Interaction of basic oligo-l -amino acids with deoxyribonucleic acid

BIOCHIMICA ET BIOPHYSICA ACTA

I45

BBA 96225

I N T E R A C T I O N OF BASIC OLIGO-L-AMINO ACIDS W I T H D E O X Y R I B O NUCLEIC ACID O L I G O - L - O R N I T H I N E S OF VARIOUS CHAIN L E N G T H S AND H E R R I N G SPERM DNA*

S E I I C H I K A W A S H I M A , SADAKO I N O U E AND TOSHIO ANDO

Department o] Biophysics and Biochemistry, Faculty o/ Science, the University o[ Tokyo, Hongo, Bunkyo-ku, Tokyo (Japan) (Received J a n u a r y 28th, 1969)

SUMMARY

The interaction of native DNA from herring sperm with oligo-L-ornithines having definite chain lengths of between 2 and 18 residues (prepared by chromatographic fractionation of a partial acid hydrolysate of poly-L-ornithine), has been studied in order to increase our knowledge of the fundamental phenomena underlying the interaction between DNA and basic nuclear proteins. DNA-oligo-L-ornithine complexes were prepared by two different procedures: (i) the direct mixing method and (ii) the dialysis method. The results are summarized as follows. I Addition of the oligomers always resulted in the stabilization of doublestranded DNA against thermal denaturation, though marked differences in melting profiles were observed, which were dependent either on the chain length of the oligomers or to the method of complex formation. 2 The binding of (Orn)n to DNA is reversible and in a state of equilibrium when n ~ 6 under the ionic condition employed. Differences in the stabilizing effect of DNA are observed for (Orn)n when n = 2-4, by comparing the values of ATm (max.); these values are comparable for (Orn)n when n = 4 6. 3 For (Orn)n, where n = 8-9, two kinds of complexes with different stabilities are formed between DNA and the oligopeptides. 4 For (Orn)n, where n = 18, binding of an irreversible nature predominates when the ionic strength of salt is low. The complex formed by the dialysis method is more stable than the one formed by the mixing method.

INTRODUCTION

The interaction of basic nuclear proteins, including histones 1-5,29 and protamines T M , with DNA has been studied to obtain both chemical and physicochemical * Presented at tlle 4oth General Meeting of the Japanese Biochemical Society (Osaka, N o v e m b e r 3rd, 1967). Presented to the University of Tokyo by S. K a w a s h i m a as a part of his dissertation for the degree of Master of Science (February 2orb, 1968).

Biochim. Biophys. Acta, 186 (1969) 145-157

146

s. KAWASHIMAet al.

information which would lead to a better understanding of both the structural and functional roles of these proteins in cell nuclei. Physicochemical approaches to elucidate the function of histones have been directed to the use of simple model homopolypeptides such as polylysine 7-1° and polyarginine 7,8,1° of high molecular weight. However, it is evident that these model polypeptides cannot completely represent naturally occurring nuclear proteins when one considers the possible primary structures of these proteins n-la. In the case of protamines, complete separation and the determination of the total amino acid sequences of the three molecular species (YI, Y I I and Z) present in clupeine, have been achieved in this laboratory 14 16 in the last few years. Each species of clupeine molecules contains several sequences of 2-4 consecutive arginine residues between groups of 1-2 (3 in one instance) neutral amino acids. All three molecules consist of as few as 3o (YII) and 31 (YI and Z) amino acid residues, of which 2o (YI and Y I I ) and 21 (Z) residues are arginine. In order to further understand both the structural and functional features of natural nucleoprotein complexes, investigations were made using basic oligopeptides of different side chain and chain lengths. In the present communication the results obtained from the interaction of oligo-L-ornithines with DNA are described. This is the first of a series of studies on these problems using oligomers consisting only of basic amino acids, and those containing both basic and neutral amino acids.

MATERIALS AND METHODS

Chemicals DNA was isolated from the testes of frozen herring by extraction with 2 M NaC1, followed by ethanol precipitation and purification according to the method of KAY et al. 17. The molecular weight of the DNA prepared in this way was 6.0. lO 8 (by viscosity determination) and e(P) = 6300. Examination by electron microscopy showed that the preparation consisted of highly polymerized fibers. CM-cellulose was a product of Pharmaeia, Uppsala, Sweden. Visking tube was washed by boiling in 0.005 M E D T A and rinsing with water before use. All the reagents used were of analytical grade.

