Vol. 63, No. 4, 1975

Estimation in acid

V.

BIOCHEMICAL

of Poly aqueous

A Secondary

H.

fijr

Structure

RESEARCH COMMUNICATIONS

from

Raman

Scattering

solution.

Gramlich,

Institut

AND BIOPHYSICAL

Klump

and E.D.

Physikalische

Schmid

Chemie

der

Universitgt

Freiburg

i. E3r . ,

Germany Received

February

10,

1975

Summary In the

frequency

region

acidic

aqueous

solutions

resolved

lines.

demonstrate of 5.73 The

of these

Raman

and 5.35

to order-disorder

(rA)

lines

that

the

Raman

consist

spectra

of several

at 725,1303,1336

hypochromic

as a function

suggest

-1

cm

of poly

Four the

results

600-1600

effect

of well-

and

of poly

(rA)

1506

cm

-1

at pH-values

of the temperature.

Raman

transitions

intensity

measurements

of aqueous

polynucleotides

are

sensitive

.

Introduction Physico-chemical (poly

(rA)

) have

and it has to those of the

studies

been

been

shown

acidic

reversible

form

spectroscopy

structural Raman were

and Thomas

published

bands

exhibits 4 acids . The

followed a , who

have

model.

The

This

they

observed.

by Small

and

Copyright o 19 75 by Academic Press, Inc. AN rights of reproduction in any form reserved.

able

been

been

published

sufficiently

shortly were

has

spectra

about

frequencies

of the

molecular

properties in terms

to make most

Peticolas5. 906

can

of the commonly occuring 596 . Laser Raman

poly(rA),

particularly

were

first

rather

potentially in aqueous

reported

comprehensive

careful

a

by several

to yield

some

of a

undergo

investigated

sensitive

bases

by a more

The

similar

interpreted

Raman

information

properties

polynucleotide

that

recently

become

been

acid l-3

laboratories

(rA)

transition

have has

solutions.

poly

of poly(rA)

methods.

of polyriboadenylic

out in many

nucleic

helical

polynucleotides

of the

that

helix-coil

spectroscopic

These

carried

of undenatured

double-stranded

valuable

of solutions

by Malt7.

study general

investigation

by Lord assignments has

been

BIOCHEMICAL

Vol. 63, No. 4,1975

In

agreement

and we

with

1508

cm

would

like

increases

to

bands

assignments

the

poly the with

that

interpreted

in

structure

of

of

is

terms

of (rA)

an in

increase is

at

260

extremly

of

compared

nm

as

similar

725,

which

the

intensity

with

the of

tt-e

of

the

increase temperature.

which of

1303

Furthermore,

a function

transition

aqueous

at

behaviour

orderilisorder acid

bands vibrations. -1 band cm

1336

temperature

an

ring

the The

intensities

there

poly

of

RESEARCH COMMUNICATIONS

chosen

as

intensity

a function

have

spectrum

temperature.

absorption

conclude

, we

(rA)

discuss

as

ultraviolet

We

in

markedly

Raman of

their

-1

AND BIOPHYSICAL

can

be

secondary

solution.

Experimental

Materials

Poly

(rA)

and

potassium

used

without of

deionized Na

water +

by

solution.

2%

(

and

acetic

w / w

),

To

acid

used

M.

Na

final

first

in

were pH

added

until

values

dropwise +

polymer

dissolved

desired

acid a

Mannheim,

acetate

The

acetic maintain

the

was

sodium

0.1

to

Boehringer,

obtatn

(rA)

and

concentrated

was

from

poly

reached

adding

NaCl

purchased

purification.

concentration

obtained

was

further

concentration

the

salt

to

concentration

were

the

polymer

of

0.15

M

in

all

solutions.

Measurements Raman

spectra

were

recorded

on

a

Coderg

PHO

Raman 9 .

0

equipped The

with

4880

were holder

% -exciting

recorded was

junction

comparing plotted poly(rA)

at

least

of

.

Model

53

line

yielded

twice.

controlled 10

Peticolas

725,

a CRL

The

similar

The

to

temperature

1336

and

them

to

against band

into

the at

1508 their

cm

laser

about

1 .5

Watt

the cell.

