Role of singlet oxygen in the degradation of hyaluronic acid

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 894-901

Vol. 115, No. 3, 1983 September 30, 1983

ROLE OF SINGLET OXYGEN IN THE DEGRADATION OF HYALURONIC ACID Usha P. Andley I and Bireswar Chakrabarti I'2 Eye Research Institute of Retina Foundation 1 and Harvard Medical School, 2 Boston, Massachusetts 02114 Received August 12, 1983

To investigate the effect of singlet oxygen on the molecular properties of hyaluronic acid, the polymer was irradiated in the presence of a dye sensitizer for singlet oxygen. Viscosity and circular dichroism techniques were used to monitor these changes. The relative viscosity of the polymer solution decreased steadily with increasing duration of irradiation, indicating an apparent decrease in molecular weight of hyaluronic acid. Circular dichroism measurements of the irradiated sample, however, did not show any appreciable change in the secondary structure, but do suggest that the generated singlet oxygen changes the tertiary structure and that this change is followed by a minor depolymerization. Hyaluronic acid molecular

primary

ride

units

structure of

alternating (I-4)

molecular

a glycosaminoglycan,

component of vitreous humor,

Its

tory

(HA),

consists

D-glucuronic

8(I+3) have

and

of

acid

8(I+4)

synovial

unbranched and

is a major macrofluid,

skin,

repeating

disaccha-

N-acetyl-D-glucosamine

linkages.

Studies

etc.

in this

with

labora-

shown that the conformation of HA in solution

weight dependent.

This

is

is evident by the fact that the

chiroptical properties of lower and higher oligosaccharides differ (4), and

and

the

conformational

in the presence of Cu 2+ This

change

of

HA

in organic

solvent

(3) cannot be detected with

cular-weight

HA.

phenomenon

to determine

the relationship

must

between

be considered

(2)

low-mole-

in attempts

conformation and biological

function of the molecule. In the aging or pathological vitreous and in synovial fluids, degradaton of HA is known change

(5,6)

to occur,

and this may

lead to a

in conformation of the molecule and thus of the structural

integrity

of

the

tissues.

It is generally

0006-291X/83 $1.50 Copyright © 1983 ~ A c a ~ m ~ Press, ~c. AHr~h~ofreproduction ~ a ~ f o r m r e s e ~ .

894

believed

that depoly-

Vol. 115, No. 3, 1 9 8 3

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

merization of HA occurs through production of free radicals during autooxidation of reductants and the reduction of molecular oxygen. McCord

(7)

(O2~)

reacts

merizing (6),

that

with

HA of

H202

bovine

degeneration

of oxygen, 102,

found

generated

to produce hydroxy radical synovial

singlet oxygen

fluid.

As

(102).

exposure

to

sensitizer singlet

sources of

oxygen

can also be generated

light,

effectsr

a photosensitizer, proceed

via

the

and

produced

oxygen.

triplet

by by

Most

state o f

the

(11).

The to

radicals

produce

free

triplet

may

radicals

or

interact radical

with

a

anions.

can produce univalent reduction of molecufurther

dismutate to H202 and react with H202 to form the hydroxyl

radical

with

Alternatively,

molecular

energy

generate

sensitizer

can

(OH').

to

tissues

it has a much longer lifetime than the excited

substrate

These product

other

various potential sources

or photooxidative

oxidations

because

oxygen

(11),

visible

state

reducing

of molecular

action

photosensitized

in many

(8-10).

species

photodynamic

anion

(OH.), depoly-

The major biological

02~ , OH., or H202 remain unknownr

Active

superoxide

can also be caused by another molecular species

have been reviewed

lar

enzymatically

oxygen,

superoxide

the

anion

sensitizer

and more

02?,

triplet

commonly

which

interacts

it involves

directly

transfer of

from the triplet sensitizer to ground state oxygen to pro-

duce singlet oxygen

(102).

In the present study, we have Used a dye-sensitized photooxidation

system

oxygen

on HA.

oxygen

is also effective in bringing about changes in HA structure

manifested

to

study

Our

the

results

effect show

for

of

photogenerated

the

first

time

species that

of

singlet

in viscosity and circular dichroism measurements.

