VOL. 14 (1975)

BIOPOLYMERS

COMMUNICATIONS TO THE EDITOR Heat of Interaction of DNA with Polylysine and Trilysine The interaction of DNA with synthetic and natural polycations, e.g., spermine, spermidine, tri-clysine, (LYs)~,poly-lrlysine, (Lys),, and various histones in dilute aqueous solution has been studied and is still being actively investigated in different laboratories, including ours.1 Our interest is mainly focused on'the thermodynamic characterization of complex formation between DNA and the above-mentioned polycations, with the aid of calorimetric measurements. Enthalpy data on the binding of spermine and spermidine by native and denaturated DNA have been already p u b l i ~ h e d ,while ~ calorimetric and spectroscopic data on DNA-histone systems should be reported in detail ~ h o r t l y . ~ Recently, calorimetric data on the interaction of 1 X M DNA (salmon sperm) in 0.01 M cacodilate buffer (pH = 7) with spermine and (Lys),, ( n = 50) a t 25°C have been reported by Ross and Shapiro.' These authors, working with the cationic reagents at concentrations such that more than one charge equivalent cation per DNA phosphate was present in solution, find that interaction between spermine and DNA occurs with no change in enthalpy while (Lys), reacts with DNA yielding A H = -300 cal/mol P. Our findings are apparently in disagreement with those of Ross and Shapiro4and we wish to summarize and discuss here a few pertinent results. Let us first recall that working with 4-9 X M DNA (calf thymus) in 2 X lop2M NaCl and 2 X M cacodilate (pH = 5.5), we have measured an enthalpy of spermine and spermidine binding of 0.3 kcal/equivalent base bound a t 25OC. Approximately the same figure has been obtained working with both sonicated and intact native DNA, independent of the ratio R between equivalent concentrations of bases and DNA up to R 0.6.2 Experimental conditions are thus different from those used by Ross and Shapiro, and we wonder whether this may be the main source of discrepancy between our results and theirs. More recently we have found, however, that also the interaction of (LYs)~ with DNA (calf thymus) is characterized by AH = 0.3 kcal/mol Lys in M NaCl and M tris buffer (pH = 7), nearly independent of R, up to R ,- 0.4. More interesting, the results reported in Figure l a show that also in the case of (Lys), the enthalpy of interaction with DNA is positive, larger for n = lo3 than for n = lo2, and practically independent of R (at least for R < 1). Experimental conditions employed by us and by the authors mentioned above are also quite different, particularly because of the much higher ionic strength used by Ross and S h a p i r ~ . We ~ suspect that this is the cause of the discrepancy between the results of Figure 1 and those of Ross and Shapiro, and propose that these differences may be due to quite different types of (Lys),-DNA complexes. It is known, for instance, that direct mixing of DNA and (Lys), solutions a t relatively low ionic strengths yields complexes with slightly distorted CD spectra.6 More distorted CD spectra would be obtained if the (Lys),-DNA complex were prepared by mixing a t high ionic strength, followed by gradient d i a l y ~ i s . ~ Our ellipticity (at 275 nm) data reported in Figure lb, which further disclose the influence of (Lys), molecular weight, are similar to those reported in the literature for complexes prepared by the latter method. More important, in all cases the complex is present in solution in an aggregated form.7~~The extent of aggregation depends on the

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1975 by John Wiley & Sons, Inc.

BIOPOLYMERS VOL. 14 (197.5)

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R (Lys/DNA) Fig. 1. (Lys),-DNA complex in 10+ M tris buffer pH = 7 and 10-2 M NaCl a t 25'C. (A) Enthalpy of interaction. (B) Ellipticity a t 275 nm. CDNA= 6-9 x 10-4 mol P/l; CI,, = 4-8 X mol/l; (0, 0)= lo3; (A, A)= lo2; ( )--DNA. An LKB 10700 batch-type microcalorimeter with glass cells and a Jouan 185 dichrograph equipped with a Xenon lamp were used.

way the complex is prepared (i.e., by direct mixing a t low ionic strength or by mixing a t high salt concentration followed by gradient dialysis) and on the value of R . Interestingly, a t R > 0.75 the higher the ionic strength, the more likely that the CD spectrum of DNA will revert to its normal shape and the more likely that the aggregation will disappear.6 We certainly need not emphasize that complex formation between (Lys), and DNA is a very complicated phenomenon. We wish to propose, however, that our enthalpy data would mostly reflect the electrostatic interactions between lysirie residues and phosphate groups of DNA. Complex formation is, in our case, entirely an entropy driven process, the gain in entropy residing in a loss of water from the hydration sheaths of interacting species. For R > 0 . 7 j and high ionic strengths a different type of (Lys),-DNA complex would be found with a negative enthalpy change, according to Itoss and Shapiro. The reasons for this effect may be manyfold, and it would be too speculative to discuss them a t the present state of knowledge. This work has been sponsored by the Italian Consiglio Nazionale delle Ricerche, Rome (Italy).

References 1. von Hippel, P. H. & McGhee, J. D. (1972) Ann. Rev. Biochem. 41,231-300. 2. Crescenzi, V., Quadrifoglio, F., Ceshro, A . & Giancotti, V. (1973) Protides of the Biological Fluids, 20th Colloq., Pergamon, New York, 483-487. 3. Bradbury, E. M., Danby, S. E., Rattle, W. E. & Giancotti, V. (submitted to Eur. J . Biochem.). 4. Ross, P. D. & Shapiro, J. T. (1974) Biopolymers 13, 41Fi-416.

COMMUNICATIONS TO THE EDITOR 5. 6. 7. 179, 8.

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Zama, M. & Ichimura, S. (1971) Biochem. Biophys. Res. Cmmun. 44, 936-942. Carroll, D. (1972) Biochemistry 11,421426. Matsuo, K., Fuke, M., Tsuboi, M. & Wada, A. (1969) Biochem. Biophys. Actu 39-42. Shapiro, J. T., Leng, M. & Felsenfeld, G. (1969) Biochemistry 8, 3219-3232.

V. GIANCOTTI A. CESARO V. CRESCENZI Istituto di Chimica UniversitA di Trieste 34127 Trieste, Italy Received August 9, 1974 Accepted October 21, 1974