Interaction of proflavine with DNA studied by colloid surface enhanced resonance Raman spectroscopy

405

Journal of Molecular Structure, 141 (1986) 405-409 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

INTERACTION OF PROFLAVINE RESONANCE

E. KOGLIN AND J.-M. Institute

WITH DNA STUOIEO

BY COLLOID SURFACE

ENHANCED

RAMAN SPECTROSCOPY

SiQlJARIS

of Applied

Physical

Chemistry,

P.O. Box 1913, D-5170 JUlich,

Nuclear

Research

Center

(KFA) JUlich,

F.R.G.

ABSTRACT The interaction of the mutagenic highly fluorescing proflavine (3,6diaminoacridine: PF) dye with calf thymus DNA has been studied by Surface Enhanced Resonance Raman Scattering (SERRS). Since the Ag-colloids almost completely quenche the strong fluorescence it is possible to obtain excellent vibrational spectra in a wide frequency range providing valuable information about the intercalation. The intercalation does not affect the vibrational frequencies of the proflavine dye. On the other hand, intensity changes are observed in some of the ring- and NH*-modes of proflavine upon intercalation. This Raman hypochromism is characteristic for ring stacking interactions and in the SERRS spectroscopy for an additional effect of the dye orientation to the surface.

INTRODUCTION Since many of the acridine

action

between

DNA and various

of fluorescence tial findings (ref.l-5).

excitement

enhancement

ment together

quality

(ref.6-8).

results

Resonance

studies,

Raman spectra

from strong

copy offers

two significant

studied

of dyes adsorbed

(SERRS)

and

resonance

(ref.9,10).

enhanceThus,

is of great interest

for

high-

chromophores. is used to obtain

dyes with DNA. SERRS spectros-

over classical

in-situ

at charged

interest

of molecular

is to show how SERRS

of acridine

and essen-

on metal electrodes

a general means of obtaining

fluorescent

by means

in recent reviews

high Raman scattering

of the fluorescence

advantages

not only to characterize

0022-2860/86/$03.50

from molecules

The combination

it offers

about the interaction

concentrations

have been summarized

activi-

the inter-

by CARS. Potentialities

in an extremely

The purpose of this communication

it allows

has been extensively

Raman Scattering

because

information

orientation

biological agents,

by lo3 - lo6 has caused extraordinary

with a strong quenching

Enhanced

biochemical

acridines

and more recently

that Raman signals

were enhanced

in recent years

and surface

have important

and bacteriostatic

by these methods

The discovery

and colloids

Surface

spectroscopy

obtained

derivatives

carcinogenes

ties, acting as mutagenes,

Raman spectroscopy:

the chemical

surfaces

identity,

structure

but also to work at low

(low7 M).

0 1986 Elsevier Science Publishers B.V.

and

406 EXPERIMENTAL Proflavine.hemisulfate (CI3HI2N3 *1/2H2S04 - H20) was purchased from Serva, Feinbiochemica, Heidelberg, F.R.G., and used without purification. AgN03 and Na(BH4) were of analytical quality and were purchased from E. Merck, Darmstadt, F.R.G. The silver colloids were prepared according to Creighton et al. (ref.11). Further details of chemica

preparation and Spex Raman instrumentation are

given elsewhere (ref.12,13 RESULTS AND DISCUSSION The SERRS effect for proflavine is illustrated in Fig. 1. The most important observation of proflavine adsorbed on Ag colloids is that SERRS

SERRS

$

PROFLAVINE

150

550

2’ c

950 RAMAN

SHIFT (cm

1350

1750

-' >

Fig. I (A) Fluorescence of proflavine. Cgnditions:A,, = 514.5 nm, power 5 mW, fluorescence intensity 4 - 10 cps, (6) SERRS spectrum of proflavine_tdsMol;rb;l o;~$ colloids. Conditions: M Tris, pH 8 Lx = 514.5 nm, power 40 mW, IO 3

407 processes

can displace

first time vibration frequency SERRS

region

could

is a powerful

spectra

be obtained.

of proflavine. spectrum

a complete

vibrational

we present

analysis

modes

of proflavine

of the carbon skeleton

skeleton.

modes

between

the acridine

and C-NH2 rocking, orange

than the

(ref.5).

Until today For this

SERRS

spectrum

wagging

modes

bands.

modes

Indeed, -1

1572, and 1638 cm

(cf. Fig. 1). On the

bands in the low frequency

dye (without

to vibrations

but must

and torsion

range

(150 to

be assigned

vibrations.

amino groups)

of the two amino

bend), 412 cm-l

The strong

that stretching

SERRS

to ring

A comparison

(ref.10 c) -1 shows that the bands at 352, 412, 594, and 1082 cm

spectrum

can be attributed 352 cm-l (C-NH2

1362, 1490,

are not due to stretching

bending

One expects

bands in the SERRS

hand, there are also strong

rock).

quality

has not been reported.

give rise to the strongest

of PF at 1322,

(arom C-C str) lead to strong

and the PF SERRS

is of better

by CARS techniques

that

Raman

interpretation of the most characteristic bands. -1 1000 and 1700 cm the strong bands can be assigned

modes

which

For the

in Fig. 1) and low-

a possible

the five stretching

1000 cm")

(not shown

to study the vibrational

spectrum

is obtained

of this ring carbon

other

dye fluorescence.