MATERIALS AND METHODS Purified hyaluronic acid (Grade I), prepared from human umbilical cord, was obtained from Sigma Chemical Co. HA solutions were prepared by dialysis at 4"C against 0 . 1 M NaCI solution, con895

Vol. 115, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

t a i n i n g the desired solvent c o m p o s i t i o n and h y d r o g e n ion concentration (2). HA c o n c e n t r a t i o n was d e t e r m i n e d by the carbazole reaction method (12). As p h o t o s e n s i t i z e r s we used m e t h y l e n e blue or riboflavin. The photosensitizer was added to the HA s o l u t i o n (0.1 to 0.3 mg/ml) prior to i l l u m i n a t i o n at 37°C with 300 foot candles of w h i t e light from a 15 W D a y l i g h t f l u o r e s c e n t bulb. Controls inc l u d e d HA plus light w i t h o u t p h o t o s e n s i t i z e r , HA plus p h o t o s e n s i t i z e r kept in the dark, and HA plus light and p h o t o s e n s i t i z e r under deaerated conditions. The samples were irradiated for v a r i o u s time periods and used for v i s c o s i t y and c i r c u l a r d i c h r o i s m measurements. V i s c o s i t y was m e a s u r e d in an O s t w a l d - t y p e Cannon v i s e o m e t e r at 25.0 ° ± 0. I°C, and intrinsic v i s c o s i t y values were c a l c u l a t e d as r e p o r t e d p r e v i o u s l y (2). To study the effect of D20 on the c h a n g e in intrinsic v i s c o s i t y of HA, the water in the p h o s p h a t e b u f f e r used for the p r e p a r a t i o n of the HA samples was replaced by D20. Circular dichroism (CD) was recorded in an Aviv s p e c t r o p o l a r i m e t e r model #100 m o d i f i e d from a Cary 60 s p e c t r o p o l a r i m e t e r w i t h a CD a t t a c h m e n t at room temperature using 0.1 to I cm quartz cells.

RESULTS

The blue

is

relative tion.0 in

effect shown

state

of o x y g e n The

superoxide to

droxyl

radical

crease

in

Mcdord 6 and

cher

which

Singlet relative

in

also

is present

and

the

oxygen-mediated because

prevented

processes of the

longer

896

not

generated in large the

irra-

neither quanti-

a known

hy-

inhibit

the

027,

used

by

quantity

to

as

enough

singlet

oxygen

dimethylfuran

of

de-

quen-

and

1,3-

in viscosity.

to be enhanced

lifetime

active

to

that

the d e c r e a s e

are known

involved

be p r e v e n t e d

Mannitol,

scavengers

was

in sufficient

did

However,

102

(5) completely

to H20,

process.

irradia-

prior

not

The

of

each

indicating

is g e n e r a t e d

the

for

HA could

catalase,

in viscosity.

azide

of o x y g e n

mixture

methylene

change.

duration

specific

of

with

viscosity

species

Enzymatically study,

the

reaction

scavenger,

viscosity.

cyclohexadiene

to the

or

solution

increasing

in v i s c o s i t y

change

in this

of

HA

inhibitors

02 - nor H202

(OH')

a decrease sodium

added

dismutase

any

an

terms with

HA,

decrease

of

molecular

of

were

radical

produce

I in

decreased

degradation

superoxide

cause

Fig.

viscosity

diation.

ty

in

illumination

To i d e n t i f y

the

by

of

in D20

iO 2 in D20.

An

Vol. 115, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

361

/",

24

o\

4°', ' \~'\%'N %

20

I

I

,

I

60

,

"~

120

180

Time of irradiation, mill

Fig. I. Viscosity change of purified HA in the presence of methylene blue (--). The samples contained 0.3 m g / m l HA, 10 -4 M methylene blue, and 0.1 M phosphate buffer, pH 7.8, in a total volume of 3 ml. The effect of addition of the following inhibitors and scavengers on the viscosity change is also shown: 15 mM sodium azide (o---l); 2 mM c y c l o h e x a d i e n e (x---x); 0.5 mM dimethylfuran (A--A); 10 u g / m l superoxide dismutase (V--V); 20 p g / m l catalase (o---~); 10 mM m a n n i t o l (*--*). The viscosity change in the p r e s e n c e of D20 (- - -) was measured by preparing the sample buffer in D20 instead of H20.

increased H20

rate

of

in the buffer Table

presence ation H20 hr.

was

I shows

in b u f f e r s

was

about

With

after This

glet

oxygen

in

When

one-third

3

with

the

result

hr, of

its

the

viscosity

before

buffer,

the

decrease

of with

intrinsic value

was

confirms HA

by

viscosity

897

in

about

the

I).

HA

irradi-

of

[n] with

In]

26%

of

with and

for

was

involvement

oxidase of

in the

irradiation

irradiation

xanthine

(Table

and after

The value

after

value

when

of HA solutions

value

the

observed

I).

