0022-

THE

1554/78/2603-0196/$02.00/0 JOURNAL

OF

HISTOCHEMISTRY

© 1978 by The

Copyright

THE

USE

OF

SULFATE

AND

Histochemical

THE

Vol. 26, No. 3, pp. 196-201, 1978 Printed in U.S.A.

CYTOCHEMISTRY

Society,

Inc.

FLUORESCENT

(ANS)

FOR

PROBE

COLLAGEN BENEDICTO

Dept.

of Cell Biology, Received

Institute

DE

of Biotogy,

for publication

March

8-ANILINONAPHTHALENE

AND

ELASTIN

CAMPOS

VIDAL

UNICAMP,

9, 1977,

and

HISTOCHEMISTRY

13100

in revised

Campinas

form

(SP),

October

14,

Brazil

1977

The reactivity of skin ehastin and collagen to ANS (8-anilinonaphthalene-1-sulphonate) dissolved in nonpolar (e.g. butanol) and polar (distified water, phosphate buffer at pH 2.8 and 8.2) solvents was compared in histohogical sections. The ehastin fibers were demonstrated to differ from collagen at the fluorescence microscopy level under certain ANS staining conditions. When treated with butanoh-ANS solutions, both fiber types exhibit a blue fluorescence with spectral emission profiles alike. However, the elastin emission values in fluorescence-relative units are larger than those exhibited by collagen. On the other hand, for fibers treated with ANS dissolved in polar solvents, elastin and collagen differ in terms of wavelengths of their emission maxima. The ANS attachment to substrate is ascribed to be predominantly nonpolar for dye butanol solutions. When the dye is dissolved in polar solvents, however, polar bindings between ANS and the stained substrate may be established that contribute to emission maximum shift to longer wavelengths. With and cence use

the

aim

dynamics

of gaining

of protein

spectroscopy of extrinsic

insight

into

molecules,

is becoming

the very

chromophores

to

the use

structure

widespread.

study

(0. Kindler, Freiburg, W. G.). Observations and measurements were carried out with a Zeiss microspectrophotometer equipped to perform microfluorometry with the III RS condenser. A monochromator ruler placed before the HTV-R 446 photomultiphier was employed to get emission spectral measurements. The relative sensibility of the photomultiphier is found in Figure 1, plotted with data furnished by Carl Zeiss Firma (Oberkochen, W.G.). A HBO-lOOw stabilized mercury lamp was used as light source. The conditions of measurements were as follows: objective Planapo, 40/0.95; area of the specimen measured, 78.54.tm2; positions I (UG 1 UV-exciter filter transmitting a A= 365/366nm exciting light, FT 420 chromatic splitter, LP 418 barrier filter) and IV (BP 546/7 exciter filter transmitting a A = 546/547nm exciting light, FT 580 chromatic splitter, LP 590 barrier filter) of the filter

of fluoresThe

structure,

in-

teractions and dynamics of proteins has been considered from general or specific viewpoints (7-11). Attempts have been made to detect the condition and degree

of

aggregation

of

histones

employing

8-anili-

nonaphthalene-1-sulphonic acid (ANS) (6, 9). In the same way, ANS has been employed to evaluate structural changes in the ehastin network (4, 5). Since no extensive research on emission spectra of fluorescent probes bound to cell and tissue components has been carried out, it seems relevant to study the conditions under which ANS binds to biological structures “in situ” and to determine the characteristics of the spectral

curves

in these

cases.

In the present work the patterns of spectral emission of the ANS bound to skin elastin and collagen were compared in histological sections. MATERIALS

AND

sets. RESULTS

The polarity and the pH of the ANS solutions influences the general fluorescence characteristics and position of the emission peaks. When stained with the

METHODS

Small pieces of human abdominal skin about 1-2mm thick were fixed in neutral 10% formalin at 4#{176}C overnight. The material was dehydrated through a series of ethanols, embedded in paraffin and sectioned with 6 .tm thickness. After paraffin removal, the

sections

were

stained

with

0.1%

solutions

of

ANS

butanol

solution,

blue fluorescence same wavelength

8-

aniino-1-naphthalenesulfonic acid sodium salt (ANS, Kodak-T 484) in non-polar and polar solvents (butanol, water, Mcllvaine buffer at pH 2.8 and 8.2) for 30 mm, after which they were mounted in Eukitte.

this

case,

that

of the

the

the

elastic

fibers

depict

a deep

with a maximum at A = 470 nm, the as that for collagen fluorescence. In collagen

elastin.

fluorescence (Figs.