Preparation o] oligo-L-ornithines of various chain lengths by chromatographic/ractionation o/ a partial acid hydrolyzate o] poly-L-ornithine Poly-L-ornithine hydrobromide was synthesized by the method of ARIELY et al. 18, F~.]589m~--71.5 ° (c 4.0 in water). We are indebted to Ajinomoto Co., for the supply of the starting compound, L-ornithine hydrochloride. An average degree of polymerization of 9 ° for the polymer was obtained by determining the N-terminal using the standard 2,4-dinitrophenyl method. Oligo-L-ornithines were then prepared by partially hydrolyzing I.O g of poly-L-ornithine hydrobromide in 25 nd of 6 M HC1 for 74 h at room temperature. The resulting mixture was fractionated by column chromatography on CM-cellulose, essentially according to the procedure described for the fractionation of oligo-L-lysine by STEWART AND STAHMANN19. A minor modification in our procedure was the use of 0.05 M acetate buffer (pH 6.0) (Fig.I). The material corresponding to each peak was pooled, and adsorbed on small columns (I.O cm × Biochim. Biophys..4cta, 186 (1969) 145-157

INTERACTION OF

(Orn)n

WITH D N A

I47

-r I

1.5

i

I q

1.0-

I

/

L ........... QS-

0 .... 0

// ~-

50

100

150 200 F r a c t i o n No.

250

300

/

,(

o lo--~2 a4 as o.'s NoC[ ( M )

Fig. I. C h r o m a t o g r a p h i c f r a c t i o n a t i o n of a p a r t i a l acid h y d r o l y s a t e of pol y-L-orni t hi ne . Poly-Lo r n i t h i n e h y d r o b r o m i d e (i.o g) w a s h y d r o l y z e d in 6 M HC1 for 74 h a t r o o m t e m p e r a t u r e . The h y d r o l y z a t e was e l u t e d on a 2. 5 cm × 65 c m c o l u m n of CM-cellulose w i t h a n e x p o n e n t i a l g r a d i e n t of NaC1 (o -+ 0.84 M) buffered a t p H 6.0 w i t h 0.05 M s o d i u m a c e t a t e , a n d w i t h a flow r a t e of 11. 5 ml per f r a c t i o n p er 7.5 m i n a t r o o m t e m p e r a t u r e . The fi rs t l a rge p e a k w a s a s c r i b e d to a c o n t a m i n a n t since t h e f r a c t i o n was n e g a t i v e to t h e n i n h y d r i n r e a c t i o n . The m a t e r i a l u n d e r t h e second p e a k w a s i d e n t i f i e d as an o r n i t h i n e m o n o m e r a n d i t s c o n c e n t r a t i o n , d e t e r m i n e d b y a n a m i n o acid a n a l y z e r , is s h o w n b y a b r o k e n line on a n a r b i t r a r y scale. The n u m b e r g i v e n to each p e a k c o r r e s p o n d s to the c h a i n l e n g t h (n) of t h e o l i g o m e r u n d e r t h e pe a k. Fig. 2. R e l a t i o n s h i p s b e t w e e n c h a i n l e n g t h a n d s a l t c o n c e n t r a t i o n i n t h e g r a d i e n t e l u t i o n of a p a r t i a l acid h y d r o l y s a t e of p o l y - L - o r n i t h i n e on a CM-cellulose c ol umn. The c o l u m n size w a s 0.9 cm × 55 cm.

3.o cm) of Amberlite CG-5o at p H 6.o, which were thoroughly washed with o.I M acetic acid. In the case of oligomers with n ~ 6, the columns were washed with water instead of o.i M acetic acid, since these small oligomers are eluted from the column by the acid. The oligomer on the resin was then eluted with o.I M HC1 and the pH of the eluate was adjusted to between 3 and 4 with Amberlite IRA-4oo (HCO 3- form). The subsequent solution was then dried by lyophilization. The degree of polymerization was determined by the following two methods: (I) the amount of ~,&di-DNP-L-ornithine separated by thin-layer chromatography from the acid hydrolyzate of dinitrophenylated oligo-L-ornithine, was determined spectrophotometrically, b-mono-DNP-L-ornithine was determined with a Hitachi KLA-3B automatic amino acid analyzer, (2) the amounts of 2,4,6-trinitrophenylated products before and after acid hydrolysis of the oligomers, were determined spectrophotometrically 2°. The results obtained by both methods were in good agreement, and showed that the fractionation according to the degree of polymerization was achieved essentially up to about n z 20. The separation, however, became gradually incomplete for the oligomers with n ~ 8. A plot of log n versus NaC1 concentration of the eluate is given in Fig. 2.

Preparation o/DNA-oligo-L-ornithine complexes DNA-oligo-L-ornithine complexes were prepared using the following two procedures which were essentially those previously described 6. Biochirn. Biophys. dcta, 186 (1969) 145-157

148

s. KAWASHIMAet al.