H20 and D20.

further

treated

control

(Fig.

HA. was

control

degradation

HA was

by D20

of

and r i b o f l a v i n

irradiation;

value.

for

blue

D20-containing

trol

blue.

of

viscosity

intrinsic

prepared

62%

of

replaced

the

of m e t h y l e n e

pronounced

(7)

decrease

16

more

the

con-

of

sin-

methylene

hypoxanthine

decreased

to

about

Vol. 115, No. 3, 1983 Table

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Intrinsic viscosity conditions

I.

[q] of HA solutions

Sample

under various

[q], ml g-i

HA + riboflavin in H20 buffer

(control)

1,380

HA + riboflavin in H20 buffer (irradiated 16 hr) HA + MB in H20 buffer

811

(control)

1,430

HA + MB in H20 buffer (irradiated 16 hr) HA + MB in D20 buffer

890

(control)

1,250

HA + MB in D20 buffer (irradiated 16 hr) HA + hypoxanthine

(0.12 mM)

HA + hypoxanthine

(0.12 mM) + xanthine

oxidase

330 1,553 510

(0.04 units/ml) treated for 3 hr

HA concentration was varied between 0.1 and 0.28 mg/ml for intrinsic viscosity measurements. Dilutions were made with the appropriate buffer. Riboflavin 0oncentration was 10 -5 M. MB concentration was 10 -4 M. Irradiations were done in white light as described in Materials and Methods. The xanthine oxidase reaction was carried out in 0 . 1 M phosphate buffer, pH 6.8. HA, hyaluronic acid; MB, methylene blue

To

see

whether

a

accompanies

the

change

we

CD

spectra

measured

two

characteristic

other was

around

190

observed

studies

in

at

ethanol/80% and

a strong

225

nm

in the with

nm

low

pH.

to pH

These

blue

spectroscopic

CD

change

(2)

with have

band

below

CD

changes (2).

undergoes

near

the

shown

of

negative

an 210

200 nm and represent

To d e t e r m i n e such

898

210

of

a change,

displays and

nm b a n d

an

solution

a positive

7

Previous

unusual

nm b a n d

the

at pH

solvent/water HA

HA

system,

nm

blue.

that

in o r g a n i c

HA

210

methylene

acidification the

solution,

one in

properties

photooxidation

aqueous

bands,

No

2.5,

of H A

in o u r

In

is o b s e r v e d

Upon

conformation

methylene

HA.

irradiation

negative

appear.

of

(1,2).

of H A

water

in

viscosity

laboratory

in the CD s p e c t r a solvent

in

negative

upon

this

change

change mixed in

20%

disappears band

around

a dramatic

alteration

whether

irradiated

the

CD

HA

spectra

at pH

VoI. 115, No. 3, 1 9 8 3

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

% -6 E

0

%

/

-2 -4 -6 I

I

210

2;0

Wavelength

'

2~0

nm

Fig. 2. CD spectra of HA before ( ) and after (---) 4 hr of ~i-at ion with methylene blue. The samples were dialyzed against aqueous (20%, v/v) ethanol, pH 2.5.

2.5

in

20%

ethanol/80%

measured. reduced

After

water

of

irradiation,

to about

one-third

control

the

its

and

positive

intensity

irradiated

CD

band

(Fig.

at

HA

225

were

nm was

2).

DISCUSSION The gen,

decrease

apparently

polymer.

in

viscosity

indicates

However,

a

change

macromolecule.

A drastic

due

to

ascorbic generally tion

of

assumed free

radicals

of

suggested

that

stage,

drastic in

the

ever,

(secondary

the

change

secondary

in

or

during

metal

oxygen.

the

free

structure,

a prolonged

off

tertiary

(13)

radicals, HA

of

related

the

to of

presence

reducing

a a

produc-

reductants CD

studies

least

at

groups,

ini-

causing

a

change

viscosity. occurred.

and (14)

the

a subsequent the

of

It was

through of

at

and

the

of HA, p r e s u m a b -

of

side

oxy-

structure)

occurred

depolymerization

899

weight

also

in

results

the

singlet

ions Fe 3+ and Cu 2+.

