Effect of heparin modification on its circular dichroism spectrum

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 973-980

Vol. 123, No. 3, 1984 September 28, 1984

EFFECT OF HEPARIN MODIFICATION ON ITS CIRCULAR DICHROISM SPECTRUM* German B. Villanueva 1 , and Nancy Allen Department of Biochemistry, New York Medical College Vallhalla, New York 10595 Received July 31, 1984

Summary. The effect of modification of the carboxyl groups of high affinity heparin was investigated. The binding affinity toward antithrombin III decreases in the following order: Heparin > heparin methyl ester > heparinylglycine > heparinylglycine methyl ester. This result agrees qualitatively with the previous studies using unfractionated heparin. Esterification of the carboxyl groups (i.e., HME) does not affect the CD profile of heparin at 210 nm but introduction of a bulkier glycine methyl ester (i.e., HGME) leads to formation of a very intense band at 235 nm. Based on reported CD analyses of uronic acid derivatives and our model building studies, it is concluded that the large difference in CD spectra of HGME as compared to unmodified heparin and HME is due to a change in ring conformation of the uronic acid moiety (i.e. 4C to IC 4 or vice versa). © 1984 Academic Press, Inc.

'

i

Heparin is a highly charged glycosaminoglycan

with various biological

effects,

the most prominent of these being its action on the blood coagulation system. Heparin functions as a blood anticoagulant antithrombin

the latter

the binding of

III with thrombin and a number of other serine proteases of the

coagulation cascade antithrombin

by accelerating

(i).

It was shown that heparin binds to lysine residues

III (2) and this interaction produces a conformational

(3) which accelerates

in

change in

its activity against thrombin.

Heparin has a CD spectrum below 220 nm with a negative trough at 210 nm and a positive peak at the 191-193 nm region. conclusively assigned to, an n ÷ ~

It has been attributed

absorption transition of either an N-acetyl

amide bond or the iduronate carboxyl group or both (4,5). lost upon periodate oxidation

(6).

to, but not

However,

These CD bands are

the ellipticity at 210 nm is almost

doubled upon acidification from pH 7.5 to 2.5 (4,5).

Supported by NIH Grant HL 23265. i

To whom correspondence

regarding this article should be addressed.

0006-291X184 $1.50 973

Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 123, No. 3, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

At present, it is not clear whether changes in this parameter are reflections of a conformational transition in the molecule or simply an acidbase property of the carhoxyl groups. use

carboxyl-modified

heparin

The objective of the present study is to

heparin to investigate the conformational

through circular dichroism.

integrity

of

Results suggest that the pH dependent

CD

spectra of heparin is due to both the acid-base property of the carboxyl

groups

and a conformational transition in the molecule. MATERIALS AND METHODS Materials. High affinity heparin was a gift from Dr. G. %Nn Dedem of Diosynth B.V., Holland. Glycine methyl ester was purchased from Sigma and l-ethyl-3-(3dimethylami~opropyl)-carbodlimide hydrochloride from Pierce Chemicals. N-methylN-nitroso-p-toluenesulfonamide was purchased from Aldrich. Uronic acid content was measured by the carbazole method (7). Nitrogen, sulfur and methoxy determinations were performed by Schwarzkopf Microanalytical Laboratory. The carbazole reaction together with nitrogen and sulfur analysis were used as the bases for determining heparin concentration and extent of modification. Heparinylglycine methyl ester (HGME) was prepared by reaction of high affinity heparin with glycine methyl ester as previously reported (8). Percentage analysis found: N, 4.0; S, 10.28; OMe, 3.21 and glycine, 8.25. Heparinyl glycine (HG) was prepared from HGME by saponification cf the latter in 0.1N NaOH under nitrogen atmosphere for 2 days at 4 ° C. Analysis found: N, 3.75; S, 10.53; and glycine, 8.1~ Heparin methyl ester (HME) was prepared according to published procedure (8,9) using ethereal diazomethane generated from alkali-treated N-methyl-N-nitroso-p-toluenesulfonamide. Analysis found: N, 3.33; S, 9.98; and OMe, 3.45. Analysis of untreated high affinity heparin found: N, 2.05; S, 10.34. On the basis of this analysis, the carboxyl groups in heparinylglycine methyl ester, heparinylglycine and heparin methyl ester were esterified to the extent of 94%, 92%, and 75%, respectively. Circular dichroism spectra were recorded in a Cary 60 recording spectropolarimeter equipped with a Cary 6001 circular dichroism attachment. The spectra are reported as mean residue ellipticity, [~], in degrees cm 2 dmole -I and utilizing a disaccharide mean formula weight of 563 (5). RESULTS The uronic acid carboxyl groups in heparin were converted either to the methyl ester or to an amide with glycine and glycine methyl ester as showc schematically below: O II C-OH

