Polynucleotides

Polynucleotides

336 BIOCHIMICA ET BIOPHYSICA ACTA VOL. 2 6 (1957) POLYNUCLEOTIDES II. PHYSICAL PROPERTIES OF SOLUTIONS OF SOME POLYNUCLEOTIDES R O B E R T F. S T ...

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336

BIOCHIMICA ET BIOPHYSICA ACTA

VOL. 2 6

(1957)

POLYNUCLEOTIDES II. PHYSICAL PROPERTIES OF SOLUTIONS OF SOME POLYNUCLEOTIDES R O B E R T F. S T E I N E R *

Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md. (U.S.A .) AND R O L A N D F. B E E R S , JR.

Robert B. Johnson Laboratory, The Children's Hospital School, Baltimore, Md. ( U.S.A .)

INTRODUCTION

In view of the uncertainty which still surrounds the molecular configuration of ribose nucleic acid, it is of great interest to examine the physical properties of simple analogues of the latter produced by enzymically polymerized single nucleotides1, 2. Of the several known enzymes which can accomplish this, that isolated from M. lysodeikticus produces polymers of sufficiently high molecular weight to be especially adaptable to physical studies *. The preparation has been described in the first paper of this series2b. The basic aim of the studies by physical methods is to establish the detailed molecular structure. It has not thus far been possible to do this unequivocally, but it has been possible to make definite inferences regarding the relative probability of various feasible structures. Some of the questions to be decided follow. It is still uncertain whether the polynucleotides in solution possess an ordered structural array, such as a helix, stabilized by secondary bonds, or whether they are simply unorganized polymers. If they should, in fact, possess a helical configuration, there remain alternate possibilities of single or double, or conceivably higher, helices3. If any ordered structure is present, there remains the problem of determining the nature of the secondary bonds by which it is stabilized. At the present state of development of the subject it is probably hazardous to take for granted that the polynucleotides produced by the various enzymes are necessarily identical in their configurational fine structure. It is desirable to state that the results and conclusions in what follows refer explicitly to polynucleotides produced by the enzyme of lysodeikticus. EXPERIMENTAL

Preparative P o l y m e r s of adenylie, cytidylic, a n d inosinic acids were prepared b y the action of the nucleotide polymerizing enzyme, isolated from M. lysodeikticus ia b y a m e t h o d described elsewhere, u p o n * The opinions or assertions contained herein are the p r i v a t e ones of t h e writers and are n o t t o be construed as official or reflecting t h e views of t h e N a v y D e p a r t m e n t or t h e naval service at large.

Re#fences p. 348.

VOL. 2 6 (1957

rOL~OCLEOTID~S II

337

the corresponding diphosphate monomers. A typical preparation of polyadenylic acid (poly-A) was as follows. To lOO ml of 0.5% ADP at p H 8.o was added 1.7 ml of o.5M Tris to p H 9.8.3 ml of o . o l M MgC11 and 3.3 ml of enzyme solution were then added and the mixture incubated at 37 ° for 28o rain. The polymer was then precipitated with concentrated KC1 and washed successively with 50 % KC1, 7o % ethanol, and 95 % absolute alcohol, followed by ether; it was dried at room temperature in vacuo. A slight variation in the isolation procedure was made for poly-I and poly-C which were precipitated with KC1 from 5° % ethanol. In several cases (poly-A III and poly-A VI) the polymer was explicitly deproteinated by emulsification with CHC1s by Sevag's procedure.

