Effect of solution composition upon poly U

Effect of solution composition upon poly U

ARCHIVES OF BIOCHEMIS'l'RY AND BIOPHYSICfl 115, 102-107 (1966) Effect of Solution Composition upon Poly U 1 RAY1\IOND A. BROWN Biolugy Division, O...

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115, 102-107 (1966)

Effect of Solution Composition upon Poly U


RAY1\IOND A. BROWN Biolugy Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Received December 20, 1965 The effect of salt concentration, ion type, pH, and solvent upon poly U melting temperature, 1'.,., has been investigated. Alcohol increases the T m of poly U. The T", depends on the type of ion present and salt concentration in a way which is different from that observed for the RNA of R17 virus. Ultracentrifuge data and the dependence of the T", upon KCl concentration suggest that the melting of poly U is a complex phenomenon.

The work of Lubin (1), Spyrides (2), and Conway (8) has demonstrated that protein synthesis both in vitro and in vivo is dependent upon the solution composition. In particular there exists a marked sensitivity to the concentration of sodium and potassium ions. Outside of the work of Gordon (4) there are few published data demonstrating the effect of ion type and concentration upon the physical properties of the synthetic polynucleotides or more complicated isolated systems. Such data would be useful for an understanding of biological and biochemical systems. The experiments described in this paper demonstrate some effects of solution composition upon the helix-coil transformations of poly U 2 first described by Lipsett (5). EXPERIMENTAL PROCEDURE A single batch (No. 47,328) of poly U obtained from the Miles Chemical Laboratories was used for the absorbance measurements and the ultracentrifnge studies. Another batch (No. 46,434) was used for viscosity and partial specific volume measurements. More than 95% of the ultravioletabsorbing material sedimented as one peak in the centrifuge, and the material had a typical absorption spectrum. The potassium salt of the polymer Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation. 2 Abbreviations used: polyadenylic acid, poly A; polycytidylic acid, poly C, polyuridylie acid, poly U.

was dissolved directly into the appropriate buffer solution. Concentrations were calculated by assuming E~i:t = 290 for the potassium salt (ll). All pH measurements were made Oil the polymer solution at 23°. Optical densities were measured in a Beckman DU spectrophotometer equipped with a thermostated cell compartment. Temperat ures within the cell compartment were regulated t.o wi thin 0.1°. Sedimentation rates were measured at an optical density of 0.70 in a Spinco model E ultracentrifuge equipped with ultraviolet optics, and the optical density tracings were obtained by means of a model R Analytrol. Molecular weights, M, were calculated by means of Eq. (1) j data were obtained from interference optics by using the short-column method of Van Holde and Baldwin (7) and the techniques of Richards and Schacbman (8). In Eq. (1), Co is the initial concentration, Cb is the final concentration at the bottom of the cell, Co is the concentration at the meniscus, Xb is the distance from the axis of rotation to the bottom of the cell, and X a is the distance to the meniscus. The other parameters are defined in Svedberg and Pedersen (9). The column length was 1 mm and the concentration was 0.4%. Partial specific volumes were determined by the procedure suggested by Svedberg and Pedersen (9). 11!f = C, - Co

-----co- (1 -



VP)(Xb2 -

Xa 2)W2



Figure 1 shows the optical density, as a function of temperature, of various poly U solutions containing 0.5 M salt buffered with 102



0.05 ionic strength phosphate pH 7.0 made up in water and in water containing 10 % by volume ethanol. The optical density profiles are dependent upon the type of ion present, with a higher melting temperature being observed with the potassium ion than with the 0.700

~ 0.600


:<:: l.JJ o -.J


u i= c..









