Fluorescence of nucleic acid — Terbium(III) complexes

Vol. 53, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FLUORESCENCE OF NUCLEIC ACID - TERBIUM(II1)

Carl

COMPLEXES

Formosa*

Division of Biology University of Texas at Dallas Dallas, Texas 75230 Received

June

29,1973

Summary Terbium(II1) binds to nucleic acids and acts as an acceptor of electronic excitation energy. The transf3$ app ears to be primarily from guanosine residues. Fluorescence from Tb bound to 5'-GMP3i+s very much orescence from either free 5'-GMP or free Tb When boun a ::;Tfsan;k shows an intense excitation band at 3Ji14 nm, where free Tb has no exlitation band. Fluorescence from bound Tb appears to be potentially useful in nucleic acid studies. Introduction At room temperature weakly used

fluorescent

(1,2).

in nucleic

used at low

in neutral

acid

studies.

(3,4),

and synthetic

from

observed

Tb 3+ bound

Materials

corrected

work

communication

acids

is

also

acids

fluorescence

fluorescent

fluorescence

bases

fluorescent

if

may introduce

enhanced This

to nucleic

of nucleic

and synthetic

natural

derivatives

(5,6).

derivatives structural

from Tb

reports

that

3+

are

only

extensively has been derivatives could

be

are

rare,

abnormalities. bound

enhanced

to the protein fluorescence

observed.

and Methods

Fluorescence pathlength

fluorescence

acid

has not been

be more appropriate

temperatures;

protein

the nucleic

fluorescence

and natural

would

fluorescent

In recent

Nhile

(2), it

used at physiological

has been

Consequently,

temperatures

have been used

solution

cuvette for

spectra

were

with

a Hitachi

instrument

obtained

at room temperature

Per-kin-Elmer

characteristics.

*Present address: Department Arizona 85721

All

of Chemistry,

Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

Model

1084

solutions

University

using

MPF-ZA,

a 3 mm

and were

contained of Arizona,

not

pH 5.6 Tucson,

BIOCHEMICAL

vol. 53, No. 4,1973

0

I

’

450

RESEARCH COMMUNICATIONS

\

I

500

AND BIOPHYSICAL

550

600

X,nm

Figure

1.

Emission

spectrum

Figure

2.

Excitation 0.008

0.01

spectra

of 10 -4 M Tb 3+ in buffer

(lower

curve)

and in

M 5'-AX-'.

M NaOAc, 0.10

Calbiochem,

of 5'-GMP:Tb3+.

M NaCl.

Sigma,

Nucleic

or Schwarz.

acid

materials

TbC13 solutions

were were

obtained

from

contributed

by Dr.

A. D. Sherry. Results

and Discussion

Figure Neither

1 shows

5'-GMP

under

the

nor Tb3'

the conditions

of emission studied

(with

here

published of the

emission in

used.

Other in

spectra peak

each of the ribonucleotides the most

base

in tRNAPhe,

the excitation found

for

yeast

While

tRNAPhe

is

characteristics 3-k

.

run range)

transfer yeast

- Tb

RNA does

(3)

acid

1085

3+

from

3+

all

the complexes

3+

fluorescence

intensity

and shows

, transfer

from

the fluorescent

in mixed quite

to previously

the relative

complexes,

contain

are

characteristics

, and similar

to Tb

component

of tRNA phe

fluorescence

1 gives

energy

minor

The nucleic

Tb

Table acid

complex.

the spectral

free

(5,6,7). nucleic

3+

5'-GMP:Tb

intensity,

seen for

a very

the

show significant

the 260-300

will

efficient.

RNA-Tb

for

for

state than

to those

emission

545 nm emission

being

the free

excitation

are similar

Tb3+

spectrum

RNA.

different

that 5'-GMP Y

In addition, from

those

at 545 nm wae negli-

BIOCHEMICAL

Vol. 53, No. 4,1973

Table

1.