Thus, we are able to demonstrate

technique

This SERRS which

In the region between to stretching

the strong

in the overtone

and simple

vibrational

reason

completely

spectra

groups

spectrum

in the proflavine

drug:

(NH2 tor), 592 cm-l (NH2 wag), 1082 cm-I (NH2 -I -1 in PF and 636 cm in acridine orange can

bands at 648 cm

be due to the C-C-C ring vibration. The SERRS

spectra

of the free and intercalated

in Fig. 2. This exhibits observe

the vibrational

the PF molecule. because

modes

conditions

spectrum

to the SERRS appear

frequencies

in the frequency

intercalation observed

solution.

strongly

to selectively

dye, in this case are unobservable

PF/DNA complex

differs

(1.6 x low7 M) in aqueous

of the intercalated

of the free proflavine

solution

(cf.

PF dye are quite

cation. -1

in inten-

The SERRS

similar

bands which

are very sensitive

to the

acid. In particular, large intensity changes are -1 bands. The ratio of the intensities of these

for the 350 and 412 cm

two bands changes PF/DNA

of the DNA molecule

range of 300 to 700 cm

with nucleic

a method

with the resonant

of the intercalated

frequencies

offers

dye are shown

are not satisfied.

sity from that of the free PF dye Fig. 2). The SERRS

spectroscopy

associated

The vibrational

the resonance

The SERRS

that SERRS modes

proflavine

affected

by a factor of two from aqueous This indicates

by intercalation

This Raman hypochromism (ref.14,15),

CARS

is characteristic

that vibrations

observed

(ref.5),

solutions

of the dye between in normal

and resonance

of ring stacking

resonance

inverse

interaction

of PF to aqueous

of the free NH*-group

are

the DNA base pairs. Raman spectroscopy

Raman spectroscopy

of proflavine.

(ref.16)

However

besides

408 Intercalation

a*

1 JD

999

550

RAMAN SHIFT

tuQ (cm

17%

-’ )

Fig. 2 (A) SERRS spectrum of proflavine (1.6.10'7 M). (B) SERRS spectrum of the PF/AO complex. Other conditions as in Fig. 1. this stacking effect an additional interpretation can be given for the intensity changes in SERRS spectroscopy. This interpretation is based on the short-range sensitivity of SERS spectroscopy (ref.10 c) and on specific orientations of PF at the silver-surface. In case of the free PF dye adsorbed on the AG colloid surface an orientation of the dye molecular plane parallel to the silver surface is possible. When the planar aromatic PF molecule intercalate between parallel adjacent basepairs the orientation of the dye is perpendicular to the colloid surface. Further works are necessary for a precise assignment of the SERRS intensity changes in the PF/DNA complex. Acknowlegements The authors thank Dr. P. Valenta for interesting and helpful discussion and Prof. H.W. Niirnbergfor his continuous encouragement.

409 REFERENCES 1 2 3 4 5

11 12

:: 15

16

A.R. Peacocke, in: Heterocyclic compounds: acridines, Vol. 9, ed. R.M. Acheson (Interscience, New York, 1973) p. 723. E.R. Lochmann and A. Michelar, in: Physico-chemical properties of nucleic acids, Vol. 1, ed. J. Duchesne (Academic Press, New York, 1973) p. 223. S. Georghion, Photochem., Photobiol., 22, 59 (1977). G. Ldber, J. Lumin., 22, 221 (1981). F.W. Schneider, in: Non-Linear-Raman Spectroscopy and its Chemical Applications, eds. W. Kiefer and D.A. Long (D. Reidel Publishing Company, 1982) p. 445: R.K. Chang, F.E. Furtak (eds) 1982, Surface Enhanced Raman scattering. Plenum, New York. R.K. Chang, B.L. Laube: CRC Crit. Rev. Solid State Mater. Sci., 12, I (1984). E. Koglin, J.-M. Sequaris: Topics in Current Chemistry, in press. B. Pettinger, Chem. Phys. Let. 110, 576 (1984). A.M.P. Alix. L. Bernard, M. Manfait (eds) 1985 Spectroscopy of Biological Molecules, Wiley-Interscience Publication, a) P. Hildebrandt, p, 25. b) T.M. Cotton, R. Holt, p. 38. c) E. Koglin, J.-M. Sequaris, p. 221. 1-A. Creighton, C.G. Blatchford, M.G. Albrecht, J. Chem. Society. Faraday Transactions 75, 790 (1979). J.-M. Sequaric J. Fritz, H.W. Lewinsky, E. Koglin, J. Coll. Interf. Sci., 105, 417 (1985). E. Koglin, H.W. Lewinsky, J.M. SBquaris, Surface Sci, 158, 370 (1985). L. Chinsky, P.Y. Turpin, M. Duquesne, J. Brahms, Biochem. Biophys. Res. Cam., 65, 1440 (1975). M. ManEit, P. Jeannesson, in: Spectroscopy of Biological Molecules, eds. A.J.P. Alix, L. Bernard, M. Manfait (Wiley-Interscience Publications, 1985) p. 42i. M.P. Mornis, R.J. Bienstock, in: Non-Linear Raman Spectroscopy and its Chemical Applications, ed s . W. Kiefer and P.A. Long (0. Reidel Publishing Company, 1982) p.543.