(z) The mixing method. To a constant volume of the DNA solution in o.o3 M NaCl-o.oo3 M sodium citrate (pH 7.o), an equal volume of the oligo-L-ornithine solution, made up with the same buffered saline, was added with constant stirring at room temperature. The concentration of DNA (usually I . I o - 4 M ) was constant throughout a series of experiments. The concentration of oligo-L-ornithine was varied to give different values for the ratio of ornithine monomer/DNA phosphorus (Orn/P) in solution. (2) The dialysis method. DNA and oligo-L-ornithine dissolved separately in 2 M NaC1, were combined by a procedure similar to that described above. The resulting solutions were successively dialyzed at 4 ° against 0. 4 M NaC1 for 4 h, o.15 M NaC1 for 3 h and finally against 0.03 M NaCl-o.oo3 M sodium citrate (pH 7.0) overnight. The procedure was similar to that first recommended by HUANG et al. a for the reconstitution of soluble nucleohistones. The concentration of oligo-L-ornithine in solution was determined by measuring the amount of ornithine released after hydrolyzing the sample in 6 M HCf at IiO ° for 24 h in an evacuated sealed tube. A Hitachi KLA-3 B amino acid analyzer was used for these determinations. The concentration of DNA was determined by phosphorus analysis and expressed as moles P per 1. Sometimes the concentration was calculated from the absorbance at 260 m# using the value of e(P) =- 6300 obtained for the standard solution of DNA. Measurements o/ melting profiles Melting profiles were measured using an Ito spectrophotometer (model QU 3) equipped with a thermally-controlled sample compartment. The temperature was measured by a Cu-constantan thermocouple inserted in a cuvette containing water and placed in the sample compartment. The samples were heated from room temperature to 98o at a rate of about 1.5°/min. No corrections for thermal expansion were made to the absorbance readings in routine experiments. The turbidity was followed by measuring the absorbance at 320 InF. The melting temperature (Tin) was defined as the temperature at the midpoint of maximum hyperchromicity attained during a transition.

RESULTS

A. Analysis o/ the precipitate When DNA-oligo-L-ornithine complexes were prepared by the mixing method (see MATERIALSAND METHODS), appreciable amounts of visible aggregates were formed for (Orn)n, when n => 5. These aggregates could be separated by centrifugation at about IOOO×g for 15 rain. In Fig. 3, the amounts of DNA in the precipitates were plotted against the Orn/P input ratio of the mixtures. Two discontinuous changes, one between n = 4 and 5, and the other between n = 5 and 6, were observed in the curves. For (Orn)n, n ~ 4, the solutions of the complexes were completely clear and no complex was sedimented by low speed centrifugation. For (Orn)5, however, a precipitatable aggregate was formed only when the Orn/P ratio exceeded unity. For (Orn)n, n _> 6, part of the complex sedimented before the Orn/P ratio approached unity, and for (Orn)9, more than 9 ° % of the DNA was found in the precipitate Biochim. Biophys..dcla, 186 (1969) 145-157

INTERACTION OF

100

n=lB ~ /

(Orn)n W I T H

n=9 . n=6

DNA

149

n=5

8C

i!il .

.

.

.

Orn/P Fig. 3. D N A precipitated b y the addition of (Orn)n at various Orn/P input ratios. The complex, DNA-(Or~)n was prepared b y direct m i x i n g of D N A and (Orn)n solutions in o.o 3 M NaC]o.oo 3 M sodium citrate.

when an Orn/P input ratio of unity was attained. Orn/P ratios in the precipitates sedimented from solutions of various Orn/P input ratios, were analyzed and the results given in Table I. Within the limits of experimental error, it can be said that, for TABLE

I

ANALYTICAL

DATA

FOR THE D N A - ( O r n ) n

PRECIPITATES FORMED BY THE MIXING METHOD

The precipitates formed by the mixing method were obtained by centrifugation at iooo × g for 15 r a i n , a n d t h e i r O r n / P r a t i o s a n a l y z e d . O r n i t h i n e w a s d e t e r m i n e d u s i n g a n a m i n o a c i d a n a l y z e r a n d t h e a m o u n t o f p h o s p h o r u s w a s c a l c u l a t e d u s i n g e ( P ) a t 2 6 o m/~ = 6 3 o o .

n

Orn/P input

Precipitated DNA (,o) o/

5

0.65 1.09 2.18 3.27 4.36 5.45

o 0 14 49 92 97

o.17 o.34 °.51 0.68 °'85 1.7o

o o 7 47 96

---1.29 0.99 1.14

8

0.25 o.49 0.75 o.99 1.23

9 18 25 91 95

-1.o 5 0.85 0.92 0.98

I8

0.22

8

0.44

18

I.OO

0.66

6o

0.80

0.88

92

I.OI

6

38

Orn/P ratio in the precipitates ---

I.OI 0.98 I.II 1.23

--

Biochim. Biophys. Acta, 186 (1969) I 4 5 - I 5 7

150

S.

