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J. Phys. Chem. 1983, 87,2148-2152
Condensation of Polyamines onto Nucleic Acids We1 S. Yen,+ Kee Woo Rhee,$ and B. R. Ware" Department of Chemlstty, Syracuse University, Syracuse, New York 13210 (Received: November 16, 1982)
The condensation of the polyamines spermidine (Sp3+)and spermine (Sp4+)onto double-stranded DNA and single-stranded poly(adeny1icacid) has been studied by using the laser Doppler technique of electrophoretic light scattering (ELS). The electrophoretic mobility reduction upon addition of Sp3+or Sp4+to solutions of nucleic acids was determined by the reduced Doppler frequency of the peak center in the ELS spectrum;changes in molecular conformation or state of aggregation were reflected in the peak line widths and line shapes. Results are interpreted in terms of the counterion condensation theory and its subsequent extension to the theory of the electrophoretic mobility. It is concluded that this theory explains both qualitative and quantitative aspects of polyelectrolyte charge reduction and conformational collapse but that electrophoretic mobility magnitudes and titration behavior are not yet predicted with the accuracy to which they can be measured.
Introduction DNA is a linear polyelectrolyte of high (negative) charge density; thus its conformation is a sensitive function of its ionic environment. Most DNA in living organisms is present in a compact form in which positively charged species such as histones or polyamines serve to stabilize the conformation by neutralizing electrostatic repulsions. The efficacy of such compounds for inducing the collapse of linear, extended DNA into more compact structures has been demonstrated with a range of techniques, including electron rnicro~copy,'-~ a number of optical and hydrodynamic techniques pioneered by Schellmann and co-worke r ~and , ~dynamic ~ ~ laser light s ~ a t t e r i n g .Wilson ~~ and Bloomfield have reported a careful set of measurements using total intensity and dynamic laser light scattering to determine the conditions for collapse of DNA molecules at high dilution upon treatment with the polyamine ions spermidine(3+) and spermine(4+)? Interpreting their data in terms of the counterion condensation theory of Manning,1° they have inferred that DNA conformational collapse occurs whenever 89-90% of the negative charges of the DNA backbone have been neutralized. They subsequently concluded that charge neutralization reduces the electrostatic repulsion to levels below those necessary to offset the attractive forces due to London dispersion interactions, thus leading to c ~ l l a p s e . ~ J ~ The theoretical formalism developed by Manning'0J2-14 describes the territorial (nonspecific) binding of counterions onto a polyelectrolyte as a condensation process which is presumed to occur whenever the ratio of the electrostatic repulsion energy to the thermal energy exceeds the reciprocal of the electrovalence of the counterion. The predictions of this theory, outlined in the next section, have thus far survived experimental tests of several sorts. The most directly measurable parameter associated with the reduction in linear charge density as a result of counterion condensation is the concomitant reduction in the electrophoretic mobility of the polyion. Recently Manning has published an explicit treatment of this effect and has noted that experimental data for the reduction of the electrophoretic mobility of a polyion, in a solution of univalent counterion at low concentration, as a function of added multivalent ion concentration would be of interest.15 In this paper we report a series of measurements using the technique of electrophoretic light scattering (ELS) to Present address: Chemical Research Laboratory, American Cyanamid Co., Stamford, C T 06904. Present address: Department of Chemistry, University of North Carolina a t Chapel Hill, Chapel Hill, NC 27514. 0022-365418312087-2 148$01.50/0
characterize charge and conformational characteristics of nucleic acids in solution. The ELS technique, invented by Ware and Flygarele and first applied to DNA solutions by Hartford and Flygare," determines the electrophoretic mobilities of polymer species through the Doppler shifts of laser light scattered from them and simultaneously provides conformational data through the diffusionbroadened widths of the Doppler-shifted ELS peaks. We have systematically studied the changes in electrophoretic mobility and diffusion coefficient resulting when the polyamines spermidine and spermine are added to dilute solutions of DNA in an NaC1-sodium cacodylate buffer. Our results corroborate the observations of Wilson and Bloomfieldgand provide new, direct information regarding the relationship of counterion condensation, reduction of the electrophoretic mobility, and induction of conformational collapse. Comparisons with the theory of Manning'O provide further support for the concept of counterion condensation and serve to indicate those areas for which quantitative agreement between theory and experiment is not yet achieved.
Theory It will be necessary during the discussion and analysis of our data to refer to certain theoretical results which we summarize in this section for the convenience of the reader. The counterion condensation theory advanced by Manning10J2-15models a linear polyelectrolyte as an infinitely thin line of average charge spacing b. Counterions are treated as point charges in the medium. The principal tenet of the theory is then stated as follows: in a solution (1)U. K. Laemmli, Proc. Natl. Acad. Sci. U.S.A., 72,4288 (1975). (2)D.K.Chattoraj, L. C. Gosule, and J. A. Schellman, J.Mol. Biol, 121,327 (1978). (3)G. C. Ruben, K. A. Mars, and T. C. Reynolds, Annu. Proc. Electron Microsc. SOC. Am., 39,438 (1981). (4) L. C. Gosule and J. A. Schellman, Nature (London), 259, 333 (1976). (5) L. C. Gosule and J. A. Schellman, J. Mol. Biol., 121,311 (1978). (6) K. L. Wun and W. Prins, Biopolymers, 14, 111 (1975). (7)B. Ramsay-Shawand K. S. Schmitz, Biopolymers, 15,2313 (1976). (8)H.Eisenberg, N.Borochov, Z. Kam,and G. Voordouw, Phil. Trans. R. SOC. London, Ser. A , 293,303 (1979). (9)R. W. Wilson and V. A.Bloomfield, Biochemistry, 18,2192 (1979). (10)G. S. Manning, Q.Reu. Biophys., 11, 179 (1978). (11)V. A. Bloomfield, R. W. Wilson, and D. C. Rau, Biophys. Chem., 11,339(1980);R. W. Wilson, D. C. Rau, and V. A. Bloomfield, Biophys. J.,30,317 (1980). (12)G. S. Manning, J. Chem. Phys., 51,924 (1969). (13)G. S.Manning, J.Phys. Chem., 82,2349 (1978). (14)G. S. Manning, Acc. Chem. Res., 12,443 (1979). (15)G. S. Manning, J. Phys. Chem., 85,1506 (1981). (16)B.R.Ware and W. H. Flygare, Chem. Phys. Lett., 12,81(1971). (17)S.L.Hartford and W. H. Flygare, Macromolecules, 8,80(1975).
0 1983 American Chemical Society
The Journal of Physicel Chemisfry, Vol. 87, No. 12, 1983 2149
Condensation of Polyamines onto Nucleic Acids
containing counterions of valence N, the fraction f of the structural charge of a polyion that will remain uncompensated by bound counterions is given by f = (N[)-'
[ > N-'
[