Measurements* All light-scattering measurements were made with a Phoenix light-scattering photometer. The sole important modification was the introduction of a battery power supply as an alternative to t h a t supplied with the instrument. Measurements over a range of angles were made using cylindrical cells with polished planar faces on the front and exit sides. These were furnished by the Phoenix Precision Instrument Company and were always used in conjunction with a pair of 2-ram entrance slits in series, furnished by the same company. The cylindrical ceils were calibrated with respect to angular symmetry with dilute solutions of recrystallized fluorescein. The technique of measurement was as follows. Each dilution, after clarification, was pipetted into a cylindrical cell and the normalized intensities as a function of angle, relative to t h a t at 9o °, were determined from0 = 3o ° t o o = I35 °. These relative intensities were corrected for scattering of the reflected beam by means of the equation 4. Go, corr.= G0,obs--o.o47 Gt80° - 0 For the purposes of this correction, deflections for angles above 135 ° were obtained by extrapolation. The absolute reduced intensity at 0 = 90 ° was determined for each dilution by pipetting directly from the cylindrical into a standard cell and measuring with the standard I cm slit. The normalized intensities, relative to 9 o°, at the other angles were converted into absolute intensities by multiplication by this quantity. The method of absolute calibration at 0 = 9°0 has been described elsewhere5. Each preparation was checked for fluorescence in the visible, which was always found to be absent. Prior to measurement, each dilution was clarified by centrifugation at IS,OOO r.p.m, in a Sorval centrifuge. Centrifugation was continued until constant scattering properties were attained. A portion of the first dilution was allowed to stand without centrifugation for an equivalent period and remeasured, as a check against possible changes with time. For the thermal degradation study at IOO° C a previously clarified polymer solution was immersed in a boiling water bath. Aliquots were withdrawn at suitable intervals and pipetted into known volumes of buffer at room temperature, where measurements were made. Concentration was determined by pipetting o.5 ml aliquots into i ml of i M KOH, heating in a boiling water bath for 1 h, diluting to 25 ml and measuring the optical density at 257 m/~ in a Beckman spectrophotometer. The optical densities were converted into concentrations of the original solution by multiplication by the factor 1.147 for poty-A or by 1.33o for poly-I. Sedimentation runs were made with a Spinco ultracentrifuge. Measurements of streaming birefringence were made with a commercial instrument furnished by the Rao Company. The refractive index increment was measured with a Phoenix differential refractometer and was assumed to have the same value for all conditions. A value of o.182 4- 0.o 5 (g/ml) was found for poly-A at -----436 m/~. Glass redistilled water and analytical grade reagents were used for all measurements. RESULTS

Sedimentation o~ polyadenylic acid at alkaline pH's A t w e i g h t c o n c e n t r a t i o n s o f p o l y - A g r e a t e r t h a n a b o u t 2 g/l in o . I M KCI, i t s s e d i m e n t a t i o n d i a g r a m c o n s i s t s o f a v e r y s h a r p line, s i m i l a r t o t h a t o b t a i n e d for h i g h l y polymerized preparations of deoxyribonucleic acid (DNA). At lower concentrations t h e r e is c o n s i d e r a b l e b o u n d a r y s p r e a d i n g a n d t h e s e d i m e n t a t i o n c o n s t a n t i n c r e a s e s m a r k e d l y . A n i n c r e a s e i n i o n i c s t r e n g t h t o o.5 c a u s e d a m a r k e d i n c r e a s e i n t h e d e g r e e * In what follows, pertinent quantities are defined in the APPENDIX.

Re/erences p. 348.

338

R . F . STEINER, R. F. BEERS

(I957)

VOL. 2 6

of spreading and reduces the concentration dependence of the sedimentation constant. An appreciable increase in the limiting value of the sedimentation constant also occurs. These points are illustrated by Fig. I and Table I. The extrapolated sedimentation constants cited in Table I and indicated in Fig. 2 were obtained by extrapolation of I/S versus concentration, which served to linearize the concentration dependence. Inasmuch as only ordinary schlieren optics were available sedimentation measurements could not be extended to concentrations lower than about 0.02 g/1. Hence some reservations must be made about the apparent limiting values of sedimentation constant. If any upward curvature occurred at lower concentrations, the values cited would be too small. From the sedimentation data it can be concluded that the degree of polymerization is quite high for all preparations studied here and that all preparations examined were somewhat polydisperse, in view of the boundary spreading at the higher ionic strengths. TABLE I LIGHT-SCATTERING

Sample

AND

MOLECULAR

Solvent

KINETIC

DATA FOR

pH

R

½

SEVERAL

M w × 1:o- ~

(A)

P A A II

o.oi M o.oi M o.oi M o.oi M o.oi M o.oi M o.oi M o.oi M O.olM

PO~= NaHCO s P O ~ , o.i M KCI N a H C O , , o.i M KCI PO~_, o.I M KC1 PO~-, o . 5 M KCI P O ~ , o . 5 M KC1 N a H C O s, o . 5 M KCI tris, o . 5 M LiC1