K A W A S H I M A e/, al.

(Orn)n, n = 5-18, the Orn/P ratios of the precipitates are unity, irrespective of the Orn/P input ratios. The results indicate that precipitation occurred as a result of a neutralization of the charges on the D N A molecules rather than the aggregation of charged D N A chains partly bound with oligo-L-ornithine. It can also be concluded that the binding of the oligomers, with n ~ 5, was of an irreversible nature•

B. Melting profiles o/ complexes formed by the mixing

method The clear supernatants, obtained by low speed centrifugation of the DNAoligo-L-ornithine solutions prepared by the mixing method, were subjected to tile 1,4

1.4[ (o)

[(b)

n=5

i

-

n= 9

i(M~x}ng

method)

_~

.

1.3

~2

1.2

It!

o ,<

0 13 .>_

1.0 60 °

1,4[(c) n = 1 8

J 70 ° 80 ° Temperoture

90 °

60 °

100 °

70 °

80 ~ [emperoture

90 °

100 °

r

T

(Mixing method)

I

!

121

l

of .~ ~'1I _si

1,QL 60 °

/

1

/

70 ° 80 ° TemperGture

90 °

100 °

Fig. 4. M e l t i n g profiles of D N A - ( O r n ) n c o m p l e x e s p r e p a r e d b y t i l e m i x i n g m e t h o d . (a) n = 5. O r n / P r a t i o s and Tm v a l u e s ( t e m p e r a t u r e in p a r e n t h e s e s ) for t h e c u r v e s f r o m left to r i g h t are: D N A a l o n e (72.7), o.22 (73.8), o.43 (75.4), o,65 (76.2), 0.87 (76.8), i . i (77.3), 2.o (78.4) a n d 5,5 (79.5). (b) n ~ 9. O r n / P ratios: i, D N A a l o n e ; 2, o.14; 3, o . 3 I ; 4, 0.56. (c) n = i8. O r n / P r a t i o s and Tm v a l u e s ( t e m p e r a t u r e in p a r e n t h e s e s ) : i, D N A a l o n e (72•6); 2, o.2o (72,6, 89.5}; 3, o-31 {73 .0 , 9o.5); 4, 0.39 (75.7, 93.0).

Biochim. f3iophys. Acta, 186 (1969) 145-157

INTERACTION OF

(Orn)n WITH D N A

i5i

thermal denaturation study. The changes in absorbance at 32o mff did not exceed 0.005 during the melting of these solutions and hence no correction was made for turbidity. Figs. 4a, b and c, show the melting profiles of the complexes formed between DNA and (Orn)n, where n ~ 5, 9 and 18, respectively. Figure 4a represents the melting profiles for the DNA-(Orn)n complexes with n ~ 6. DNA-(Orn)n complexes of 11 = 8 and 9, gave the melting profiles shown in Fig. 4b. A typical result for complexes formed with higher oligomers is reproduced in Fig. 4 c. The main features of these 3 types of melting profiles are as follows: Type I: n =< 6. Here oligo-L-ornithine contributes towards raising the Tm values of all the molecules or every portion of DNA in the solution (Fig. 4a). It is noted that the shape of the melting curves for the complexes are similar to the curve for free DNA and Tm only shifts to higher values with increasing concentrations of oligoL-ornithine. Type II: n = 8- 9. The melting profiles are represented in Fig. 4 b. This type can be distinguished from Type I in the following ways: (i) the melting curves of the complexes are not parallel to that of free DNA; (2) the complexes melt with a higher Tm than that of DNA, but do not exhibit total hyperchromicity below ioo°; (3) the shapes of the melting profiles are not symmetrical, and the Tm values obtained from the midpoint of maximum hyperchromicity are merely apparent ones since these melting profiles cannot be ascribed to the transition of a single ordered structure. These features of the melting curves m a y be explained by assuming the presence of three species of molecules (or portions of molecule) in the solution, i.e. (a) free uncomplexed DNA (melting at the initial parts of the curves), (b) a less stable complex (melting at the final part of the curves with broad transition widths) and (c) a stable complex which does not melt below IOO° (not shown in the figure). Type III: n = approx. 18. The melting profiles show biphasic transitions when the Orn/P ratio is below unity. Each of the first transitions shows approximately 1.4

i

(a) n :7 [Mixing method)

i

i

,

1.4~

n = 11

~

:

1,3l(MIxing m e t h o d ) ? ~ ~ ~

1,3 i

1.2

1.2

o

o

.>O_1.1 !/ 1,60 o0'

70 °

......... k

80 ° Temperature

90 °

100 °

1,0 =

60 °

I

70 ° 80 ° Temperature

I

90 °

I

100 °

Fig. 5. Melting profiles of (a) D N A - ( O r n ) ~ a n d (b) D N A - ( O r n ) n c o m p l e x e s p r e p a r e d b y t h e m i x i n g m e t h o d . O r n / P ratios: (a) I, D N A alone; 2, 0.39; 3, o.53. (b) i, D N A alone; 2, 0.2o; 3, 0-39; 4, 0.59. Biochim. Biophys. Acta, 186 (1969) 145-157

152

s. KAWASHIMAet al.

the same Tm value as that of free DNA, while the second transitions correspond to the melting temperature of the complex. In the case of (Orn)n, n approx. 18 (Fig. 4c), the Tm values of the first transition in the complexes with Orn/P ratios of 0.20 and o.31, were identical to the Tm of free DNA within the limits of error, while the Tm shown by the complex with Orn/P = 0.39 was somewhat higher, and the transition was rather broad. The second transitions are sharper with increasing Orn/P ratios, and the apparent Tm values shifted to higher values. It is noticeable that one type of DNA-(Orn)8_ 9 complex has a higher Tm than the complex of DNA-(Orn)x 8. When this fact is considered in relation to the results obtained with complexes formed by the dialysis method (see Section C), it is seen that the complex of DNA-(Orn)~ 8, formed by the mixing method, is not the most stable one formed between DNA and (Orn)18. It is of interest that the melting profiles for n = 7 and n = I I (Figs. 5a and 5b) can be placed in intermediate positions in the above classification. There is, however, a possibility that these oligomers are contaminated by neighbouring fractions of oligomers which belong to the different types, since the fractionation becomes incomplete for oligomers of these lengths.

C. Melting profiles o/complexes/ormed by the dialysis method It seems to have been established that basic polypeptides form complexes with DNA under reversible conditions in high salt concentration, and that the binding becomes irreversible when the salt concentration is lowered by dialysis 6,10. The melting profiles of DNA-(Orn)n complexes formed by the dialysis method (see MATERIALS AND METHODS) were therefore examined to gain an insight into the properties of these complexes. Solutions of complexes formed by this method, in contrast to those of complexes formed by the mixing method, had an appreciable turbidity, especially at high Orn/P ratios. The most turbid sample of DNA-(Orn)I s (Orn/P = 0.66) had absorbances of 0.335 at 260 m# and o.125 at 320 mE~ at room temperature, the latter changing to O.lO5 after complete thermal denaturation. It is preferable, of course, to correct for the turbidity, but this correction has been discussed by some authors in similar studies without unequivocal success 1°. Hence no correction for turbidity was made for the present melting experiments since this cannot be done by any simple procedure, particularly since the particle containing the ehromophore is, at the same time, the origin of the turbidity. Figs. 6a and 6b show the melting profiles of DNA-(Orn)n and DNA-(Orn)I 8, respectively. For any Orn/P ratio below unity, a single transition with a Tm approximately identical with that of free DNA was seen at the initial portion of each curve, and the complexes did not melt below IOO° under the solvent conditions used. Thus it is noted that the complexes formed by the dialysis method are more stable against thermal denaturation than those formed by the mixing method. The reversibility of the binding of oligo-L-ornithines to DNA was also analyzed from the dialysis experiments in which the Orn/P input ratio before dialysis was adjusted to unity, and the Orn/P ratios as well as the melting profiles of the inner solutions, were examined after dialysis. These oligo-L-ornithines were completely removed from the dialysis bag when they were dialyzed in the absence of DNA, only the molecules which were bound to DNA remaining in the dialyzed solutions. The results of these experiments are given in Table II. Perfect reversibility was ohBiochim. Biophys. Acta, 186 (1969) 145-157

INTERACTION OF 1.4

I

(Orn)n WITH D N A i

153 1.4

i

(o) n: 11 (Dialysis method) / , . , f / ~ - - ~

1.3

1.3

1.2

1.2

~n=18 ' (Dialysis m e t h o d )

/

o o

e~ .~ 1.1

_o

4

I.C60 °

70 °

L

80 °

Temperoture

90 °

100o

1.060 °

70 ° 80 ° Temperature

90i °

lOrO o

Fig. 6. Melting profiles of (a) D N A - ( O r n ) n and (b) D N A - ( O r n h B complexes p r e p a r e d b y the dialysis method. O r n / P ratios which were determined on the dialyzed solutions, and Tm values for the first t r a n s i t i o n ( t e m p e r a t u r e in parentheses): (a) I, D N A alone (74.2); 2, o.41 (75.0); 3, o.66 (76.2): 4, 0.75 (77.5). (b) i, D N A alone (73.2); 2, 0.20 (73.7); 3, 0.37 (74.6); 4, 0.66 (73.8).

