 5 m*/g with an error limit of + 5 %. The first sample was used for adsorption of DNA and the other for RNA adsorption. Each set of our adsorption experiments was carried out at a given p H and I 0.05 b y suitable addition of HCI and NaCI. At p H 6.5, 7.8 and 9.0, buffer solutions were used to maintain I at 0.05. To 20 ml of each solution in a set, 2 g of alumina powder were added. The solution was stirred intermittently for 4 h in a thermostatic b a t h at 25°+o.1 °. The solutions were left without disturbance for a further 20 h. The clear solution from the top was decanted off and, on measuring its absorbance at 320 m y with a Beckman DU Spectrophotometer, it was found to be completely free of solid particles 3. The absorbance of the solution was then measured at 260 m y from which the concentration c of the nucleic acid after adsorption was calculated from a standard absorbanceconcentration curve at each p H used. From the difference in the nucleic acid concentration before and after adsorption, the amount X of the acid in mg adsorbed per g of alumina powder was calculated. Both c and X are found to be reproducible within 4%. In Fig. I, X is plotted against DNA concentration c, at p H 6.5, I 0.05, using NaCI, phosphate and acetate buffers. The nature and shape of the curves possibly indicate the Langmuir type of monomolecular adsorption; in fact the Langmuir plot of c / X versus c has been found to be satisfactorily linear. For NaC1 at p H 6.5, the in' 0.75
A c e t a t e burfeo~r
'pN-25 ~pH-'3. 6
( t r [ s buffer)
Equilibrium c o n c e n t n 3 t i o n of D N A , m g / 1 O O m l
pH 6 5
O 1D 2.O &O '&O Equilibrium c o n c e n t r a t i o n of D N A , m g l l O O m l
Fig. I. A d s o r p t i o n of D N A onto a l u m i n a p o w d e r at 25 °, p H 6.5, I 0.05 a n d w i t h s o l v e n t as indicated. Fig. 2. A d s o r p t i o n of D N A o n t o a l u m i n a p o w d e r at 25 °, I 0.05 a n d w i t h p H as indicated. F o r the c u r v e s at p H 6.5, 5.o a n d 4.0, the electrolyte consisted of NaC1 w i t h HC1 added as required.
Biochim. Biophys. Acta, 161 (1968) 561-563
tercept and slope from such a Langmuir plot were O.lO674-O.Ol 5 and 2821±412 , respectively, calculated b y the least squares method. The m a x i m u m amount adsorbed (Xm) per g of the solid powder obtained at higher values of c is fourid from Fig. I to be significantly dependent on the composition of the salt in the solution. F r o m the known values of Xm and the specific surface area of the solid powder, the area per adsorbed DNA molecule in the presence of NaC1 solution was found to be 35.1" lO 7 A 2. If it is assumed that the DNA molecule remains on the surface, statistically as a spherical gel bead* of radius rG, its projection area a rG2 m a y b e taken to be 35.1" lO7 so that r G -- 1.o57" lO4 ,~. This value of r G agrees satisfactorily with that obtained for a dissolved DNA moleculO. In Fig. 2, the adsorption isotherms at I 0.05 are shown for various values of p H ranging from 2.5 to 9.0. All these data are found to fit satisfactorily in the linear Langmuir plot of c / X against c. From a scrutiny of each curve in Fig. 2, it m a y be noted that Xm took a minimum value at p H 6.5 but its value increased with increased acidity and alkalinity of the medium. I t is possible that adsorbed DNA undergoes different amounts of structural change in the presence of acid and Tris buffer.
"~ ~ j//• i / 1"01--~
~ .,~/ ~ ..~.~/
nat ured RNA r _ / ~ O ' q z ~ c i d denet u ed ~', -DNA , Alkali denotured.... DNA
i. . . .
0 2.5 5.0 7.5 10.0 Equilibrium concentrotion of nucleic acid,rag/lOOml
Fig. 3. A d s o r p t i o n of n a t i v e a n d d e n a t u r e d D N A a n d 1RNA o n t o a l u m i n a p o w d e r a t 25 °, p H 6. 4 6.5 a n d I 0.05 w i t h NaC1.
In Fig. 3, the adsorption isotherms of native and denatured DNA are compared at p H 6.5 and 1 0.05. The method of preparation of heat-denatured DNA was described earlier 1. The p E of two concentrated solutions of DN'A were, respectively, brought to p H 2.5 and 12.5 b y the addition of HCI and N a O H and after 24 h the p H values of these solutions were brought back to 6.4-6. 5 again b y suitable addition of acid or alkali. The adsorption onto solid powder was measured spectrophotometlically. From Fig. 3, it m a y be noted that the adsorption of DNA increased b y 5- to Io-fold at a given nucleic acid concentration due to the denaturation by heat, acid or alkali. This result indicates that, using an alumina column, it m a y be possible chromatographically to separate native DN'A from its denatured form. It is also noted with Biochim. Biophys. Acta, i 6 i (1968) 561-563
interest from this figure that at higher values of c, X for heat-denatured DNA begins to increase sharply with concentration without reaching any limiting value. This is obviously a feature of multi-layer adsorption possibly indicating the formation of DNA aggregates at the solid-liquid interface. In Fig. 3, the isotherm for native RNA is also included. Unlike DNA, the curve for RNA does not show a maximum adsorption region at very high nucleic acid concentration. The isotherm for the heat-denatured RNA also suggests multi-layer adsorption. We are thankful to Professor N. DAVIDSONof Caltech (U.S.A.) for suggesting the investigation of this problem. One of us (S.N.U.) is grateful to the Indian Council of Medical Research for a research fellowship and we wish to thank Dr. NAGRAJAN RAO of the Clerkson College of Technology, Potsdam, for surface area measurement of alumina powder.
Chemistry Department, Jadavpur University, Calcutta-32 (India) I 2 3 4
S.N. UPADHYAY D . K . CHATTORAJ
D. K. CHATTORAJ, P. CHOWRASHI AND K. CHAKRAVARTI, Biopolymers, 5 (1967) 173. P. CHOWRASHI, D. K. CHATTORAJ AND K. CHAKRAVARTI, Biopolymers, in the press. H. B. BOLL, Biochim. Biophys. Acta, 19 (1956) 464 • N. DAVIDSON, in J. BONNER AND P. O. P. TS'O, The Nucleohistones, Holden-Day, San F r a n cisco, 1964 .
Received February 26th, 1968
Biochim. Biophys. Acta, 161 (1968) 561-563