6.95 8.o 6.5 9.4 7.9 6. 5 7-3 8.o 8. 5

124o 1434 lO7O 967 926 765 662 716 828

3.0 i lO% 3.6 3.5 3.5 3.0 2. 7 2.8 2.8 3.1

PAA III

O.OlM P O ~ , o . 5 M KCI O . o l M tris, o . 5 M KC1

7.8 8.2

551 560

1.2 i.o

P A A IV

O.OlM tris, o . 5 M KCI

8.2

508

I.i

PAA V

0. 5 M o.oi M O.olM O.olM

6.8 6.8 7.i 7.I

lO3O 167o

3.6 4.0

KCI PO~ P O ~ , o . I M KCI P O ~ , o . 5 M KC1 H~O o.oi M N a H C O 3

PREPARATIONS

OF

PAA

c = o

Lbire]ringenc¢

× ro~S

(A)

14.9

16. 4 17.5 7,ooo-1 I,OOO 5 , o o o - 7,ooo

8. 5

* T h i s q u a n t i t y s h o u l d be c o m p a r e d w i t h Vz-6 ./R2~, ½ for t h e c o r r e s p o n d i n g conditions. k

g/

Streaming bire[ringence A few streaming birefringence measurements were made upon preparations poly-A V and poly-A VI, The results are summarized in Fig. 2 and Table I. The magnitude of the birefringence increases with increasing gradient. The only theoretical treatment available for fitting the variation of extinction angle with gradient requires the assumption of a prolate eUipsoidal shape 6. The curves of extinction angle as a function of gradient were fitted to theoretical lengths using the theory and computations of ref. n. It was found to be impossible to fit the data to a theoretical curve for a single length, a range of lengths being indicated. While of the same order Re[erences p. 348.

VOL. 26 (1957)

POLYNUCLEOTIDESII

339

45 S20* 20.0

40

15.0

3oI

lO.O

2O

5.0

Io Q



I

t.o

.....

t

i

E.O CONC. (GMSIL]

Fig. 1. Concentration dependence of s e d i m e n t a tion c o n s t a n t for P A A V a t t w o ionic strengths. • : o.oI M P O ~ , o . s M KC1, p H 7.1 ; O : o . o x M PO~, o, I M KC1, p H 7,i.

I

I

200o

I

t

40o0

t

[

t

6o0o

G Fig. 2. E x t i n c t i o n angle as a function of gradient for P A A V, 0 , 0 : H~O p H 8.9, conc. = o.3o 5, o.o76,respectively(g/1 ) ; @, @ : o.oz M NaHCOs, p H 9,I, c o n c . = 0.3o5, o.152, respectively (g/l).

of magnitude as the extensions computed from fight-scattering data, the birefrigence lengths are definitely greater. This is reminiscent of the findings for the case of DNA 1°. The apparent lengths cited in Table I were computed on the assumption of an axial ratio of 300, which follows roughly from fight-scattering data. However, the computed lengths are very insensitive to this parametern. The assumption of an ellipsoidal shape is certainly a rather crude approximation to the mean shape of the coiled poly-A molecules in solution. In view of this, the agreement in the apparent length as computed from streaming birefringence with that from light scattering is probably as good as could be expected. The variation in extinction angle with gradient is significantly different in water and in O.olM NaHCOs, the apparent length being shorter in the latter medium. In both cases, no significant variation was observed with concentration for the range of very low concentrations studied.

Molecular size and shape o/polyadenylio acid at alkaline pH's The principal method employed in studying the molecular parameters of polyadenylic acid was light-scattering. The usual angular method of plotting and extrapolation developed by ZIMM was used to compute molecular weights and radii of gyration7. While the limiting slope and intercept of the reciprocal reduced intensity as a function of sin2 B/2 at zero angle and zero concentration are theoretically related unambiguously to the weight average molecular weight and the radius of gyration, Re]erences p. 348.

34o

VOL. 2 6 (1957)

R . F . STEINER, R. F. BEERS

in practice, for polydisperse systems, the presence of downward curvature can make the extrapolation from the usual range of angles measured (3o°-135 °) somewhat uncertainS, 9. Inasmuch as all the samples of poly-A studied were obviously polydisperse from their sedimentation patterns at low concentrations, the question of the reliability of the extrapolations merited detailed consideration. K

c RO

i p~l Mw

m a y be written in logarithmic form (logK +logMw) +log

"~0

+logPo

= o

Thus for a particular system log (c/Ro) as a function of angle 0 differs from log Po only by a constant additive term. Inasmuch as for monodisperse systems of rod or coil-like macromolecules or for polydisperse systems of coils following a " r a n d o m " distribution 9, 4 n/R z\½ PO = [(x), where x --