2 and

was 3). Values

weaker of 94.12

than and

44.26 fluorescence arbitrary units were detected for elastin and collagen, respectively. Sections stained with the ANS aqueous solution disclose a deep blue fluorescence of the collagen bundles and elastic fibers, 196

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BRIEF

197

REPORTS

446 ip

FIG.1 FIG. 1. Relative microfluorometers.

sensibility

curves

of the

1 P 28 and

both with an emission peak at A = 470 nm. (Position I of the filter set, A = 365 nm exciting light) (Fig. 3). The elastic fiber fluorescence exhibited very light orange fluorescence chroma with a peak positioned at A = 690 nm when excited with green light (position IV of the filter set, A = 546 nm exciting light) differing from collagen bundles which depict a faintly red fluorescence.

The

slides

stained

with

ANS

solutions

at pH

yielded a bright blue fluorescence for the elastic fibers, and a greenish one for the collagen bundles. These spectral emission curves are shown in Figure 4. Examination of these slides with the filter set position IV (A = 546/547 nm exciting range) reveals no differences between the fibers, which are pale red. In the material stained at pH 8.2, differences are found when collagen and elastic fibers are compared with one 2.8

VIDAL,

B.C.

HTV-R

446 photomultipliers

that

were

used

in Zeiss

another, the latter depicting a greenish orange fluorescence at about A = 520 nm (filter-set-position I, A = 365 nm exciting light). (Fig. 4). This fluorescence is about 15 times weaker than that of the collagen bundles stained at pH 2.8 and measured at the same wavelength. The collagen bundles showed deep blue fluorescence with an emission peak at A = 470 nm. By using the filter set at position IV (A = 546/547 nm exciting light) the elastic fibers showed a very strong red fluorescence with a maximum value detected at one of the monochromator ruler extremities; A 700 nm. The fluorescence of the elastic fibers at a pH of 8.2 is about 4-5 times more intense than that of same fibers stained at pH 2.8. Table I summarizes the wavelength data at which the fluorescence emission peaks are found in elastic fibers and collagen bundles.

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BRIEF

198

Fic. indicate

2.

The not

Fluorescence elastic fibers.

spectral

curves

corrected

for

However, =

the

365

nm)

raphy

image

and,

of the

close

to

addition,

with

in the

sensibility

wavelength in

accordance

shown the

is very

of a section

the

reports

from

human

present

work

were

of

apparatus.

excitation

curves

in

the

A cited

the the

REPORTS

ray

in the

(Am,

idues

are

bic

in

DISCUSSION

The

fluorescence of

solutions

proved

between If the

it is possible

type

regions

to take

literature

(6, 10,

with

the

were

shifted

to A

polar

behavior, their

residues.

in the

bundles

(3).

There

of lysine

nm

11).

found the

and

stained

hydroxylysine.

In an

im-

in collagen res-

allow

types

in

as peptide

self

as-

rigidity

may

(1, 9). In the

collagen

fibers

showed

a maximum

at

under

may

collagen

molecular

2.8

these

stained

the A

preswith

most 520

=

conditions

intense nm.

It

the

ANS

is

hydro-

evidence sites

74.10

for

indicating

binding

3 g of elastin, fact

agrees

sites

indicated. achieved

is

ANS

the of

use An by

of

ANS

butanol

the

examination

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mobility

which (2, 4).

and

in

than

by considering in hydrophobic-

collagen,

of

improvement

and/or

literature

view

site efficiency

fluorescent

understood is richer

hydrophobicity the

fluorescence more

than

from point

estimating

the

has

1 binding

polarity

elastin

for data

elastin

about

environment that

morphological tures,

that

in butanol can be elastin composition with

that

ANS,

and

on the

The

binding and

in fact

the

is sufficient

collagen that the of

the

of

explain

groups There

namely,

oriented

that

such intensity

at pH

with

and

and

contrib-

character

binding should be effected through their sulfate in available NH2 groups of the collagen bundles.

(4).

reported

in hydrophobic

solution

depends

a different

plays

for

ANS

maxima

compositions more

instance,

work,

the

chain,

between

binding

For

hydropho-

can

collagen factors

fluorescence

ent

phobic

in

from had

ionic

chains.

al(I)

knowledge

with

the

hypothesized

butanol

spectrum,

of molecules

are differences

470

were

interaction

aggregation

(10,

reports

elastin has

hydrophobic

role

=

expected

Collagen

way,

same

as suitable

emission

of the

different

groups

dyes

sections

and

ANS,

the

Arrows

more

hydrophobic

This

Other

fluorescence

with

non-polar

to

polar

portant

at A

the

is also collagen

sterically

bind

with the

side

due

influence

substrate

with

solution,

which

aspect,

polar

When red

probably

clustered the

11).

nm,

In this

peak

accordance

solvent to the

520

=

data.

related

stained is in

sociation,

collagen

than the

solution.

much

of differences

sections.