TABLE II (Orn)n

RECOVERED

AS A C O M P L E X A F T E R D I A L Y S I S ( I N P U T RATIOS OF

O r n / P = I)

AND THE EFFECT

ON T H E M E L T I N G T R A N S I T I O N

Oligo-L-ornithines a n d D N A were mixed in 2 M NaC1 w i t h i n p u t ratios of O r n / P = i and dialyzed as described in MATERIALS AND METHODS. Analyses of O r n / P ratios and melting profiles were carried o u t on the solutions inside the dialysis bags.

n

Orn/P recovered

Tm

Hyperchromicity produced at zoo ° (% o//ree DNA )

Free D N A 2 3 4 5 6 7 8 9

o o.o3 o.oi 0-o7 0.o6 0.40 0.27 0.68 0.65

72.6 ° 72.60 72.8 ° 72.6o 74.9 ° 76.3 ° 75.5 ° ---

IOO 99 99 99 81 77 85 o o

served up to n = 4. For (Orn)5_7, the reverse reaction was very slow, and for (Orn)8 and (Orn)9 , binding was practically irreversible. It should be noted that for (Orn)8 and (Orn)9 , soluble complexes having 0 r n / P ratios of about 0. 7 were formed by this method, and the complexes did not exhibit appreciable hyperchromicities below IOO°. For oligomers larger than n = I I , soluble complexes could not be formed by this method and all the DNA was recovered in the precipitates when the Orn/P input ratio was unity. Biochim. Biophys. Aela, 186 (1969) 145-157

154

s. KAWASHIMAet al.

DISCUSSION

Although cationic substances of small molecular weight such as spermine 21 24, spermidine21, 2a, diamines 21,25,2°, polyamines v, Mg2+ (see refs. 21, 25 and 27) and Na * (see refs. 21, 25-27), have been reported to show stabilizing effects on DNA, these substances differ from the basic nuclear proteins, at least with respect to their effects on the heat denaturation of DNA. In the mode of interaction of the proteins and DNA, there must be some minimum structural requirements which distinguish the basic nuclear proteins from these small cationic substances. A careful analysis of the melting profiles of DNA-(Orn)n in which n is between I and 18, leads to the following conclusion concerning the mechanism of complex formation and the properties of complexes made by the mixing method. The effect of oligo-L-ornithines of n G 6 on the melting profile of DNA is apparently similar to that observed for these small cationic substances, and can be ascribed to a reversible interaction between DNA and oligo-L-ornithine. This conclusion, drawn from the thermal denaturation experiments with (Orn)5 and (Orn)6, appears to be inconsistent with the conclusion that the interaction is irreversible, obtained from the observation of precipitate formation as described in Section A of the RESULTS.By considering the results obtained from these two different types of experiments, it seems most reasonable to conclude that, for these oligomers, the interaction at lower temperatures is nearly irreversible and rearrangements (i.e. migration of the oligopeptide from one binding site to another) take place slowly. Nevertheless, the time needed for the rearrangement at elevated temperatures is much shorter than the time scale involved in melting experiments. Of course, the rate of rearrangement is temperature dependent, thus the interaction is practically irreversible at lower temperatures (precipitate formation) and becomes reversible at elevated temperatures melting phenomena). r~ ,[a

--

8

~-~

6i

0

. . . . . . n=5

no6

2

4

6

8 Orn/P

10

2,0 t

th=4

---

12

14

16

b n=2/

~

,n=3 ,'

/ / / / ,' /

7

n=4

i

18

,'

0

1.0 (Orn/P) i

2.0

Fig. 7- a. R e l a t i o n s h i p s of z]Tm to O r n / P ratio, b. P l o t s of zJTm -1 versus (Orn/P) -1. The s y m b o l s on a n d n e a r t h e lines i n d i c a t e t h e p o i n t s of o b s e r v a t i o n . Some more v a l u e s r e a d from t he c u r v e s in Fig. 7 a f a c i l i t a t e d t h e d r a w i n g of s t r a i g h t lines in Fig. 7 b w i t h m o r e a c c u r a c y .