' ~ '/

0

s i n - -2, a p l o t o f l o g

c

(~-0)

versus log sin 0/2 should differ from a plot of log Po versus log x for the appropriate model only by additive factors in the above cases and hence should be superimposable upon some portion of the latter by a translation parallel to the rectilinear axes. In the case of a " r a n d o m " distribution, (R~} ½ is replaced b y the corresponding Zaverage quantity. It was found that for all preparations of poly-A discussed here at ionic strengths c of o.oi or greater and p H ' s > 6.5 the plot of log vR~\Sc=o versus log sin 0/2 was

superimposable upon the corresponding plot of log Po versus log x for a single-peaked system of polydisperse coils of size distribution such that Z = I (in Zimm's notation) o r M z : M w : MN = 3 : 2 : I. This distribution, which is quite common for m a n y unfractionated polymers, predicts a linear relationship for K c/Ro as a function of sin 2 0/2. Figs. 3 and 4 show, respectively, the conventional Zimm plot for poly-A I I in o.5 M KC1 and the plot of Po -1 (at zero concentration), as a function of sin s 0/2. The latter was computed using the intercept obtained by linear extrapolation. As can be seen from Fig. 4, the angular variation of Po -1 fits weu the one expected for the above type of polydisperse system, which will henceforth be designated as a " r a n d o m " distribution, following the practice of DOTY et al. 9. If the distribution is indeed " r a n d o m " then the linear extrapolation of K c/R o presents no difficulty. However, we have not as yet considered the effects of stiffness of the coils which might result in appreciable deviation from Gaussian behavior. A molecular weight of 3" lOS, which is close to the average obtained for poly-A I I and poly-A V, corresponds to a degree of polymerization of 8,500. This corresponds to a completely extended length of 29,000 A, assuming for convenience a single helical model, or 14,ooo A for a double helix, taking the nucleotide separation as 3.4 AI°The observed length of 1,7oo A (2,IOO for the monodisperse model at an ionic strength of 0.5) is thus only 1/6 to 1/12 the m a x i m u m possible extension. TRELOAR has concluded from a study of aliphatic chains that deviations of the Re]erences p. 348.

(I957)

VOL. ~

341

POLYNUCLEOTIDES II

'

/

/

MONODISP.~RSE

o c

,5

.o

JO .05 !

2 . 0 3 " 0 ~

=

1.0

2[0

J

3.0

Fig. 3- Angular-concentration grid plot for preparation PAA II in O.olMphosphate, o.5 M KC1, p H 6.5.

t.O

.25

.50

.75

Fig. 4- Comparison of experimental and cornputed curves of p - x for PAA II under the conditions of Fig. 3.

distribution of endlengths from the Gaussian only become serious when the end-to-end length exceeds I/3 the contour length xl. It is thus quite likely that any effects of coil stiffness are unimportant here insofar as they affect the angular dependence of scattering. It appears, therefore, that the present system at high ionic strengths may be well approximated by a polydisperse system of Gaussian chains obeying a distribution close to the "random" one. Thus considerable confidence may be placed in the linear extrapolation in this case. Similar statements may be made for the other poly-A samples discussed in this paper, although the mean molecular sizes vary widely. The fight-scattering results would appear to indicate that at pH's > 6.5 and at ionic strengths greater than o.oi, the Gaussian model is probably adequate. However, the streaming birefringence results in water in the absence of added electrolyte seem to point to such high extensions that the particles must be approaching a rod-like character under these circumstances. Thus the maximum length of Ii,OOO A for poly-A V in water is well over 1/3 the maximum extension for both the single and double chain models and, indeed, approximates that quantity for the latter model. Fig. 5 and Table I summarize the effects of pH (in the neutral and weakly alkaline range) upon the molecular parameters of poly-A. At constant ionic strength no important change in the radius of gyration outside of experimental uncertainty occurs between pH's 6. 5 and 9.4. The substitution of Li + for K + resulted in a slight apparent expansion, which is probably not significant. A detectable change was, however, observed upon varying the ionic strength, a definite decrease in radius of gyration being obtained at higher ionic strengths. The shrinkage amounted to about 40% between ionic strengths of o.oI and 0.50. Re/erences p. 348.

342

R.F. STEINER, R. F. BEERS

VOL. 26

(I957)

.20

1250

.15

c

iooo

Ro .10

750

6 o I

i

i

.25

.50

.75

SIN z

500



2~5

J .50

Fig. 6. Curves of c/Re as a function of sin S 0/2 for preparation PA III in three different concentrations of CaCI2. The solvent is 0.5 M KC1 plus o.oI M tris, pH 8.2. The concentration of PAA is 0.337 g/l. O: No added Ca++; ~ : o.oo~4 M Ca++; Q : o.oo71%/Ca++.

