Fluorescence quenching and conformational changes of proteins

Vol. 39, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FLUORESCENCE QUENCHING AND CONFORMATIONAL CHANGES OF PROTEINS

B. Arrio,

F. Rodier,

Laboratoire

C. Boisson

de Biologie

and C. Vernotte

Physico-Chimique

91 - ORSAY,France.

and Laboratoire

Received

March

de Photosynth&se

du CNRS, 91-GIF-SUR-YVETTE,France.

26, 1970

summary. A fluorescence quenching technique for conformational studies of proteins is described. The dynamic quenching of fluorescein by iodide ions is sensitive to the molecular weight and the charge of proteins, and is suitable for following the conformational change of trypsin in the presence of a substrate. Introduction.

When a quencher

dye,

of the fluorescence

is

the ratio described

by the relation F +

where is

is

the rate

the

Volmer the

constant

(see

quenching", the

If

isomerizes

a protein

may induce proteins,

we have

rescence and is (6)

quenching known

quenching

of the non fluorescent

excited

The product

dye.

fig.

1).

the

second

The first 'I the

of the

ions

of dyes in solution

term

dye-quencher

Stern-

of fluorescein

and Bowen (7). 589

represents

(see

bound

relation

has already

".

of a protein to the protein.

conformational

(FITC),

analyzed

quenching

changes the

(l),(2),(3).

has been

(1)

or collisional

isothiocyanate type

K

K T is the well-known

molecules, constants

complex, t is

of relation

dynamic

the quencher,

dye and the quencher,

of a dye covalently

quenching

dynamic

the

and with

conformational

small

fluorescein

by iodide

to be of the

the

quenching

by binding

chosen

quencher

(1)

without

between

fluorescence

variations

or without

K T (Q) )

by diffusion

was to follow

by measuring

cence

intensities

of the

Our intention

with

of a fluorescent

:

constant

(K)

intensities

fluorescence

of encounter

" static

to a solution

) . ( 1 t

equilibrium

the mean lifetime

added

= (1 + K'(Q)

F 0 and F are the

K’

is

(1)). because

change TO label the

fluo-

been studied,

The mechanism by Weller

of fluores(4)

Weber

(5),

Vol. 39, No. 4,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

pH=%4

t 1.03 -

0

Figure

First,

1

4

2

Stern-Volmer plot for trypsin-FITC quenched by iodide ions, (trypsin concentration 10-5 M). assuming that the quenching of bound FITC fluorescence by

iodide ions is also diffusional,

we have followed the variation

of the

Stern-Volmer constant with the molecular weight of various proteins. Second, with an enzyme, trypsin, we have shown differences between the Stern-Volmer constants when trypsin is in the presence of a low molecular weight ethyl ester (ATEE), and in the absence of this substrate : N acetyl-l-tyrosine Substrate.

Material

and methods. FITC was bound to glutathion,

lactoglobulin

B, ovalbumin, bovine serum albumin and trypsin,

given by Nairn (8). Each protein buffer

lysozyme, lactoglobulin

A,

using a method

was incubated in a 0.5 M carbonate bicarbonate

pH 8.2 with FITC at the sameconcentration

as the protein.

After

one

hour, the free FITC was eliminated by chromatography on Sephadex G 15 or G 25. For trypsin, O-2 N butylamine was added to the buffer to protect trypsin against autolysis. The number of bound FITC was determined spectrophotometrically, using extinction coefficients given by Tietze (9). Fluorescence intensity and polarization were measured with an apparatus built by the authors. Decay time was measured with a T R W flash. Results and discussion.

Table I (10) gives the Stern-Volmer constants of free

FITC and bound FITC. Except for trypsin, the Stern-Volmer constant decreases as the molecular weight increases. There is no simple relation between molecular weight and the Stern-Volmer constant, as one could expect if the rate of encounter between the FITC-protein

complex and the quencher was dependent 590

BIOCHEMICAL

Vol. 39, No. 4, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

I

M.W. FITC

400

Glutathion-FITC

900

Lysozyme-FITC

K(M-l) 20 5.8

17 000

5.0

Lactoglobulin

A-FITC

35 000

4.3

Lactoglobulin

B-FITC

35 000

4.3

40 000

3.9

Ovalbumin-FITC Bovine

Serum Albumin-FITC

Trypsin-FITC

on the molecular and positive hindrance

charges clear

trypsin

studied

the

results

indicate l/

a very

Decay times 0.5 ns

(pH

the

3/ Perrin's temperature

it;

steric

effect

charges.

with

trypsin,

and FITC.

is To justify

we have

The following

:

FITC and trypsin-FITC

in phosphate curves

are

the

same :

buffer,

p

0.1).

of free

FITC and trypsin-FITC

q

plot

for there

trypsin-FITC

by varying

are

two relaxation

at least

viscosity

at constant for

times

FITC (11).

2.

rotates

freely

charged

protein,

Similar

effects

Hodges

positive

: negative

.