II I

O II C-N-CH2-C-O-CH3

OH O , I II C-N-CH 2-C-OH

H I

H I

H I

HA-Heparin

OH

Heparlnylglycine methyl ester (HGME)

Heparinyl glycine (HG)

974

0 II ------~-O-CH3 H i

Heparin methyl ester (}{ME)

Vol.

123, No. 3, 1 9 8 4

Previous studies

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(8,10,11) have monitored the effect of carboxyl group

modification of heparin on antithrombin III binding by using heparin cofactor assay.

Although this measures the immediate neutralization of thrombin by

heparin-antithrombin

III, it is not a direct measure of the binding of heparin

to antithrombin III.

In the present study, the binding of heparin and modified

heparin to antithrombin III is investigated directly by monitoring the intrinsic fluorescence enhancement and ultraviolet difference spectrum which are generated when heparin binds to antithrombin III.

Figure IA shows the intrinsic

fluorescence of antithrombin III as a function of muccpolysaccharide

| 2.0

!

i

i

~

|

i

250

300

i

ii

"

HA-H ~1.5 ~x

/ / / ~

HME

~I.0

0.5

////~

o

•

50 1

100 150 200 Mucopolysaccharide,~m! i i i

F 1.2

1.1

HG

ME-~ ''~'''''~

/

-

.

~

/

1.0 I 10

I I I 20 30 40 Mucopolysaccharide, ~glml

I 50

I 60

Figure I. Fluorescence Enhancement and Ultraviolet Difference Spectral Parameters of Antlthrombin III as a Function of Mucopolysaccharide Concentration. (A) UV difference spectra at 290 nm. (B) Intrinsic fluorescence enhancement at 340 nm with excitatiin at 280 ran. (-41-m--), high affinity heparln; (-O-O-), heparin methyl ester; (-Q-t-), heparinylglycine; and (-O-O-), heparinylglycine methyl ester.

975

Vol. 123, No. 3, 1984

concentration.

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

Ken

the binding is compared based on the amount of

mucopolysaccharide required to reach half saturation, decreases in ~ e

order: Heparin > ~ E

> HG > H ~ E .

the binding affinity

This order of reactivity

agrees with the results obtained by heparin cofactor assay (Ii), namely that H~E

is inactive and M E

is relatively more active than HG.

the fluorescence enhancement due to M E

The magnitude of

and HG, however, suggests that they

should be more potent than the 13% and 2.6% observed respectively by heparin cofactor assay.

The results obtained from the ultraviolet difference spectra

also support the trend observed from the fluorescence data and heparin cofactor assay (Figure IB).

H~E

has minimal difference spectra and H E

approaches the

control ~ - h e p a r i n more closely than HG. ~e were far

circular

dichroism spectra of heparin and carboxyl

modified

heparin

investigated in order to assess the contribution of carboxyl groups to the ultraviolet

heparin a ~

HE

electronic

transition.

The circular

dichroism

at pH 7.4 and 2.5 are compared in Figure 2.

the CD minima of heparin and M E HA-he~arin and ~ E

spectra

of

It can be seen that

at 210 ~. have comparable magnitude at pH

7.5.

have ellipticities of 1600 +_ 150 degrees cm2 dmole-i and 1750

0.5

~

1 . 0

1.5

H A - Heparin p H i 5

x

HME,pH2.