Y

Fig. 5. Variation of < R ~ ¥' with ionic strength for P A I I . O: o.orM PO, plus KC1, pH 6.5-7-3; : o.oIM NaHCO s plus KCI, pH 9.4-8.0; • : o.oxM tris plus LiC1, pH 8.5. P o l y a d e n y h c acid thus displays to some extent the electrostatically induced expansion at low ionic strengths characteristic of polyelectrolytes, although the effect is less m a r k e d t h a n for some synthetic polyelectrolytes, such as polymethacrylic acid. I t is possible t h a t this m a y reflect the fact t h a t for this system all or most of the negative charges are adjacent to the valence angle, which limits the extent to which electrostatic stress m a y be reheved b y expansion.

Effect o~ calcium I t was found t h a t increasing amounts of calcium added to solutions of P A A at p H 8. 5 resulted in a m a r k e d alteration in light-scattering properties, as Figs. 6 and 7 show. The initial slope versus sin 2 /9/2 decreased markedly, corresponding to about a 40% drop in radius of gyration at o.oo7M Ca++. At about this molarity the intercept began to drop appreciably, and at higher Ca ++ concentrations a visible turbidity was produced. All measurements were carried out in the presence of o . s M KC1. Under these conditions the second virial coefficient was negligibly small, and an extrapolation versus concentration was unnecessary. All of the preceding refers to measurements made soon (within x5 min) after the addition of calcium. T h e y are consistent with, and suggest, a m a r k e d shrinkage of the poly-A coils upon binding of Ca ++, accompanied b y aggregation. It must also be pointed out t h a t the observance of the shrinkage effect depends upon the avoidance of a local excess of Ca ++ with concomitant aggregation which is only slowly reversible. In the above experiments o.I M CaCI~ was added in stepwise quantities not greater than 0.5 ml, with vigorous stirring.

Re/erences p. 348.

VOL. 26 (I957)

343

POLYNUCLEOTIDES II .20

c R$

750

.15

.tO

250 .05

I

.0025

I

!

.OO5tr MOLARITY Co

.O075 I

concentration of added CaCI s. T h e s o l v e n t is

o.5M KC1 plus o.oi M tris, pH 8 . 2 . 6 : PAA III, o.337 g/l; O : PAA II, o.io4 g/1.

I

.50 Sin 2 ~ + I0C

Fig. 7" Variation of ( R ; ) ~4 computed on t h e basis of a "random" size distribution, with

1.0

Fig. 8. Angular-concentration grid plot for PAA at t w o pH's. O : O.OlM phosphate, pH 6.8; • : o . o 2 M glycine, pH 3.I.

Effect ol acid pH Figs. 8 and 9 and Table II summarize the effect of acid pH's upon poly-A and poly-C. To minimize aggregation, which became very pronounced at higher ionic strengths, measurements below pH 5 were carried out in o.o2M glycine buffer and at initial concentrations > o.I g/I, aggregation having been found to be minimal in this buffer. A decrease in pH from neutrality to 5 or below resulted in a very pronounced decrease in the angular dependence of scattered intensity and hence in a marked shrinkage Z4s/lZ$

RgQ° .700

f.75

.600 1.50

.500 1.25

3.0

.400

I

I

I

I

4.0

5.0

6.0

7.O

x I0 ~

pH

Fig. 9- Dependence of dissymmetry (Zull~) and Roo* upon pH for poly-A VII in o . o i M NaAc. ® g~ll. A R~.

Relevences p. 348.

344

R. F. STEINER, R. F. BEERS

VOL. 26 (1957)

TABLE II EFFECT OF ACID pH UPON POLYADENYLICAND POLYCYTIDYLICACID Preparation

Solvent

pH

\/ "0 g2 \/V 2

Mw × zo-*

Poly-A V*

o.o2M o.o2M o.o2 M o.o2 M o.oIM

glycine glycine glycine glycine PO~

3.I 3-I 3.5 4.2 6.8

521 471 537 Io5o I67O

5.6 :~ lO% 5.6 5.4 5-4 4.0

Poly-C I

o.oI M PO~ 0.02 M glycine

7.0 3. i

948 i IO

1.3 4.5

Poly-A VI

o. 5 M KCI, o.oI M tris

8-3

547

2.o

Poly-A VI

This sample was dissolved in o.ooiM KC1, plus o . o o z M KHzPO,, titrated to pH 2.5, and let stand 15 min. bIaHCO 8 was then added to o.oi M and KC1 to o.5M. The final pH was 7-4

