Metachromasia: An explanation of the colour change produced in dyes by heparin and other substances

THROMBOSIS Printed

RESEARCH in the United

LETTER

METACHROMASIA: PRODUCED

vo1.2, PP* Pergamon

States

9 1973

Press,

Inc.

TO THE EDITORS-IN-CHIEF

AN EXPLANATION

IN DYES

3’77-382

BY HEPARIN

OF THE COLOUR AND OTHER

CHANGE

SUBSTANCES

A.

Wollin and L. B. Jaques Hemostasis-Thrombosis Research Unit, Department College of Medicine, University of Saskatchewan, Saskatchewan, Canada S7N OWO

(Received

6.12.1.972; Accepted

by

in revised Editor A.L.

of Physiology, Saskatoon,

form 16.3.1973. Copley)

Al 3STRACT Previous explanations given for metachromasia have been the dyestacking model of Bradley and Wolf and the pairing concept of Young, Phillips and Balazs. The special properties of heparin and heparinoids in producing metachromasia are reviewed. To explain these observations, a model combining the dye-stacking and pairing concepts is proposed. The dye-dimer interacts with suitable ionic sites provided by the chromotrope. The loose ionic interaction alters the electron mobility of the dye-dimer, and this results in the colour change. This is illustrated with the absorption spectra recorded as.differences between dye and dye-chromotrope in a double beam recording spectrophotometer. A metachromatic band shift was found with the dye in 3.8 M NaCl so that the chromotrope can be either a polyanion such as heparin or an ion cage formed by a strong electrolyte.

The colour

change in dyes,

well known to readers cells

and Wolf anion,

of Thrombosis

and with mucopolys

for the phenomenon

Supported

accharides

Research, Two

,

by a dye-dye

by a grant-in-aid

interaction interaction

from

explanations

is assumed for

the Medical

377

is a phenomenon

as associated

many

between adjacent

Research

with mast

have been proposed

In the dye-stacking

of metachromasia.

(1) an electrostatic

followed

known as metachromasia

model dye

of Bradley and poly-

dye molecules

Council

of Canada.

378

METACHROMASIA

resulting

in a change in absorption

cept of Young, rise

Phillips

as a result

ion site which changes of the dye.

Phillips

ious experimental heparinoids

ces

mental oids,

of practical and mast

dye with acid-base sorption

spectra

of the dye. The position

concentrations

of heparin

on paper Mass

Law relationship

times

the concentration

tween a series according

an excess

concentration

by inorganic sion from

an increase

(6, 15).

absorption

(lkg/ml)in

concentration

in excess

(8).

strength

Dehydration to the ti -band

suppresses (16).

(6),

a simple

is incr eas ed ten

an interaction

for

but this requires

The reaction

is suppressed

(12, 13, 14) but suppresion in the medium

than with heparin

metachromasiawith obtained

be-

would be suppressed

heparin

and hydrogen

Products

me-

solution

of that required

temperature

with other mucopolysaccharides

producing

there is no change in the

of a thousand-fold

in ionic

wave-length.

gel (11) and shows

by excess

and increasing

characteristic

aqueous

interaction

present

ab-

of heparin-heparin-

which involves

dye molecules,

on

can be seen with very low

for equilibrium,

hypothesis

instudies

those by the same

at a shorter

is suppressed

electrolytes

much greater oids

required

from

in the presence

When heparin

(6, 12).

experi-

and heparin-

reaction

for the substance

(10) and in agarose

The M-band

substan-

(6). As is well-known,

aA-band

and heparinoids

of adjacent

inducing

and B-bands,

The reaction

to the amount of heparin

equilibrium.

shifts

produces

sulfate..

For the stacking

M -band.

and

we have made the follow-

different

showd,-

about 120 nm less

chromatograms

system

Heparins

with heparin

publications,

is characteristic

of heparin

to a-

fail to explain var-

to metachromasia.

change is definitely

95 nm for dermatan

models

to those using this colour

of the dye in solution

8, 9),

in the chromophoric

to other metachromatic

In previous

of the/.+band

tachromasia(7,

symmetry

and oxidation-reduction

Addition

con-

between the dye and the polyan-

on metachromasia

importance cells.

change is assumed

attention has been paid to the numerous

reported

The colour

ing points.

oids,

little

In the pairing

this colour change at very low concentrations

unfavorable

However,

the colour

relating

Vo1.2,No.4

of the dye.

out that both these

observations

observations

heparin

the energetic

are able to produce

(4, 5).

(2),

interaction

(3) points

and under conditions

spectrum

and Balazs

of electrostatic

BY HEPARIN

is

and heparin-

a shift of light

from heparin

by enzy-

METACHROMASIA BY HEPARIN

vo1.2,No.b

matic

degradation

disaccharide

(17) examined

fragments

Full metachromatic arin hexasaccharide.

solution.

tion or moderate tration

slightly

occurs,

depends

of electrolytes

cepts by suggesting

thatmetachromasia

combining

by being interposed

posed by them.

Due to the resonance

with the negatively

charged

group in the thiazine

dyes from

and thus reduces

electron

causes

the dye-‘dimer.