structure

thereby

time,

is

autooxidation The

by

molecular

tertiary

noted

the

cleaved

induced

in the v i s c o s i t y

was

generated

randomly

in

depolymerization

molecular

change

after

that

as

viscosity

and the t r a n s i t i o n

reduction

tial

in

depolymerization,

acid

HA,

decrease

change

conformational

ly

a

of

HowIn

the

Vol. 115, No. 3, 1983

current

study,

significant molecule, ly

singlet

indicating

of

HA

(2)

that

to

a

the

the

weight do

not

disappearance)

of a change absence

between

the

of

suffered

225

molecule,

similar

to the change

(14),

structure

major

that

the

research

of

cannot of on

be

the

singlet

the

effect

of

decrease that the

the pos-

be considered. structure

it is likely

that

structure

of the

of Cu 2+ and

ascor-

secondary

tertiary

may occur. show

that

In the

present vessels

in the

only

properties role.

enough on

can

normal

of eye

the

of HA,

The eye

presence

singlet

evidence

species

not

in diabetic of

strong

900

The

of

and

both

body.

cules.

samples

in

the

this

the

On prolonged

some positive

is

provided

depolymerization.

Moreover,

oxygen

probably

must

in t e r t i a r y

photogeneration

ignored.

change,

indicate

samples,

has

blood

as a test

in s e c o n d a r y

in the m o l e c u l a r

riboflavin

from ruptured

possibility

generation

as

such

of

virtual-

reported

change. may

the

was

enough;

structure

is to

changes oxygen

It

in the presence

report

here

solvent,

band

of

depolymerization;

changes

by a minor

organ

used

difference

alteration

cause

photoreceptor

vitreous

some

this

singlet

products

CD

by d e p o l y m e r i z a t i o n of

anion

tosensitizer heme

drastic

purpose

superoxide also

a

followed

The

but

followed

been

large

and u n i r r a d i a t e d

caused

irradiation

nm

of a significant

102 has

acid

is

nm)

by a

conformational

structure.

significant

generated

bic

the

organic

in the tertiary

irradiated

of

conformational

the

(210

remains

a conformational

polymer

this

band

structure

has

HA

acidic

the

in

sibility the

not

(2)

was not followed

CD

phenomenon

solvent

show

has

the

in

of

molecule

In

the s e c o n d a r y

undergoes

helix,

change

intrinsic

integrity

molecule

4-fold

<10,000

the

that

in o r g a n i c

determining

molecular

in

Howeverr

for

(not

oxygen-induced

alteration

unchanged.

change

MW

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is a

a pho(5)

or

retinopathy, oxygen

(8-10)

for

to warrant

biological

in

the

in vivo further

macromole-

Vol. 115, No. 3, 1 9 8 3 ACKNOWLEDGMENTS: pr0ject EY04342,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Supported by NIH grant EY01760, and Charles A. King Trust Fellowship.

NIH

pilot

REFERENCES 1 •

2. 3. 4. 5.

6. 7o

8. 9t 10. 11.

12 13. 14

Chakrabarti, B., and Park, J.W. (1980) CRC Crit. Rev. Biochem. 8, 225-313 (and references therein). Park, J.W., and Chakrabarti, B. (1978) Biopolymers 17, 13231333. Figueroa, N., and Chakrabarti, B. (1978) Biopolymers 17:24152426. Chakrabarti, B, (1981) In Solution Properties of Polysaccharides, Brant DA (ed.). ACS Symposium Series, No. 150. American Chemical Society, Washington, D.C.F pp. 275-292. Berman, E.R. and Voaden, M. (1970) In Biochemistry of the Eye, Graymore CN (ed.). Academic Press, New York, 1970, pp. 373-471. Furthmayr, H. and Timpl, R. (1976) In International Review of Connective Tissue Research, Hall, D.A.. and Jackson, D.S. (eds.), Academic Press, New York, vol. 7, pp. 61-99. McCord, J.M• (1974) Science 185, 529-531. Fridovich, I. (1976) In Free Radicals in Biology, vol. II, Pryor, W.A. (ed.). Aca-demic Press, New York, pp. 239-287. Fridovich, I. (1978) Science 201, 875. Halliwell, B. (1978) Cell Biol. Int. Rep. 2, 113. Foote, C.S. (1976) In Free Radicals in Biology, vol. II, Pryor, W.A. (ed.). Academic Press, New York, pp. 85-133. Bitter, T., and Muir, H.M. (1962) Anal. Biochem. 4, 330-334. Harris, M.J., Herp, A., and Pigman, W. (1971) Arch. Biochem. Biophys. 142, 615-622. Figueroa, N., and Chakrabarti, B. (1977) Invest. Ophthalmol. Vis. Sci. 16(ARVO Suppl.), 67.

901