Relative

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

at 545 nm in 10w4M Tb +3

Emission

Compound

Concentration

Excitation Maximum

Fluorescence Intensity

5'-GMP

4.10-5M

290 nm

1.0

7*10-5

285

0.7

5'-IJMP

5*1o-5

268

6.10-*

5'-CMP

7*10-5

280

3.1o-2

5'-AMP

4.10-5

s270

2.10

G

6-1O-5

%290

2.10-3

yeast

gible

RNA

for

tion

each of the

was necessary

each from

complexes

for

the

Preliminary

quantitative

of the

nucleotides

four

3+ is

5'-GMP:Tb

of the bases,

the

given

fluorescence work is

1, and except

indicates

for

that

the

10 3 M-1 .

association

Thus

the

no correc-

structures

of the

constant

fact

can be due to the different

different

G,

Tb 3+ .

of free

around

largest

to possibly

in Table

-2

that

fluorescence

electronic

complexes,

for

properties

or to both

causes. The results that

binding

While

the excitation

Tb3+ fer cation

is

through

be occurring escence than

that

in

from

the

is

also

are ruled

Tb

3+

terbium

the nucleic

acid

of 5'-GMP

alone

important

out,

the

In the

but

in view

is

small,

would

work

of

that

trans-

on nucleotide-

be suprising. energy

(7).

transfer

This

The intensity

two orders

max at 330 nm) so that

1086

a change

concentrations

not

of the donor

about

quite

of past

- Tb 3+ complexes. is

of excitation

case of G, collisional

has indicated level

indicate

transfer

and at higher

to the base

3+ complex (emission

in

no phosphate,

enhancement

larger.

complexes triplet

contains

observed;

binding

an excited

5'-GMP:Tb

which

fluorescence is

effects

(a),

Work on other Tb3+

the

be completely

complexes

(G),

itself

spectrum

and of G these cannot

guanosine

to the base

to Tb 3+ .

energy in

with

may also of fluor-

of magnitude it

to

is

likely

larger that

BIOCHEMICAL

vol. 53, No. 4,1973 if

transfer

is

It

is known

that

weak

from

level,

the phosphorescence

so that

(2),

a singlet

AND BIOPHYSICAL

the same argument

RESEARCH COMMUNICATIONS

the transfer

is

of the bases

at room temperature

should

hold

if

over

a very

transfer

is

short

from

distance. is

very

a triplet

level. Though Tb3+,

it

electronic

was found

perturbed

excitation

the electronic

to 5'-AMP.

concentrated

are

shown in Figure

interaction

between

further

5'-AMP

in

favor

- nucleic

and structural

acid studies;

other

interactions;

spectrum

with

the

solution,

free

Tb

This

3-b

states

of the base to the base

to be potentially it

for

Tb 3+ , monitored

a very band.

is

360 nm

large These

considerable

and those

of Tb 3+ , and

itself.

useful

may have

and may be useful

shows

there

to

are greatly

two in the

has no excitation that

much energy

of free

largest

however,

suggests

of binding

from Tb 3+ appears

Emission

with

2.

the electronic

evidence

peaks

transfer

of Tb 3+ itself

states

The excitation

peak at 414 nm, where

results

action

of 5 '-AMP does not

at 545 nm, shows several Tb 3+ in

region.

cation

that

on binding

by emission

is

excitation

applications

monitoring

in the study in nucleic

of

conformational acid

inter-

molecules.

Acknowledgements I am grateful to Dr. A. D. Sherry for discussions and for sharing his supply of terbium with me, and to Drs. Sherry and Cottam for a copy of their paper prior to publication. This work was aided by Grant AT-503 from the Robert A. Welch Foundation (to Dr. D. M. Gray). References 1. 2. 3. 4. 5. 6. 7. 8.

M. J. K. J. C. A. A. R.

Daniels and W. Hauswirth, Science 171, 675 (1971). Eisinger and R. G. Shulman, Science l&, 1311 (1968). Beardsley, T. Tao, and C. R. Cantor, Biochemistry 2, 3524 (1970). R. Bario and N. J. Leonard, J. Amer. Chem. Sot. 95, 1323 (1973). K. Luk, Biochemistry lo, 2838 (1971). D. Sherry and G. L. Cottam, Archives of Biochem. Biophys., in press. Heller and E. Wasserman, J. Chem. Phys. 42, 949 (1965). M. Izatt, J. J. Christensen, and J. H. Rytting, Chem. Rev. 2, 439 (1971).

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