594

2.0

9I 4 574 I87

I.i 1.5 1.5

Poly-A VII*

o.oiM PO 4 o.oi M NaAc o.oi M glycine

6. 7 5.0 3.5

* The concentration of poly-A at which the pH was altered to an acid value was 0.07 g/1. of the coils. The radius of g y r a t i o n fell to a b o u t I/3 of its value at n e u t r a l i t y at p H 3. The effect was somewhat complicated b y a n increase in molecular weight owing to aggregation which, was not, however, sufficient to m a s k the shrinkage effect u n d e r these conditions. The behavior of poly-A is thus in some ways analogous to t h a t of D N A in the acid TM. No change in the scattering with time over a period of four hours at 25 ° C was observed at p H 3. The molecule is thus considerably more stable w i t h respect to acid t h a n to alkali. The sensitive of the a p p a r e n t radius of g y r a t i o n to small degrees of aggregation makes it a n i n c o n v e n i e n t p a r a m e t e r to delineate the precise p H region i n which the change of molecular state occurs. The d i s s y m m e t r y coefficient Z45/135 a n d the reduced i n t e n s i t y at 0 ~ 9 °0 are less sensitive to aggregation a n d hence more suited to a "light-scattering t i t r a t i o n curve". I n Fig. 9 these p a r a m e t e r s are plotted as a function of p H for poly-A V I I in o . o x M NaAc, to which o . I M HC1 was added dropwise, with stirring. The surprising feature of Fig. 9 is the relatively high p H range at which the change in light-scattering properties occurs. The region of most rapid change was in the v i c i n i t y of p H 5-5, as compared with p H 3 for DNA. However, t i t r a t i o n studies u p o n poly-A indicate t h a t adenine is t i t r a t e d in this region*. The effect appeared to be reversible, w i t h i n e x p e r i m e n t a l u n c e r t a i n t y . A r e t u r n to n e u t r a l i t y , after exposure to p H ' s as low as 2.5, resulted in a regaining of the original molecular properties as Table I I shows. Polycytidylic acid also showed an even more m a r k e d collapse at acid p H accompanied b y considerable aggregation. * Subsequent work has considerably clarified the nature of the structural change occurring at acid pH's 1~. R e # f e n c e s p. 348.

VOL. 9.6 (I957)

POLYNUCLEOTIDESII

345

Thermal degradation o/poly-A At sufficiently high temperatures poly-A was found to undergo a rapid degradation at slightly alkaline pH. A single run was made at ioo ° C and pH 8.3, solutions being cooled to room temperature prior to measurement. As Fig. IO shows, there was no sign of any initial lag period. The variation of (R~/2)~/M as a function of time has been included in Fig. IO. The latter parameter appears to remain constant in the early stages of the reaction, while undergoing a marked rise in the later stages. The initial constancy of this quantity would appear to exclude any irreversible thermally induced drastic change in shape or configuration under these conditions. The later rise probably reflects the presence of aggregates to w h i c h / R ~ ) is very sensitive. I.oa

-

(R~)

.75

XZM

!

.

_

25

,o.o 3.0

~ I tO00

I 2000

XlO'l 3000

4000

TIME

Fig. IO. Variation in relative molecular weight and i n / R z\t2= M with time for t h e r m a l hydrolysis k g/'" at ioo ° C for P A V in o . 5 M KC1 plus O.OlM tris, p H 8. 3. O : ratio of mol. ,art. to initial mol. wt.; • : \,/R2\I,~ ~/~ ~ M •

Comparison o/polyadenylic and polyinosinic acids Inasmuch as the monomers of polyadenylic and polyinosinic (PIA) acids differ only in the replacement of the 6-amino group of the latter by a hydroxyl group, it is of considerable interest to compare their physical properties. As the solubility of poly-I is quite low at neutral pH's in the presence of salts, all measurements were carried out at pH 9.5 in carbonate buffer. As the pK of the 6-hydroxyl group is in the range 8-9, most of the latter will be ionized under these conditions. Fig. I I shows a typical angular-concentration grid plot for the single sample of poly-I studied. The curvature is here very marked. It proved impossible to superimpose the log c/Ro versus log sin 8/2 plot upon the theoretical curve for a random distribution. Hence, it is probable that the polydispersity is greater than for a random distribution. Under these circumstances the extrapolation of the Zimm type plots to zero angle is somewhat uncertain, and the values of molecular weight and radii of gyration should be regarded as only approximate. However, the agreement in the apparent Re/erences p. 348.