We can visualize group

rate

The charge

obtained

trypsin

weak interaction

7.2

shows that

See fig.

has numerous of K

this

or increase

it.

which

titration

same pK 6.7

can lower

influence

between

of free

can modify

value

of interraction

2/ Fluorimetric give

may also

of the high

state

parameters

by the protein

shape

with

3.9 43.0

Other

carried

interpretation

4.5 ns +

only.

and molecular

particularly this

weight

70 000 23 500

(3)

of trypsin

the situation around

which

have been

and Holmstrijm FITC,

its

attracts

by assuming

covalent the

described

(23 % to 27 %) and the decrease

(12). the

About

increase

of lifetime

591

with

the

quencher

and Lamer the

fluorescent

the highly

charged

by Umberger

and Tegner

we have observed

bound

negatively

that

(11,

quenching

of fluorescence (4.5

ns to 3.4 ns)

positively molecules. Weller

(41,

mechanism polarization in the

Vol. 39, No. 4,197O

---I 6wp

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 3

t 1 zone Pi-k%24

5_/cc

g/---Q-

,+---&-

-_--

_ -----

---

--cl---

TRYI%+-FIK

I 5

1

Figure

I 15

I 10

I 20

I 25

I 35

I 30

1,10-' I 1 40

Perrin's plot for trypsin-FITC (concentration 10-6 M) in various buffer glycerol solutions at constant temperature (corrected curve).

2

piesence of the quencher (iodide lo-'M

at pH 7.2 and 25OC). This result is quenching. The plot of the reciprocal of the

in agreement with a diffusional polarization

versus the ratio

of the lifetime

without quencher to the

lifetime with quencher has not been represented because we have not precise -6 measurementsin the range of 10 M to 10m5Miodide. The quenching of trypsin kind,

is therefore

suitable

FITC by iodide ions, being of the diffusional

for following

conformational

changes induced by

substrates.

. +ATEE 0 -

ATEE

K

b

Figure

3

b

8

7

9

0

PH

Stern-Volmer constants for trypsin-FITC 10-G M), ATEE 7.10-3 M, 25OC. 592

(concentration

BIOCHEMICAL

Vol. 39, No. 4, 1970

In figure with

or without

free

and bound

(3)

are represented

ATEE. FITC.

We have ethyl

same. The esterasic that

change

encounter

Conclusion

.

changes

of proteins.

fluorescence

quenching

cules.

Our results

lation

at low

of small active

encounter

show that

concentration

molecules site between

The binding

nearly

the

same. We a conforma-

by binding

another sites

to distinguish and that

bound

of

ATEE.

on the conformational

on the binding

because

The

FITC can be affected

modified,

has described

5.10-')

the

follows

around

information

is possible q

FITC is

constant

charges

(13)

(KM ATEE

can be observed of trypsin.

are

and without

on

FITC.

constants

low,

pii

BAEE

the

information it

on free

is

quenching

Winkler

to get

like

effect

activity

can give

Recently

of trypsin esterasic

and fluorescein

Such experiments

effect

the

repartition

iodide

at various

follows

on ATEE with : the

between

substrates

of the

constants

ATEE has no quenching

have a quenching

hydrolysis

variation

Stern-Volmer

that

constant

activity the

of trypsin

and the

the

ester)

and at the pH where

can conclude tional

verified

of the Stern-Volmer

trypsin

the

The more specific

(N benzoyl-1-arginine variation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

technique of small

a molecular long

range

molepopu-

effects

FITC has no interference

of ATEE causes

of

the modification

with of the

FITC and iodide.

Acknowledgments : the authors are greatly J.LAVOREL for the many helpful discussions during acknowledge Professor J. TONNELAT for suggestions the manuscript.

indebted to Professor this work. We gratefully and for the preparation

of

References. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Umberger J.Q. and Lamer V.K., J. Amer. Chem. Sot. 2, 1099 (1945). Williamson B. and Lamer V.K., J. Amer. Chem.Soc. 2, 717 (1948). Hodges K.C. and Lamer V.K., J. Amer. Chem. Sot. z, 722 (1948). Weller A., Discussions of the Faraday Society w Energy transfer with special reference to biological systems It, 21, 28 (1959). Weber G., Trans Faraday Sot. E, 185 (1948). Weber G., Biochem. J. 47, 114 (1956). Bowen E.J., Barnes A.W. and Holliday P., Trans. Faraday Sot. 5, 27 (1947) Nairn R.C. "Fluorescent protein tracing " Ed. E. and F. Livingstone Ltd Edinburg London (1954). Tietze F., Mortimore G.E. and Lomax N.R., Biochim. Biophys. Acta 2, 336 (1962). Vernotte C., C.R.Acad.Sc. Paris 264 D , 1892 (1967). Wahl P. and Weber G., J. Mol. Sic%, 371 (1967). Holstrijm B. and Tegner L., Photochem. Photobiol. z> 207 (1966). Winkler M.H., Biochemistry 8, 2586 (1969).

593