2.5

~..j.qF~----- HA-He~rin, pH 2,5 I

200

I

2~0

I

220 in nm

I

230

240

Figure 2. Circular Dischroism Spectra of High Affinity Heparin and Heparin Methyl Ester.At Neutral and Acid pH. m e solution at neutral pH is in water while the solution at acid pH is in 0.5M NaCI.

976

Vol. 123, No. 3, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/

\.

2

o~0 "o

%

HG, pH 7.5

~2 HG, pH 2.5 r~

4

x

I HGME, pH Z5

101-

\

/ ~ F - - H G M E

210

220

230 Xin

, pH2.5

240

250

260

nm

Figure 3. Circular Dichroism Spectra of Heparinylglycine and Heparinylglycine Methyl Ester. All other conditions are the same as in Figure 2.

150 degrees

cm 2 d m o l e -I ,

ellipticities enhancement HME

in H A - h e p a r i n

cannot

It is evident

derivatives

minimum

centered

(+250

are enhanced 150 degrees

carboxyl

groups,

are

at 215 nm

~ 45 degrees at pH 2.5

this

cm 2 dmole -I to +500

~I00

from heparin.

degrees

) has appeared

~ 45 degrees

cm 2 dmole -I at 215 nm.

more

At p H

cm 2 dmole -I

Since CD

interesting of these

two

7.5, HG has a

) and a new positive

at 235 nm.

These

two bands

cm 2 dmole -I at 235 nm and

At pH 7.5, HGME has CD bands w h i c h

977

The

groups.

3 that the CD ellipticities

different

in HME.

the

the pH dependent

of the carboxyl

of HG and HGME provides

in Figure

(-i000

that

enhances

the same extent.

to 1.4 times

indicates

to the p r o t o n a t i o n

totally

Acidification

to a p p r o x i m a t e l y

times as compared

of the CD spectra

heparin

band

is 1.6

be due solely

Examination observation.

at 210 nm.

of both m u c o p e l y s a c c h a r i d e s

has no ionizable

spectra

respectively,

-3800 are more

Vol. 123, No. 3, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

than an order of magnitude

larger than those of HG at these wavelengths,

ellipticity being +6000 degrees cm2 d~cle-1

the

~ 500 degrees cm 2 dmole -I at 235 nm and Ii,000 ~ 700

at 215 nm.

Within the range of experimental

bands in HGME are only slightly sensitive

to pH.

error,

these

Similar studies done on HGME

prepared from commercial heparin gave spectra which were only five times greater than those of HG at 235 r~n (Data not shown). DISCUSSION The results of the present studies confirm previous virtually no antithrombin groups,

III activity and that ~ E ,

with no free carboxyl

although less active than unmodified heparin,

than HG which has free carboxyl groups.

findings that H~ME has

is relatively more active

Although it has Dot been so

demonstrated

that carboxyl groups are essential for the binding of heparin and

antithrombin

III, these results suggest that the relative charge orientation of

heparin must play an important role, because esterification displacement of this charge by one methylamino group

(i.e. HME) and

(i.e., HG) causes dramatic

loss of heparin cofactor activity. Comparison cf the CD spectra of unmodified heparin and HME provides interesting observations. spectra

(Figure 2).

some

HME and unmodified heparin have very similar CD

The pH dependence of the CD spectrum of heparin has been

known for a long time (4,5).

From titration studies

(5) it was suggested that

the variation of optical parameters of heparin with pH was due to the acid-base property of the carboxyl group rather than a conformational contrast with these previous groups still exhibits

change.

However,

in

studies, HME, which has no ionizable carboxyl

this pH dependence.

The acetamido n ÷ fi transition in

heparin is not expected to be pH dependent.

Thus,

it is concluded from this

study that the pH dependence of the CD spectra of heparin must be predominantly due to conformational

changes,

of the carboxyl groups. studies

and to a lesser extent to the acid-base property

This is in agreemeet with results from hydrodynamic

(12) which indicate that the heparin molecule behaves as a random coil

at pH 2.5 and as a fully extended rod at neutral pH. that these two phenomena are inseparable,

978

It is possible,

however,

i.e., that the conformation of heparin

Vol. 123, No. 3, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

may be a function of the integrity of the carboxyl groups.