The nature of the binding reaction is analyzed from the curves given in Fig. 7 a where the values of ZlTm (ZITm = Tm (complex)--Tm (free)) are plotted against Orn/P and the double reciprocal plot in Fig. 7 b. An empirical relation (Eqn. I) similar to that employed by MAHLER AND MEHROTRA26 in the analysis of interaction of DNA with diamines, is applicable to the present saturation effect on ZJTm (Fig. 7a). dl Tm (observed) --

Z] Tm ( m a x . ) . r Kr+ r

Biochim. Biophys. Acta, 186 (1969) 145-157

(I)

INTERACTION OF (Orn)n WITH D N A

I55

where r -- Orn/P, ATm(max.) = A T m (as r approachesoo) and K , = (r)aTm when ATm = ATm(max.)/2.

Eqn. I can be converted into the double reciprocal form: i

i

zl Tm (observed)

A Tm (max.)

i+

(2)

The straight lines in Fig. 7b indicate that the relationship between these parameters fits the equations. It can be concluded from Fig. 7 that for (Orn)n, where n = 2-4, the K , values (the negative reciprocal of the intercept on the abscissa) differ appreciably: ATm(max.) = 2 ° for n = 2, ATm(max.) = 3 c for n = 3 and ATm(max.) = IO° for n = 4- Thus, interactions between (Orn)2, (Orn)a and (Orn)4, and DNA, take place to the same extent when K~ is equal or approximately equal to 7, but the stabilization effect decreases in the order (Orn)4 > (Orn)3 > (Orn)2. For (Orn) 5 and (Orn) 8, A Tm (max.) values are identical with the value for (Orn) a, indicating that the stabilization effects are comparable among these oligomers. However, A Tm values reach saturation at much smaller Orn/P ratios, and K, is smaller than I for (Orn)5 and (Orn)6, while for (Orn)4 the value of K r is about 7. For the DNA-(Orn)n complex where n = 8-9, the presence of two kinds of complexes which differ in stability, must be considered. Thus the "less stable complex" melts below IOO° and shows a broad transition (Fig. 4b), indicating that the time required for the rearrangement is comparable with the time scale involved in melting experiments under the conditions used. The other complex does not melt below IOO°. A definite conclusion cannot be made as to the structural difference in these two kinds of complexes at the present stage. However the following possibilities can be conceived. (I) The mode of binding of oligo-L-ornithine with DNA is different for these two complexes, one type of binding being more stable than the other. (2) The distribution of oligo-L-ornithine on the DNA molecule is not random and there are two species of DNA (or segments of DNA) in the solution. One is abundantly bound with oligo-L-ornithine and the other is poorly bound. It is likely that the former species is a more stable complex. Neither of these two possibilities is completely independent of the other, but one could be the cause of the other. The type of binding which characterizes the "less stable complex" could be "defective binding", in which not all of the charged groups of oligo-L-ornithine are effectively neutralized by the phosphate anions of DNA. One more consideration which should be taken into account here is the formation of an intermolecular association of the complex. Such an aggregation could represent essentially the same molecular species as the precipitate but a part of the aggregation could be kept in solution either by its limited solubility or by the effect of the soluble uncomplexed moieties which are present in the same molecule. It should be noted that the formation of the more stable complex is enhanced with increasing Orn/P ratios. In the case of DNA-(Orn)ls, the complex formed by the mixing method has a Tm below IOO° and is less stable than the complex formed by the dialysis method (compare Fig. 4c with Fig. 6b). Neither can the structural difference in these complexes be defined. It is a noticeable fact, however, that the more stable complex is formed by the dialysis method, in which the binding is at first reversible and later Biochim. Biophys Acta, 186 (1969) 145-157

156

s. KAWASHIMAet al.

becomes irreversible. The binding of DNA and (Orn)l 8 in the mixing method is irreversible judging from the occurrence of a typical biphasic transition in the melting profile. The difference in the stability of the complexes depending on the conditions of formation, has already been observed and discussed in the case of DNA-clupeine 6. These observations on the mode of interaction between oligo-L-ornithines and DNA can be discussed in relation to the interaction of natural basic nuclear proteins with DNA. For example, the interaction of (Orn)l s with DNA is very similar in some respects to that of clupeine with DNA, as has already been mentioned in the above discussion. This is to be expected since clupeine has 20-21 arginine residues per molecule. In other respects, the interactions between DNA and these basic peptides, which have comparable chain lengths and similar numbers of charged groups, show some differences. For instance, the values of Tm differ for the complexes D N A (Orn)18 (Tm = 88 °) and DNA-clupeine (Tin = 93°), formed by the mixing method, showing that the nature of the side chains of the basic peptides is also responsible for the stability of the complexes. Similar studies using oligo-L-arginines of various chain lengths have been completed and the results will be published elsewhere. Throughout these experiments, none of the basic oligopeptides added to DNA gave melting profiles such as those given b y DNA-histone complexes, which have been reported to show broad, mono-phasic transitions 3. It is therefore evident that in the case of nucleohistones, the peptide moieties composed of nonbasic amino acids, play an important part in determining the physical and possibly biological properties of the nucleohistones. Studies using various types of model peptides composed of both basic and nonbasic amino acids with different side chains, are considered to be promising for the elucidation of the structure-function relationship in both nucleoprotamines and nucleohistones.