346

R.F. STEI~ER, R.

F.

.20

BEERS

tO00

.i5

VOL. 26 (1957)

O

(%),& t

c__ R0

JO

,.500

.05

_I

.500

LO00 SIN z

t.500

I

.05

I

.10

* IOc

Fig. II. Angular-concentration grid for PIA-I in o.olM COy, o.o2M KC1, pH 9.3.

Fig. 12. Variation in ~-~(,R~)½ with ionic strength for PIA-I. The buffer is o.o~M CO3- plus KC1 to t h e indicated ionic s t r e n g t h at p H 9.3-

molecular weight, which was 1.8. Io 6 (--~- 10%) for a considerable range of incident slopes, would appear to make it unlikely that any very marked errors have been introduced. Subject to the above reservations, the apparent radii of gyration have been evaluated and plotted in Fig. 12. For the sake of consistency, they have been computed on the "random" model to facilitate comparison with the corresponding data for poly-A. The most salient feature of Fig. 12 is that the dependence of radius of gyration upon ionic strength is more marked than for poly-A, a nearly 2: i change being observed between F = 0.03 and F = 0.i.

The e]ect o/]ormaldehyde upon poly-A FRAENKEL-CONRAThas observed a reaction occurring at neutral pH between formaldehyde and nucleotides containing a free amino group TM. The reaction presumably is of the Schiff type resulting in formation of a - N = CH 2 group and m a y be followed spectrophotometrically. After completion of the reaction, which requires about 48 hours at room temperature, a shift in the maximum position of about 5 mF to longer wavelengths and an increase in the extinction at the maximum of about 20% occurred. No reaction was observed for DNA, the inference being that the 6-amino groups of adenine in DNA are not available for reaction, being blocked as a result either of strong hydrogen bonding or inaccessibility from steric causes. The effect of formaldehyde upon the U.V. extinction of poly-A is summarized

.Re/erences p. 348.

v o L 26 (1957)

347

POLYNUCLEOTIDES II J

in Table III. Reaction appeared to occur about the same extent with poly-A as with adenylic acid. The maximum was shifted from 257 mt~ to 262 m~. No reaction was observed with DNA from the same bacteria or with IMP. It would thus appear that most of the 6-amino groups of poly-A are available for reaction with formaldehyde at pH 7. TABLE I I I Subsl,a~e

pH

A~.max

Po~-A AMP DNA IMP

7.2 7 .2 7.2 7.2

+5 +5 o o

Aem(%) 26 20 o o

U.V. e x t i n c t i o n r e a d i n g s w e r e m a d e after 4 8 h in o.oi M p h o s p h a t e a n d 1.2 .% H C H O .

DISCUSSION

All of the data thus far presented are consistent with a representation of these preparations of polyoA and poly-I as polydisperse coils at ionic strengths > o.oi and pH's > 6.5. Although the degree of coiling apparently is sufficient to render valid the assumption of a Gaussian distribution of end-to-end separations, the degree of extension is still considerable and these polymers must be regarded as rather stiff coils even at high ionic strengths. Any involvement of most of the 6-amino groups in hydrogen bonding at alkaline pH's must be such as not to affect their accessibility to reaction with formaldehyde. In any event, any hydrogen bonding in which they are involved is probably not analogous to the DNA case. The marked structural change at acid pH's is not inconsistent with a hydrogen bonded structure involving amino groups, but it may well prove unnecessary to invoke anything more than the weakening of electrostatic stress caused by titration of 6-amino groups and consequent neutralization to account for it. A similar statement might be made about the shrinkage in the presence of calcium. Another fact which deserves consideration is the increased extensibility at low ionic strengths of polyinosinic acid as compared with polyadenylic. However, at the pH at which measurements were made (9.5) the 6-hydroxyl groups should be largely ionized. As these charges are located on the purine rings and are well separated from the phosphate primary bonds, it is to be expected that the resultant electrostatic stress could be more readily relieved by expansion of the polyion~at low ionic strengths. Whether hydrogen bonding has any part in stabilizing the structure at alkaline pH remains somewhat obscure. In view of the constancy of in the early stages of thermaJ hydrolysis, it would appear that any such bonds present are either very stable or else are readily reformed upon cooling. Perhaps the evidence most suggestive of an ordered structure at acid pH is the, rather sharp character of the drop in Z~5/ 1~ with pH. This is reminiscent of the DNA case and suggests something of the nature of a disruption of an ordered structure upon going from acid to alkaline pH.