It is only in this

context that the results of the titration studies and chemical modifications can be reconciled.

The second aspect of the present study deals with the CD spectra of HG and HGME.

While esterification of heparin (i.e., }{ME) does not have a significant

effect on the CD of heparin, attachment of a glycine moeity or a glycine methyl ester (i.e. HG, HGME) produces a new CD spectrum at 235 nm.

This new band

cannot be due entirely to the glycine amide bond transition because while this transition is obviously present in both HG and HGME, the CD maximum at 235 nm is more than ten times stronger in HGME than in HG.

This long wavelength band has

been previously observed in a number of disymmetric a-hydroxy carboxylic acids (13).

It was observed to occur in copper complexes of heparin to a lesser

extent and opposite in sign (14).

On the monomer level, it has been observed in

glucuronoside but not in iduronoside dependence,

(15) and because of its marked solvent

it was assigned to the n ÷ D

transition of unsolvated molecules and

the band at 210 nm to the same transition as molecules hydrogen bonded to water. In the present case however,

solvent interaction can be ruled out because this

band is not affected by the presence of 6M GdmCl in HGME.

Listowsky et. al. (15)

and Morris et, al. (13) independently observed that the common feature exhibited by uronic acid derivatives with

peculiar long wavelength band at 234 nm is the

presence of a 0-(4) equatorial orientation.

Thus, only the "normal" 210 nm n ÷ ~

transition is observed for uronic acids with the 0-(4) axial orientation while the 210 nm and 234 nm bands are seen with uronic acids containing the 0-(4) equatorial orientation.

Model building studies done in our laboratory indicate

that the methyl ester group

(i.e., }{ME) can easily be accommodated in the sugar

ring structure, but the glycine and glycine methyl ester units

(i.e., HG, HGME)

cannot be oriented in space without interfering with the highly charged sulfate groups in the molecule.

We therefore believe that the appearance of this long

wavelength band in HG and HGME is due to ring inversion (i.e., IC 4 to 4C I or vice versa) of the uronic acid moiety.

It is only through this kind of rotation

of the chromophore against a disymmetric environment of the rest of the molecule

979

Vol. 123, No. 3, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that large changes in the CD spectra can be effected. course, tentative at this time.

This conclusion is, of

Additional physical monitoring methods,

involving nuclear magnetic resonance and X-ray studies, will be necessary to identify the specific conformers of uronic acids which predominate in these modified heparins. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.

Rosenberg, R.D. (1977) Sem. in Hematol. 14, 427-440. Rosenberg, R.D. and Damus, P.S. (1973) J. Biol. Chem. 248, 6490-6505. Villanueva, G.B. and Danishefsky. I. (1977) Biochem. Biophys. Res. Commun. 74, 803-809. Stone, A.L° (1977) Fed. Proc., 36, 101-106. Park, J.W. and Chakrabarti, B. ~-[977) Biochem. Biophys. Res. Commun. 78, 604-608. Ching Ming Chang, M. and Ellerton, N.F. (1976) Biopolymers 15, 1409-1423. Bitter, T., Muir, H.M. (1962) Anal. Biochem. ~, 330-334. Danishefsky, I. and Siskovic, E. (1972) Thromb. Res. i, 173-182. Fales, H.M., Jaouni, T.M. and Babashak, J.F. (1973) Anal. Chem. 45, 2302-2303. Danishefsky, I. and Siskovic, E. (1971) Carbohyd. Res. 16, 199-205. Danishefsky, I., Ahrens, M. and Klein, S. (1977) Biochem. Biophys. Acta. 498, 215-222. Lasker, S.E. and Stivala, L. (1966) Arch. Biochem. Biophys. 115, 360-372. Morris, E.R., Ress, D.A., Sanderson, G.R. and Thom, D. (1975) J. Chem. Soc. [Perkin II] 1418-1425. Mukherjee, D.C., Park, J.W. and Chakrabarti, B. (1978) Arch. Biochem. Biophys. 191, 393-399. Listowsky, I., Englard, S. and Avigard, G. (1969) Biochemistry 8, 1781-1785.

980