11

were

was peak

The

band

by

as

reference

of

the

cm lines.

and a

bands

at

by

heights 925

Small placing

normalized

at

spectra cell

by

heights and

All

thermostated

normalized

acetate

907

the

sample.

measured

measured

used

the

described

The

15OC. The

-1

of

probe

were at

at

arrangement

the

height

cm

ion

the

-1

temperature.

1100

argon

temperature

of

a thermocouple

1303,

A

Spectrometer

were -1

and

the

BIOCHEMICAL

Vol. 63, No. 4,1975

pH to

values

were

0.01

pH

standard

and

Figure (rA)

in

aoueous

(rA)

at have

growth in

created as

the

the

is

due

to

by

independent

the

the

that

the

meter

with

lines

helix

3 the has double

same

shifted helix

of

the

of intensities

however,

that at

1336

The

1508

cm

-1

.

was 5

this

the

37OC.

and

Peticolas

.

These

authors

experimental

band

effect

pronounced,

result

effect”

more

plotted

25OC

the

In

temperature

coil

in

was

the

against

only

slightly

40°C

range

of

the

of

of

stacking

decreases

plot

is

a pH

of

corresponding

(rA) as

at

the

pH of

908

the

This

increase

was

shown

is

increase

partly

is due

ordered

single-

temperature.

where to

lower

a function

to

spectroscopy

increasing

5.35

the

transition

stabilization

a increased

value. temperature

rather

25OC

intensities

This

in the with

as

the

as

are

ultraviolet

in

bases

the

at

40-55OC,

as

solutions.

(rA)

from

polymer

increase

unstacking

shown

the

normalized

bands

temperature.

such

poly

all

of

with

transition

the

the temperature

intensities

methods

poly

solution

hypochromic -1 1336 cm

neutral

to

aoueous

and as

temperature

and

of

26OC

spectrum

the

distinctly

. Above

degree

an

1303, of

were

Below

in

freouencies

same

spectra

temperature.

increase

found

the

for

of

2.

at

the

725,

Small

“hyper-or

four

noncooperative

Figure

the

at

by

recorded

see,

in

physico-chemical

In

behaviour

the

3,12

to

pH

performed

Raman

clearly

a function

the

double

The

of

can

are as

intensities

strands.

interval

One

temperature.

calorimetry

similar

was

Kopenhagen)

two

for

increased

term

Figure to

at

Peticolas

.

intensity

of in

5.73 are

Ramanhypochromic

heights

40°C,

pH

increasing

independent

to

the

. The

is shown

26,

meter

Ramanhypochromism

to

To

and

solution

term

by

peak

Small

intensity

a paralell

make

pH

of

pH

here

increases

the

affected

at

shown

neutral

UV-spectra

solution

markedly

of

observed

stripcharts

neutral

pronounced

The

the

original

bands by

intensities

or

two

reported

most

of

(type

solutions.

The

poly

a Radiometer

RESEARCH COMMUNICATIONS

Discussion

37,5OC.

those

with

Calibration

1 shows

poly

of

units.

buffer

Results

at

measured

AND MOPHYSICAL

The

general is

similar

to

Vol. 63, No. 4,1975

BIOCHEMICAL

1600

1100

Raman 37,5OC,

Fig.1

Fig.

that

2

cm-l

in Figure

increase

I

I

I

15

30

50

(rA)

increase

of the

1

70

T°C

(w/w)

at

of the bands at 725, 5.73 vs .temperature

2. The

of cooperativity

RESEARCH COMMUNICATIONS

600

spectra of 2 % poly pH = 5.73

Plot of the intensity in oly (rA) at pH 8 15 C)

shown

AND BIOPHYSICAL

is even

bases.

