ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 1, November,

Optical

Properties

DULAL

AND BIOPHYSICS pp. 393-399, 1978

of C&II) Complexes Glycosaminoglycans

with Heparin

C. MUKHERJEE,*, ’ JOON W. PARK,*, BIRESWAR CHAKRABARTI*, t ’

’ Department of Retina Research, Eye Research Institute of Retina Foundation, 02114 and t Department of Ophthalmology, Harvard Medical School, Boston, Received

May

10, 1978; revised

July

and Related

*

AND

Boston, Massachusetts Massachusetts 02115

18, 1978

Absorption and circular dichroism measurements have been carried out to obtain information regarding the stability and the nature of complexes between Cu(I1) and heparin, and between Cu(I1) and related glycosaminoglycans. In the presence of Cu(II), all glycusaminoglycans, except keratan sulfate, show a characteristic absorption band near 237 nm, which we assign to charge-transfer bands involving ligands to metal ion. From the absorbance values, the formation constants of Cu(II)-heparin and Cu(II)-N-desulfated heparin have been determined to be approximately 1 X lo4 and 2 X 10’ mol-‘, respectively. The large difference in the stability constant values is attributed to the difference in the charge density of the polymers, and to involvement of more than one ligand in the case of heparin. The CD characteristics of the Cu(II)-heparin complex suggest that both carboxyl and sulfamino groups are involved as ligands. The appearance of extrinsic CD bands in heparin, heparan sulfate, dermatan sulfate, and N-de&fated heparin at pH > 5 is ascribed to asymmetry of chelate rings. Absence of CD change in chondroitin sulfate and N-desulfated heparin (pH < 5) in the presence of Cu(I1) suggests that only the carboxyl group is involved in those complexes. The differences either in iduronic acid conformation (C-l vs. I-C) or in intersaccharide linkages between dermatan sulfate and heparin (or heparan sulfate) are revealed in the difference CD spectra between the complexes and the polymers. The change in the intrinsic Cotton effect on complex formation is accounted for as a change in spatial orientation of the ligand groups rather than as a major conformational change of the polymers.

Glycosaminoglycans are closely related acidic polysaccharides. These polymers are all generally described (1) as linear-chain polymers consisting principally of a sequence of disaccharide repeating units of hexosamine (glucosamine or galactosamine) and hexauronic acid (iduronic or glucuronic acid). One exception to this is keratan sulfate which bears D-gahXtOSe instead of uranic acid. The hexosamine component is either N-sulfated, i.e., heparin and heparan sulfate, or N-acetylated. Besides the carboxylic group in the uranic acid moi’ On leave of absence from cutta, Chemistry Department, ‘Present address: Korean Chemical Technology, Daeduk, ’ To whom all correspondence

ety, all GAG4 but hyaluronic acid contain O-sulfate groups. It has been recognized that the nature of glycosidic linkages and the substituents in the sugar rings determine the optical characteristics (2-4), x-ray diffractograms (5), and interaction properties (6) of GAG. The reactive groups such as sulfamino, carboxyl, and O-sulfate could be determining factors of the molecular properties, and hence the physiological function (2), of the polymers such as the anticoagulant activity of heparin (7). The reactivity of groups may be studied in terms of equilibria of interaction with

the University of CalCalcutta, India. Research Institute of Korea. should be addressed.

4 Abbreviations used: GAG, glycosaminoglycans; KS, keratan sulfate; HP, heparin; HPS, heparan sulfate: ChS, chondroitin sulfate; N-DSHP, N-desulfated hepariq DS, dermatan sulfate. 393 0003-9861/78/1911-0393$02.00/O Copyright AU rights

0 1978 by Academic of reproduction in

any

Press, form

Inc. reserved.

