Changes in the raman spectrum of frog sciatic nerve during action potential propagation

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CHANGES IN THE RAMAN SPECTRUM OF FROG SCIATIC NERVE DURING ACTION

POTENTIAL

PROPAGATION

B. Szalontai, Cs. Bagyinka, and L.I. Horvath Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences H-6701 Szeged, P.O.B. 521, Hungary Received

March

14,1977

Summary: Raman spectra of frog sciatic nerves were recorded in different states of functioning. During excitation reversible changes were observed in the Cqo-carotenoid peaks enhanced by the resonance Raman effect. This change can be explained by transient carbon-carbon bond equalization of the polyene chain. Possible biological consequences are also discussed. Introduction:

Several

biomembranes.

The carbon

of

9-11

conjugated

by the

in the modes

resonance

The resonance

to

being

membrane

environment ough it

Copyright All rights

(at chain

using

rise

consis to an intense

476 nm) and, are

thus

strongly

blue

of

the

enhanced

excitation

(l-6).

of C40-carotenoids are dominated -1 -1 1525 cm and 1158 cm which were

it

since the

very of

was suggested

(i)

minute

resonance

were done that

membrane

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

amounts

to local

carotenoids

yield

situ

(5).

re-

well-resolved

and (ii>

changes

molecules

respec-

as in

enhancement,

carotenoid

investigations

that

can be utilized

sensitive

the

vibrations,

was suggested

constituents

through is

give

and v<=C-C=)+q(CH>

Recently

molecules

hancement

carbon

at

v(-C=C-)

(3-5).

spectra

which

region

Raman effect

bands

tively

porter

of the

constituents

of C40-carotenoids

bonds

blue

natural

Raman spectra

by two intense assigned

are

skeleton

double

'I~+-+v transition vibrational

carotenoids

in the The firt

on erythrocyte

ghosts

perturbations

bring

the

en-

microthor(71, about

and dras-

660 ISSN

0006-291

X

BIOCHEMICAL

Vol. 76, No. 3, 1977

tic

changes

Frog

in the microenvironment

sciatic

pattern

nerve

(81,

excitation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

also

and we here

upon the

of the

reporter

known to exhibit report

carotenoid

on the effect

Raman spectra

B-carotene. Raman

of biological

of carotenoid

molecules.

Experimental: Sciatic nerves were dissected from the frog During the preliminary experiments we found Rana esculanta. that the nerve fiber was very sensitive to the intense laser light. To prevent its damage we designed a special sample holder in which the center part of the fiber exposed to the laser beam was bathed in Ringer solution. This enabled us to detect Raman spectra during simultaneous electric stimulation as well. Raman spectra were taken at room temperature with a Cary 82 spectrometer using an argon ion laser at 514.5 nm and 200 mW. Variability in the condition of the animals may account for slight variations in optical responses. Nerves were stimulated by suare pulses of max. 1 V and 0.1 msec width action potential could be observed at 200 Hz. Good quality for 2-3 hours without scanning the laser beam on the nerve. Results

and Discussion:

Raman spectrum ably

similar

of the to that

the peak positions conclusion

that

In the resting of

these

(5),(Fig.l).

ta

that

we found

was shifted

from

carotene-solvent observed

at

the nerve

of

(5j.J

remained laser

resonance

case of

enhanced with

the

are almost neither

fact

structure

in the membrane is

more or less

that

mode was

with

the

even after

B-carotene

proved

661

molecular with

from nor the

as yet

known

the membrane-bound

intact

its

that

Raman spectra

indistingushible the

on

C40-carotenoid

that

da-

v(-C=C-1

we concluded

interact

of nerve

by the

Although

whereas

should

comparison

justified

the v(-C=C-> nerve

modes of previous

S-carotene

resting

from

we drew the

Since

to mention

light

intensities

interaction.

carotenoid-binding interesting

In fact

1520 cm-1 depending

(This is

(5,101.

the

was remark-

1527 cm-1 to

C40-carotenoids

other

in the

C40-carotenoid(s)

b-carotene

nerve

In accordance

-1 1520 cm in the

environment.

sciatic

relative

are the

C40-carotenoids (5)

frog

carotenoids

and the

-1 cm region

900-1700

role

of

it

is

carotenoid

prolonged to be very

each

exposure unstable

to in

Vol. 76, No. 3, 1977

BIOCH’EMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ti

f

J c t &Jo

Fig.

1

lecithin

the

characteristic (i>

excitatory

and,

Horvgth

l&O

’

cm’

process

to be published).

the

w(-C=C-)

band was shifted

as revealed

11520/Il,5,

exhibited

thus,

’

mode exhibited

changes:

1518 cm-'

spectra

(L.I.

The v(-C=C->

(ii)The

’

Raman spectrum of frog sciatic nerve dominated by resonance enhanced C40-carotenoid peaks. Instrumental setting: slit 4 cm-1 I scan speed -1 -1 1 cm set pen period 1 sec. Signs + and - denote spectra tak& in excited and resting state of the nerve, respectively. Note the overall deterioration of the spectra during the experiments.

multi-bilayers

During

’

all

a slight 11520/Il157

ratio

from

by high was

(although values

662

1520

cm-l

resolution

decreased

scans. (Fig.1).

apparent) were

to

corrected

The

deterioration for

simulta-

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A 1.2 t

f

1.0

.a I 2% I,,,, .6 .L .2 I In

rl

-+ 107 .%

1Jxl .04

ID7 .97

ul7 95

110 .96

I& .8L

ux .Ol

104 .96

114 .99

Ic9 .97

InI,

Fig.