ACKNOWLEDGEMENTS

This work has been supported in part by grants from the Ministry of Education of Japan. After this paper was ready for publication, we became aware of the publication of a paper by OLIOS et al. 28, in which they described the interaction between a number of oligo-L-lysines and calf thymus DNA. The results obtained were, in part, very similar to ours. REFERENCES I 2 3 4 5 6 7 8 9 io II

J. BONNER AND R. C. C. HUANG, J. Mol. Biol., 6 (1963) 169. E. W. JOHNS AND J. A. V. BUTLER, Nature, 28 (1964) 853. R. C. C. HUANG, J. BONNER AND K. MURRAY', J. Mol. Biol., 8 (1964) 54" L. S. HNILICA AND D. BILLEN, Biochim. Biophys. Acta, 91 (1964) 271. Y. OHBA, Biochim. Biophys. Acta, 123 (1966) 76. S. INOOE AND T. ANDO, Biochim. Biophys. Aeta, 129 (I966) 649. E. RAUKAS, Biokhimiya, 3 ° (1965) 1122. IV[. LENG AND G. FELSENFELD, Proc. Natl. Acad. Sci. U.S., 56 (1966) 1325. M. TSUBOI, K. MATSUO AND P. O. P. T s ' o , J. Mol. Biol., 15 (1966) 256. D. E. OLINS, A. L. OLINS AND P. H. VON HIPPEL, J. Mol. Biol., 24 (1967) 157. D. M. P. PHILLIPS, ill J. BONN~R AND P. O. P. Ts'o, The Nucleohistones, H o l d e n - D a y , San Francisco, 1964, p. 46.

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DNA

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12 K. IWAI, K. ISHIKAWA, T. SENSHU AND H. HAYASHI, Proc. I8th Symp. Protein Structure, Osaka, 1967, p. 25. 13 K. IWAI, K. ISHIKAWA AND H. HAYASHI, Proc. 29th Syrup. Protein Structure, Tokyo, 1968 , p. I. 14 T. ANDO, I'~. IWAI, S. ISHII, M. AZEGAMI AND C. 1NTAKAHARA, Biochim. Biophys. Acta, 56 (1962) 628. 15 T. ANDO AND K. SUZUKI, Biochim. Biophys. Acta, 121 (1966) 427 . 16 T. ANDO AND K. SUZUKI, Biochim. Biophys. Acta, 14o (1967) 375. 17 E. R. M. KAY, N. S. SIMMONS AND A. L. OOUNCE, J. Am. Chem. Soc., 74 (1952) 1724. 18 S. ARIELY, M. WILCHEK AND A. PATCHORNIK, Biopolymers, 4 (1966) 91. 19 J. w . STEWART AND M. A. STAHMANN, J. Chromalog., 9 (1962) 23320 I~. SATAKE, T. OKUYAMA, M. OHASHI AND T. SHINODA, J. Biochem. Tokyo, 47 (196o) 654. 2i H. TARBOR, Biochemistry, I (1962) 496. 22 IV[. MANDEL, J. Mol. Biol., 5 (1962) 435. 23 S. Z. HIRSCHMAN, M. LENG AND G. FELSENFELD, Biopolymers, 5 (1967) 227. 24 A. M. LIQUORI, L. CONSTANTINO, V. CRESCENZI, V. ELIA, E. GIGLIO, R. PULITI, M. DE SANTIS SAVIVO AND V. VITAGLIANO, J. Mol. Biol., 24 (1967) 113. 25 G. ]~ELSENFELD AND S. HUANG, Biochim. I?,iophys. ,4cta, 37 (196o) 425 26 H. R. MAHLER AND B. D. MEHROTRA, Bioehim. Biophys. Acta, 68 (1963) 211. 27 W. F. D o v e AND N. OAVIDSON, J. Mol. Biol., 5 (1962) 467 . 28 D. E. OLINS, A. L. OLINS AND P. H. VON HIPPEL, J. 2~10l. Biol., 33 (I968) 265. 29 Y. OHBA, Biochim. Biophys. Acta, 123 (1966) 84.

Biochim. Biophys. Acta, 186 (1969) 145-157