This

26OC

1303, (ratio

and

1336 and 1508 cm to intensity at

more

distinct,

result

agrees

due to an well

with

W-measurements. One can see relative This

changes is shown

dependence at three

all

to 70°C of the

2 and 3 that

of the more

intensities

clearly

of two selected different

We assume for

in Figures

that

pH values in this lines

one can find

(725

at 70°C figure. cm

-1

a similarity,

lines

in the same 4 where -1 at 725 cm

show

different

temperature

interval.

in Figure

the temperature

lines

and

pH values, the degree

different

pH 7.0,

pH 5.73

of structural

in detail,

and 1336

cm -l)

viz .large

of the

we normalized

Though,

the variations for

intensity

909

cm

-1

is plotted

and pH 5.35.

ordering

and therefore

1336

different increases

polymer the

peak

is identical heights

in intensities pH ‘s , differ occur

a lot in the same

Vol. 63, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-

7.0

---

5.73

-.-.-

5.35

I

PH

--3 .O

1.0 I

r,15oe

--2 .O

Ir,T336

.5

0

1.

I 15

I

I

1

30

50

70

T°C

Fig.

3

Plot

of

2

Fig.

4

The

of

intensity

stretching

mode pH

values

dependence. has

a much

and

In

of

the

of

larger

,

single

contrast

cm

is

nearly

5.73

and

this

we

in

the

molecule

the bands 5.35 vs.

acid

pH-values

-1

line,

5.35, conclude

single has

is

1303, (ratio

line

different

shows the

double

strand.

J 70

1336 and to intensity

1508

cm

-1 1336 cm in poly (ratio to intensity

(rA)

predominantly

independent

that

on

725,

at 725 and temperature

which

this

I 50

at

coil

This

7,O.

However,

temperature

of

this

transition

suggests

relative

pH

a strong

intensity

helix

a C5-N7

that

the

conformations

line

(1336

than

on

the

C5-N7 in

cm

bond

double

helix

of

line

strand.

to

the

behaviour

of

the

1336

-1

.

temperature

dependence

ordering

adenine

the

1336

From

degree in

the 13

for

of and

I 30 T OC

of the bands at 5.35 vs .temperature

Plot of the intensity at pH ‘s 7.0, 5.73 at 70° C)

intervals

the

intensity rA at pH )

ypc;

temperature

at

the

I 15

1

0

-.-.-.-.-.-.-.-*

cm

910

-1

line,

the

intensity

the

-1

)

BIOCHEMICAL

Vol. 63, No. 4,1975

at

725

that

-1

cm the

is

intensity

strand

as

These

investigations

useful

tool

The

the

well

as

dependent

of

this

line

in

the

double

show

for

authors

H .W.

temperature

studying

the

for

at

depends

on

once

more,

the

the

three

pH

values.

degree

of

ordering

that

Raman

changes

Deutsche

reading

all

RESEARCH COMMUNICATIONS

This

means

in

the

single

helix.

conformational

thank

Wilson

AND BIOPHYSICAL

spectroscopy in

nucleic

and

for Mrs

be

acids.

Forschungsgemeinschaft manuscript

may

financial

.I .Ziegert

for

support,

drawing

figures.

References 1.

J .R.

Fresco

and

P .Doty,

2.

DN.Holcomb

3.

H .Klump

, T .Ackermann

4.

P.

B.

5.

E.W.Small

6.

N .N

7.

R.A.Malt,

8.

R.C.

9.

E.D.Schmid, Phys.Chem.

and

Doty,

I.Tinoco

and

and

L.

1o .

E . W.

Small

11 .

G. J . Thomas, 324 (1973)

12.

M.J.Massoulie,

13.

M .Tsuboi,

and

S . Rice,

and

Chen

120

Jr.,

Biopolymers

7 (1969)

69

(1970)

590

461

Spectrochim

H . Berthold,

(1971) 3

(1966)

423 (1958)432

10

.Acta B.Brosa,

and

, Biopolymers

23A Ber

K .A.

Hartman

260

(1965)

10 ,Biochim

37 Compt.Rend private

121

(1967)

2551

.Bunsenges.

149

W . L . Peticolas M . C.

(1965)

Macromolecules

Thomas

3928

Proc.Nat.Acad.Sci.US.44

.Acta

G.Berthold, 75 (1971)

(1957) 3

Biopolymers

J. Koenig,

G.J.

.79

Biopolymers

W.L.Peticolas, and

.Soc

E , Neumann,

Biochim.Biophys Lord

.Chem

Jr., and

I3 .McGill

.Aylward

J .Am

communication

911

5554

(1971)

1377

. Biophys

.Acta