2

a - Corrected 11520/11157 values of different nerve tissues: Each column-pair represents the average of 4-6 measurements on a nerve in stimulated (+), and resting (-1 state. Bars indicate the standard deviation. m is the correction factor for simultaneous peak amplitude measurement. r 2 is the correlation of m ; note that those nerves which exhibited no effect have poor correlation with varying deterioration. bThe logarithm of uncorrected band intensities versus time: The intervals represent successive measuring cycles in the resting (-1 and excited state of the nerve. (I 1115,: A-h-A, 1520 : o-o-o, O-D-0) 3005:

neous peak amplitude Fig.2a) rate

could and the

collected

measurement.

be calculated scan speed.

in Fig.2a

This

from the Finally

grouping

all

data

correction

observed I

factor

(+I

(m,

deterioration

values were 1520"1157 of each individual nerve

663

Vol. 76, No. 3, 1977

tissue

in

BIOCHEMICAL

a column-pair.

teristically

Since

depends

have

something

tion

arose

on the

to explain.

whether rate

uncorrected

band intensities

could

deterioration

rate

Note

that

the

lines

in

Fig.2b,

sity

of the

confirmed in the

results

which

was responsible

by the

excitation)

is

shift

of

dicates

the that

ed to changing less

(if

the

which

describes

state ization

out.

Raman intensity the

mixing

wavefunctions properties should

under changing

manifest

finding

inten-

was also

(51.)

longer

delocalized

(5).

lower

Since

other

changes

in

However

depends

of

S1 (first

vibrational

664

where

also

should are

considerably prob-

polarizability on the

and,

mixing

excited)

term

state

perturbation

elec(9).

r-electron in

the

in-

be connect-

rTl++~ transition

due to transient changes

chains

frequencies

the

also

(during

The observed

peaks

the

is prov-

ratio

polyene

alone

ratio.

Raman intensity 11520~Il15,

of

From band

11520/11157

decreasing

through

the

v(-C=C-1

Raman intensity

affected

functioning.

and 11157/Iloo5 1005 cm-1 band was

was the

mode towards

order.

possi-

to two paralel

This

changing

strongly

can be ruled

thus,

tronic

bond

it

to that

of

a uniform

of

reference

observed

decreasing

at all)

abilities

the

v(-C=C-> the

the

the

similar are

exhibited

de-

11520/Iloo5

that

for

that

r-electrons

ratios

the

this

excitation

was lowered.

as an internal

for

fact

bands

ques-

logarithm

(Fig.2b)

states.(The

we concluded

An explanation

the

the

'and resting

these

the

during

the

affected

on the state 1520 cm -1 band fit that

band

to be taken

assumption

time

all

charac-

we clearly

depending

indicating

excited

functioning

Plotting

since

of the

v(-C=C-)

ratio

excitation

versus

out

not

point

11520/11157 of

spectra.

by comparing

suggested

ided

the

be ruled

state

biological

terioration

bility

the

As a resonable

the of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

resonance

S1

delocalen-

Vol. 76, No. 3, 1977

BIOCHEMICAL

hancement.

In

may change

selectively

bands

are

lenth

(5).

sign

the

this

case the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

enhancement

since

the

of the v(-C=C-1 band 1157 cm-1 and 1005 cm-1

known to be insensitive On the

basis

transient

to partial

to delocalization

of this

reversible

carbon-carbon

argument decrease

and chain

we tentatively of

bond equalization

the

as-

v(-C=C-1

of the

band

polyene

trough. Raman optical tential

responses

propagation

carotenoids proposed

and the

were

found

to operate

(10).

In view

bond

equalization

raise

the

are

involved

stimulating

to be changed.

our

(i.e.

possibility

Acknowledgements:

bond order

as electronic

of this,

in

were observed

the

conduction

We thank

discussions

the

in

on transient

membrane-bound

been

biomembranes carbon-carbon may

carotenoids

impulses.

T. Lakatos

and their

have

of m-electrons)

of nerve

Drs.

po-

of membrane-bound

conuctors

finding

action

Carotenoids

rearrangement that

during

encouraging

and F. Jo6

for

interest.

References: 1. Behringer,

J.,and Brandmiiller, J.,(1959) Ann.Phys., 4, 234-238. 2. Popov, E.M.,and Kogan, G.A., (1964) Opt.Spectrosc., 17, 3. Tric, C., (1969) J.Chem.Phys., 51, 4778-4786. 4. Rimai, L., Kilponen, R.G.,and Gill, D., (1970) J.Amer.Chem.Soc., 92, 3824-3825. 5. Rimai, L., Heyde, M.E.,and Gill, D., (1973) J.Amer.Chem.Soc., 95, 4493-4501. 6. Inagaki, F.,and Tasumi, M., (1974) J.Molec.Spectrosc., 50, 286'303. 7. Verma, S.P.,and Wallach, D.F.H., (1975) Biochim.Biophys.Acta, 401, 168-176. a. Larsson, K.,and Rand, R.P., (1973) Biochim.Biophys.Acta, 326, 245-255. 9. Albrecht, A.C., (1961) J.Chem.Phys., 34, 1476-1487. lO.Tien, H.T., (1974) in Bilayer Lipid Membranes (BLM) Theory and Practice pp. 444-448, Dekker, New York.

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