FIG. 1. Optical density at 2nD mu as 1.1 function uf temperature in: (a) 0, 0.50 M NaCl, 0.0.5 J.l strength sodium phosphate pH 7.0; (b) e, same as (a) but in 10% by volume ethanol; (e) 6, 0.50 M KCI, 0.05 ionic strength potassium phosphate pH 7.0; (d) .A., same as (e) but in 10% by volume ethanol.

sodium ion; and contrary to the results obtained by Herskovits et al. with salmon testes DNA (10), addition of alcohol favors the ordered form of the molecule. It is convenient to take the maximum slope of the optical density versus temperature curve as the melting temperature ('1'm)' Using this convention it is possible to measure T« with a precision of ±O.3°. A study of the effect of pH upon melting temperatures is shown in Fig. 2. '1'", changes very little, if at all, over the pH range 5.5-8.0. It was convenient to use a pH of 7.0 in the balance of the studies. In order to overcome solubility problems in nonaqueous solvents the salt concentration was reduced to 0.15 M and the phosphate buffer to 0.02 ionic strength. The melting temperatures shown in Fig. 3 were obtained in solutions containing different proportions of methanol and ethanol. Again one sees a difference in T", with sodium and potassium ions and an increase in 'I'm produced by the nonaqueous solvents. Solubility problems and low melting temperatures made it impossible to obtain comparable data with formamide. However, with the addition of 10% formamide to solution 2 of Table I there was only a 4 % decrease in optical density at 0.7°, which implied a melting temperature less than 0°. In contrast to the alcohols, formamide decreases the melting temperature of poly U. The observed sodium-postasium effect appeal'S to be a part of a general ion specificity.


o 5.0





FIG. 2. Melting temperature of poly U phosphate pH 7.0.


a function of pH


0.50 M NaCI, 0.05 IJ. sodium



In Table I are listed the observed melting temperatures of poly U in several different solutions. In solutions 3 and 4 the replacement of phosphate ion by chloride ion results in a significant decrease in the melting temperature. There is an increase in the 1'm upon replacing potassium ion with cesium. By using cesium chloride and 20 % ethanol it is possible to raise the 1'm to 11.3°. The dependence of 1'111 upon KCl concentration is shown in Fig. 4. The curve appears to break down into three separate regions. To further investigate this matter, ultracentrifugal studies were made of poly U at three different KCl concentrations and at two different temperatures, 1° and 23°. Since the optical density at 23° was independent of ionic strength, and since 90 % or more of the increase in optical density change took place between 1° and 23°, the ultracentrifuge data obtained at 1° and 23° should be characteristic of the ordered and disordered phases, which give rise to the melting phenomena. The ultracentrifuge results, along with the measured partial specific volumes and reduced specific viscosities, are shown in Table II. The measured parameters are a function 10



Solu tion composi tion M NaCI; 0.05 ionic strength sodium phosphate buffer pH 7.0 0.15 M KCl; 0.05 ionic strength pot.assium phosphate buffer pH 7.0 0.15 M KCl; 0.05 ionic strength pot.asaium phosphate butTer; 0.01 !VI tris (hydroxyrnethyl )ami nomethane 0.18 M KC]; 0.01 M 'I'ris-chloride buffer 0.18 M KCl; 0.01 M 'I'ris-chloride buffer; solvent 10% f'ormamide, 90(/0 water (v/v) 0.15 M CsClj 0.05 ionic strength porussium phosphate buffer 0.15 M CsCl; 0.05 ionic strengt h pot.assium phosphate buffer; solvent 20% ethanol, 80% water

1. 0.15

2. 3.

4. 5.

6. 7.