ANS

distinguish

different

(3).

of ANS

butanol

contains to

detection

com-

are

to

of the

emission

solutions

easily

proteins

and sites

preparations

ANS

of extent

this

non-polar

characteristic the

to

various residues

interaction

fiber

ANS

a2-chain

I collagen

with

fibers.

of sufficient

of the

the

treated

adequate

collagen

non-polar

of

tissue

be

and

and

probes in

to

elastic

accessible

The

connective

0.1%

considerably

tissue

characteristics

the

the the

aminoacid

the

ponents

with

on

uting

(4, 5, 7, 8).

literature

stained

example,

bibliog-

obtained

skin

for

the

the

mentioned

solutions of this of the

closely

From

the

purpose

of struc-

is specially

technique

can

stained

prepara-

be

BRIEF

tions

with

a polarizing

microscope

equipped

199

REPORTS

by an analyzer, it is possible to detect a strong blue birefringence due to collagen and a weak gray-yellowish fluorescence provided by the elastic fibers (Vidal, unpublished data).

to perform

fluorescence microscopy. Under these conditions with use of a blue light excitation filter which transmits a A = 435-490 nm light, and substituting the barrier filter

F

110

100

go

80

70

60

50

40

30

20

10

0 420

440

460

480

500

520 540

560

580

600

620

FIG. 3. Emission spectral curves for human skin sections. O-O, bundles both stained with 0.1% ANS aqueous solution; 0 0 elastic solution. All measurements were made by using the filter set position ANS/H20; the microfluorimetry was done with filter set position IV.

640

660

680

700

nm

Elastic fibers and, x-x, collagen fibers stained with 0.1% ANS butanol I. +-+, Elastic fibers stained with

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200

BRIEF

REPORTS

F

120

110

100

go

80

70

60

50

40

30

20

10

0 420 FIG. 4. Emission spectral 0.1% ANS solutions. x-X, bundles and, #{149}#{149}, elastic

440

460

480 500

520

540

560

580

600

620

640

660

nm

curves of fluorescence of human skin section stained with buffered collagen bundles and,#{149} #{149}, elastic fibers (staining at pH 2.8); x fibers (staining at pH 8.2).

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solutions of x, collagen

BRIEF

TABLE Wavelengths

of the Emission

Peaks

for Elastic Elastic

Dye

Solvents

Filter

set

Mcllvaine

pH 2.8 Mcllvaine pH

buffer

at

buffer

at

I

and

Collagen

set

Fb

470

61.56 45.27

520

Stained

with

690

8.12

ANS

Collagen

Filter

460-470

Fibers

Fibers

I (A = 365/366

Wavelengths

Water

201

REPORTS

700

546/547

Filter

set

F

Wavelengths

8.31

470-480

Solutions Bundles

I (A = 365/366

nm)#{176} F 59.32

520

122.78

470

25.10

470

44.26

8.2

Butanol

470

A, wavelengths F, fluorescence

of the excitation arbitrary units;

LITERATURE

94.12

light beam. average from

10 measurements.

CITED

1. Beyer

CF, Craig LC, Gibbons WA: Structural requirements for binding of the fluorescent probe TNS with peptides. Nature (New Biol) 241:78, 1973

2. Bigelow proteins

CC: On the average hydrophobicity of and protein structure. J Theor Biol 16:187, 1967 3. Fietzek PP, Kuhn K: The primary structure of collagen. Int Rev Connect Tissue Res 7:1, 1976 4. Gosline JM: The physical properties of elastic tissue. Int. Rev. Connect Tissue Res 7:211, 1976 5. Gosline JM, Yew FF, Weis-Foch T: Reversible structural tin, as

changes indicated

by

in a hydrophobic fluorescence

protein, elasprobe analysis.

Biopolymers 14:1811, 1975 6. Laurence DJR: Interaction of calf thymus fractions in aqueous solution with naphthalene-1-sulphonic 1966

acid.

Biochim

histones 8-aniinoJ. 99:419,

7. Parker CW, Godt SM, Johnson MC: Fluorescent probes for the study of the antibody-hapten reaction II. Variation in the antibody combining site during the immune response. Biochemistry 6:3417, 1967 8. Parker CW, Yoo TJ, Johnson MC, Godt SM: Fluorescent probes for the study of the antibodyhapten reaction I. Binding of dimethyl aminonaphthalene-1-sulfonamido group by homologous rabbit antibody. Biochemistry 6:3408, 1967 9. Penzer GR: 1-Aniinonaphthalene-8-sulphonateThe dependence of emission spectra on molecular conformation studied by fluorescence and protonmagnetic resonance. Eur J Biochem 25:218, 1972 10. Stryer L: The interaction of naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol 13:482, 1965 11. Stryer L: Fluorescent probes provided insight into the structure interactions, and dynamics of proteins. Science 162:526, 1968

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