The strength

Polyanions

reported

The dye-dimer

with the negatively

the new absorption

band.

and the charge density metachromatic

activity

the metachroma-

via the amino groups which partially

charged

associated of heparin

blocks system

delocalization.

of anionic

sites

coils

of the structural

observed,

for the extent of the band shift.

with

to reduce has been

on each end coil can induce arrangement

with the coil are responsible and heparinoids

of hep-

for interaction

charge

in

at a short-

activity

groups of the heparin

features

of the

is reflected

coils with heparin positive

the N-

The loss

light energy

metachromatic

with a partial

Special

of the bonding is responsible

one would expect

We believe

to absorb

for loose

as pro-

and allow looseinteraction

have a tendency to form loose Evidence

reacting

and Geddes (18), although

of the binding to the N-group

arrangement

repulsion.

interacting

d-electron

con-

of water molecules

This interaction

We explain the exceptional

intermolecular (20).

molecule.

the dye molecule

arin as due to the suitable

by a dye-dimer

being part of the conjugation

dye chromophore

in the band shift.

and pairing

in the dye molecules,

of this interaction

chromophore.

(19).

concen-

consistentwith

between the two dye molecules

heparin

are part of the dye’s

er wave-length

bysheppard

on each end of the dimer

tic band shift to be the result

mobility

the dye-stacking

The presence

unit as suggested

charge to form

dehydra-

The limiting

in a manner

is produced

manner.

not necessarily

a partial

(6,12).

phenomenon

we propose

the dimer

by ethanol,

com-

on the nature of the polyanion.

these observations,

stabilizes

of dye-polyanion

is not destroyed

To explain the metachromatic

with the polyanion in a loose

was given by the hep-

between dye and polyanion which does not

This interaction

of electrolyte

to heparin,

in the precipitation

concentrations

and

with the dye with no metachromasia.

as compared

Interaction

lead to metachromasia plex from

by us showed that the monosaccharide

reacted

activity

379

for the high The strength Interaction

METACHROMASIA BY HEPARIN

380

without development an ion-pairing Fig. between Three those

of a metachromatic

of dye and polyanion

1 illustrates

some

the absorption

types

spectra

named these d, B and ,q -bands dimer

and metachromatic

ute azure A solution creased

transmission)

an increase

Addition

tion for both band.

with appearance

the change in in the first

4.7,qg/ml

shoulder

dye

of 0.5

(Curve

due to reduction

the*-

2) reduced

fortt and

of dimer,

Increasing

of the dye-

light absorp-

now occurring

at theA B -bands,

not to changes heparin concentrac

and p -bands, FIG.

(in-

at the 6-band,i.e.

at the expense

was the same

further

M NaCl to a dil-

of the #-band

of light absorption

of the monomer.

(Curve 3) decreased

dyes.

to

them to the dye monomer,

with light absorption

b -band was mainly

substance.

(21)

Addition

of heparin

recorded

which corresponded

light absorption

in light absorption

vibrational

were

Michaelis

of the dye-dimer

of 0.7Aglml

As thedecrease

tionto

1) reduced

U - and b-bands

Differences

and related

of the dye.

of

interaction.

isolated,

and attributed

in the concentration

monomer.

clearly

of thiazine

form

(Curve

the result

of dye and of dye-metachromatic

bands were

of the absorption

is conceivably

without dye-dye

of these points.

spectra

of spectral

colour

Vo1.2,~0.4

andincreased

1

Spectral Differences in Light Absorption Between Azure A andAzureAWith NaCl or Heparin.

01.

450

.

.

500

*

*‘**a.’

550 WO”.I.npth

600 (ml

700

Spectra recorded with a double beam spectrophotometer (Beckman DK-2). A deflection below 100% transmission indicates increased light absorption in the sample cell; above 100% transmission, decreased absorption. Reference cell: azure A solution 10 Mg/m.l. Sample cell: R - azure A solution 10 fig/ml; 1- plus 3.1 x 104,c(g/ml (0.5 M) NaCl; 2 - plus 0.7Ag/ml heparin; 3 - plus 4.7 &g/ml heparin; 4 - plus 2.2 x lo5 ,qg/ml NaCl (3.8 M). Azure A (National Aniline, certificate NAz 19) purified by the method of Pal and Schubert (22). A series of heparin preparations were studied. Results reported for Heparin Lederle Lot 1804.

MBTACHROMASIA BY HEPARIN

vo1.2,~0.4

the light absorption The formation

475 nm (Curve not essential

4) with the dye in 3 M NaCl.

band shift results charge

system

similar

from

of the dye-dimer, to that produced

were

utilized

and

in sequence;

only and the dye-

equilibrium.

in the recording

of a r~(-band

It is evident that polyanions

as is usually

a great number

band.

of both dye-dimer

the dye-dimer

to maintain

a new observation

for metachromasia,

partial

removed

was reduced

1 also reports

at the expense

that the dye species

of the M-band

concentration

Fig.

occurred

It is probable

i. e. the formation monomer

in the /rc-band with no shift in the metachromatic

of the ~-band

dye-monomer.

38 1

thought.

of ions forming

thus inducing

an effect

We suggest

at are

this

a cage around the on the conjugation

by the polyanion. REFERENCES

1.

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