3.1 2.9

1.4 <0.0


11 .3

of the KCl concentration, and along with the T 111 data they suggest that the physical properties of poly U are related in a complicated way to the salt concentration. DISCUSSION

The partial specific volume of poly U in 0.30 and 0.60 M KCl was higher than that observed in 0.15 M KC1. The most reasonable explanation for this difference is a change in the charge on the molecule with a concomitant change in electrostriction. The observed molecular weights of a highly charged molecule such as a polynucleotide will be substantially in error if not corrected for charge effects (11). In Eq. (2), adapted from the paper of Johnson et al. (11) M* is the apparept molecular weight of a charged polymer; V, its partial specific volume; and z, the total charge on the polymer, M*(l - Vp)

FIG. 3. Melting temperatures of poly U in 0.15 M salt solutions buffered with 0.05 p, phosphate pH 7.0 and containing different volume percents 01 alcohols. (a) 0 I potassium salts in ethanol solntions; (b) e, potassium salts in methanol solutions; (c) 0, sodium salts in ethanol solutions; (d) 6, sodium salts in methanol solutions.


= lVI(l

- 'z2 M

- Vp)

± (1- V ± p);


J.l1 ±, the molecular weight of the supporting electrolyte; and V±, its partial specific volume. For a polymer with one charge per residue it is convenient to rewrite Eq. (2) in the form of Eq. (3), where R is the molecular weight of the monomer unit:










0040 M KCI

FIG. 4. Melting tempe ra ture of poly U stre ngt h potasaium phosph ate pH 7 .0.

HS !\

funct ion of K CI concent ra ti on in 0,02 ionic



Te mpera tur e

0 .15 M K CI j 0 ,02 iouic stren gt h phos pha te buffer pH 7 ,0 0 .30 M K CI; 0.02 ionic stre ng th phospha te buffer pH 7.0 O.GO M KC]; 0.02 ion ic streng th ph osph a te buffer pH 7.0

23 1 23




23 1



S.. 5 .28 !). OO 6.43 9 .54: 8 .51 8 .71



" X 1Q-'


1.25 (l. 44a ) 2. 17 (2. 55" ) 1.52 2.09 2. 05 2.36

0 . 54 0 .54 0.58 0 .58 0 .57 0 .57

7}apl c

0 .31

0.29 0 .31

Corrected fur ch arge effect .

( l - M *) M± (l-V±p ) - -jl-r- = 2R - (1 - VP)


If one assumes that each phosphate on t he poly U carries on e electronic ch arge, one m ay use Eq. (3) to correct the observed mo lecular weights. The values in' p arentheses arc t he corrected values for t he molecular weights in 0.15 M KCl. Correcte d values for the 0.30 and 0.60 i\I KCl solutions have not b een computed, since it app ears likely t hat ion pairs are form ed in the more concentrated salt. The difference in observed sedim entation rat es in 0.15 and 0.30 M KCI at 23 0 probably reflects a change in t he com pactness of the random coil resulting from a decrease in electrostat ic repulsion between the residues. In 0.60 M K CI t here appears to

be some asscciation of the poly U without any change in ultraviolet absorption. Although the molecular weights increase at the lower temperature, there is never the doubling or larger increase in the molecular weight which would be expected if the structure at the lower temperature were made up of two or more strands. At 10 there is a suggestion of a minimum in the uncorrected molecular weights in 0.30 M KCl. The minimum is more definite when the corrected v alues are compa red. Although the sediment at ion rates and molecular weights are not d irectly comparable because of the difference in concentrat ions, a. reasonable con clusion is t hat the poly U configuration is more sym metri cal in 0.30 1\[ KCl than in the R eI solutions of higher ancllower molarities. The