~R~/M

Re/evences p. 348.

348

R . F . STEINER, R. F. BEERS

VOL. 2 6 (1957)

SUMMARY I. Three different t y p e s of enzymicMly produced polynucleotides h a v e been examined by light scattering, s e d i m e n t a t i o n , a n d s t r e a m i n g birefringence, a n d h a v e been s h o w n to have the configuration, in a q u e o u s solution at ionic s t r e n g t h > o.oi, of r a t h e r stiff coils, coiled to ~/6 or less of their m a x i m u m possible extension. 2. The coils are m o d e r a t e l y flexible and m a y be contracted m a r k e d l y b y increasing the ionic strength, decreasing the p H below 6, or adding calcium. 3. Most of the 6-amino g r o u p s of poly-A are available for reaction with formaldehyde at p H 7. 4. T h e r m a l degradation at neutral p H proceeds w i t h o u t an initial fall in /R~>/M upon cooling. 5. Poly-I a p p e a r s to show a more p r o n o u n c e d dependence of extension upon ionic s t r e n g t h t h a n poly-A. 6. A p r o n o u n c e d s t r u c t u r a l change occurs for poly-A in the vicinity of p H 6.

REFERENCES 1 M. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Biochim. Biophys. Acta, 20 (1956) 269. 2a R. F. BEERS, Jr., Nature, I77 (1956) 790. 2b R. F. BEERS, Jr., Biochem. J , 66 (1957) 686. 3 j . D. WATSON AND F. H. C. CRICK, Nature, 171 (1953) 737. 4 K. A. STACEY, Light Scattering in Physical Chemistry, Academic Press Inc., New York, 1956. 5 p. DOT'/ AND R. F. STEINER, J. Chem. Phys., 18 (195 o) 1211. * H. A. SCHERAGA, J. T. EDSALL AND J. O. GADD, Jr., J. Chem. Phys., 19 (1951) ilOt. 7 B. H. ZIMM, J. Chem. Phys., I (1948 ) lO93, lO99. H. BENOIT, J. Polymer Sci., i i (1953) 507. 9 A. M. HOLTZER, H. BENOIT AND P. DOTY, J. Phys. Chem., 58 (1954) 62410 M. E. REICHMANN, S. A. RICE, C. A. THOMAS AND P. DOTY, J. Am. Chem. Soc., 76 (I954) 3047 . 11 L. R. G. TRELOAR, Proc. Phys. Soc. (London), 55 (1943) 345. 12 M. E. REICHMANN, B. H. BUNCE AND P. DOTY, J. Polymer Sci., 1o (1953) lO9. 13 A. CHARLESBY, Proc. Roy. Soc. (London), ,4, 224 (1954) 12o. 14 C. A. THOMAS, Jr., J. Am. Chem. Soc., 78 (1956) i86i. 15 C. A. THOMAS, Jr. ANn P. DOTY, J. A m . Chem. Soc., 78 (1956) 1854. 16 H. FRAENKEL-CONRAT, Biochim. Biophys. Acta, 15 (1954) 307 • 17 R. F. STEINER AND R. F. BEERS, J. Polymer Sci., in the press. R e c e i v e d M a y 6 t h , 1957

APPENDIX DEFINITION

O

OF

SYMBOLS

USED

IN

TEXT

= angle b e t w e e n direction of view and incident b e a m

GO,corr = g a l v a n o m e t e r deflection a t angle O, corrected for reflectance Ge,obs c

RO Mw

PO K no

dn/dc ;t

Re, Rg2 V~ u3 ]

= = = = =

uncorrected g a l v a n o m e t e r deflection weight c o n c e n t r a t i o n reduced intensity at angle 0 w e i g h t average molecular weight factor b y w h i c h scattering is reduced at angle B, owing to internal interference

= 2 ~'~n~ Idn/dc)'~/2 4 N = = = = = = =

refractive index of solvent refractive i n c r e m e n t of p o l y m e r wavelength radius of g y r a t i o n r o o t m e a n s q u a r e r a d i u s of g y r a t i o n Z-average degree of p o l y m e r i z a t i o n n u m b e r of chain elements b e t w e e n interchain cross-links.