reduced specific viscosities measured at a concentration of 0.2 % slso suggest that the poly U at lOis more symmetrical in 0.30 M KCl. The most reasonable explanation of the data is that there are two different structures at the lower temperature, one made up of more than one strand and the other of a single strand folded back onto itself in a manner first suggested by Fresco et ol, (12). In 0.30 M KGl there appears to be more of the single-stranded structure. Since for each of the six conditions listed in Table II there was only a single sedimenting boundary, it is felt that the boundary must represent a heterogeneous collection of species, both with regard to molecular weight and conformation. One would infer from the dependence of T 1li upon KGI concentration that the states of poly U change in going from 0.15 to 0.30 M and then to 0.60 M KCl. The ultracentrifuge data, partial specific volumes, and specific viscosities support this inference. The effect of nonaqueous solvents upon biological macromolecules is a complicated subject which has been reviewed by Singer (13). Herskovits (14) has presented evidence for the importance of hydrophobic" interactions in stabilizing the DNA double helix. Fasman et al. (15) have presented similar evidence for the helical form of poly C. Since ethanol and methanol increase the T m of poly U and decrease the T m of DNA and poly C, one may question the importance of hydrophobic forces in stabilizing the structure of poly U. T m in solutions containing methanol, ethanol, (water), and formamide are correlated with the dielectric constants 24, 33,80, and 109, respectively. This observation would suggest that the ordered phase of poly U is stabilized more by a charge-charge or dipole-dipole interaction than by hydrophobic forces. However, Graves ei al. (16) have shown that certain proteins are stabilized at low temperatures by nonpolar solvents, and the possibility exists that the effect of nonpolar solvents upon hydrophobic interactions is different at low temperatures than at the higher temperatures usually studied. Alkali metal ions interact with the RNA extracted from bacterial virus R17 (4) in a 3 The definition of hydrophobic forces given by Fusman ei al, (15) is the one used in this paper.

way different from the eay they interact with poly U. For the RNA of R17 the order of 'I'; is Na+ > I{+ > Cst. For poly U it is Gs+ > K+ > Nat. The T m of the RNA of R17 is a linear function of the logarithm of the salt concentration. The dependence of 'I'« of poly U upon KCl concentration appears complicated. One concludes that there are substantial differences in the structures of the two molecules. In explaining the specific effect of different ions upon protein and nucleic acid denaturation, other workers (17,18) have either attributed them to a general interaction with the water or to an interaction between the ion and a specific group in the macromolecule. It is noteworthy that the Na", I(+, and Cs+ ions all have the same effect upon protein denaturation but have a specific effect upon the nucleic acids. This would suggest thn,t the ions interact with a specific group in the macromolecule. Recent publications suggest that the structure of RNA in solution is a matter of considerable complexity. On the basis of lowangle X-ray scattering, Witz and Luzzati (6) have suggested that poly A in solutions below pH 6.0 has a structure similar if not identical to that observed for the oriented fiber of poly A by Rich et al. (19). This structure consists of more or less rigid doublestranded helices with bases perpendicular to the fiber axis. Timasheff ei al. (20) have suggested that ribosomal RNA is statistically double-stranded and is in the form of small rods. Witz et al (6) have suggested that at neutral pH poly C, poly A, and poly U (the latter at low temperatures) have similar structures and that the structure is doublestranded with the bases interleaved and stabilized by hydrophobic forces. Although the latter two structures (the ribosomal type and that proposed for the synthetic polynucleotides) are statistically double-stranded, there remains the question whether the overall structure has been formed from a single strand folded back onto itself or whether two or more strands are involved. Poly A and poly C at neutral pH clo not show a sharp melting temperature. Poly U does have a sharp melting temperature. On the basis of this and other data quoted above,


one must cond ude that there are important cliffcrences between the structure of poly A and poly C on one hand and t hat of poly U. The biological and biochemic al data would suggest that the ability of messenger R NA t o perform its func tion is strongly dependent upon the composit ion of th e solution (1- 3). A possible explanat ion for t his dependence is t hat t he configuration of t he messenger is im portan t and t hat the configuration is sen sitive t o t he solution composi tion. Nirenberg and Mattaei (27) have shown that DNA, like double-stranded RNA, is inactive in the in vitro peptide-synthesizing system. The results quoted above would suggest that the configuration of poly U, which is highly active in the in vitro assay for messenger, is sensitive to solution composition. P oly U is the simplest biological macromolecule for which a sodium-potassium spe cificity has been shown to exist. If may be useful for st udying ion specificities in biology. REFERENCES 1. 2,

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