394

MUKHERJEE,

PARK,

small molecules or ions. The interaction of metal ions with the biological macromolecules are either structurally or functionally important, or both. The effect of metal ions on the hydrodynamic properties of HP (8-10) and optical properties of hya.luronate (11-13) have been studied extensively. A reversible conformational change of HP (10) and hyaluronate (13) was suggested on binding Cu(I1) ion. The biological significance of the interaction between copper ion and protein is well understood, but the interaction involving GAG is not. It has been suggested (7), however, that the binding of Cu(I1) may form the basis for assaying the anticoagulant activity of heparin. The equilibria between Cu(I1) and GAG are of particular interest because of the stability and the unique spectral properties of the complexes formed. The present communication describes the results of absorption and circular dichroism measurements of complexes formed between Cu(I1) and GAG. The studies provide a better and simpler method for the determination of stability constants of these complexes, and the spectral characteristics also reveal information regarding the nature of ligand groups and their spatial disposition relative to one another, and thus, the conformation of the polymer. MATERIALS

AND

METHODS

HP of USP unit 170 and chondroitin sulfate were purchased from Sigma Chemicals. Pyridine salt of HP was hydrolyzed in 95% MezSO-5% water at 5O“C for 2 h to yield N-desulfated heparin. Standard samples of HPS (33% iduronic acid, 41% N-sulfate), dermatan sulfate, and KS were kindly provided by Prof. M. B. Mathews, University of Chicago. Analytical quality cupric sulfate, calcium chloride, sodium chloride, and sodium cacodylate were used. Standard stock solutions of GAG were prepared by dissolving vacuum-dried samples in glass-distilled water. Solutions of different pH values were obtained by mixing two solutions having the same Cu(II)-polymer concentration but different pH values, in different proportions. When well defined pH values of the solutions were desired, 0.01 M cacodylate-buffer was used. Difference absorption spectra of the complex were recorded in a Cary-15 spectrophotometer as described (12). All CD spectra were recorded in a Cary60 spectropolarimeter using cells of different path lengths (0.2 to 1.0 cm). Concentrations of the polymers were determined by the modified carbazole reaction

AND

CHAKRABARTI

method, as described previously (13) and calibrated with standard solutions. The molar ellipticity values were expressed in terms of the disaccharide repeating unit, and the average dirneric formula weight for HP and HPS wereassumed to be 563 and 500, respectively. Structural heterogeneity was not considered for other samples. No correction was made for the refractive index of the solvent. RESULTS

Absorption Spectra and stants of the Complexes

Stability

Con-

Upon addition of Cu(II), alI GAG, except KS, exhibit an absorption band around 237 nm in difference spectra. At a given pH and HP concentration, the absorbance of difference spectra increases monotonically with increasing concentration of Cu(I1) and reaches a limiting value; for a 0.4 mg/ml HP solution at pH 4.6, no significant change in absorption was observed when the concentration of Cu(I1) was higher than 1.5 X 10e3 M, suggesting that virtually all of heparin is complexed with Cu(I1) under this condition. The variation of absorbance of HP-Cu(I1) and N-DSHP-Cu(I1) with pH is shown in Fig. 1. In the inset of Fig. 1, we have illustrated the difference absorption spectra of HP-C@) complexes at two different pH values. Since decrease in pH results in the protonation of the anionic sites, the dependence of absorbance on pH can be explained in terms of competitive reactions

PH

FIG. 1. Variation of [AA]zsi of 0.4 mg/ml HP and 0.4 mg/ml N-DSHP with pH in presence of 5 X LO-% Cu(I1). Inset is difference absorption spectra at pH values shown.

OPTICAL

PROPERTIES

OF

Cu(I1)

395

COMPLEXES

between H+ and Cu(I1) to the same anionic sites. Generally, when a metal ion, M, forms a 1:l complex with an anion, A-, of the acid, HA, the equilibrium relations between these species can be represented (disregarding the charges of the complex and the metal ion) as

@+][A-1 Ka = ([Alo - [A] - [AM])

VI

and

[AMI

(2)

KC = [A-l@410 - [AM]) where K, is the dissociation constant of the acid HA, and K, is the formation constant of the complex AM. [A]0 and [M]o denote the total concentration of the acid and the metal ion. If [M]o >> [Alo and pH < pK,, equations [l] and [2] can be combined and rearranged in an appropriate form

FIG. 2. Plot of log (1 - f)/f from data in Fig. 1.

vs pH for HP-h

[2] for the midpoint, pHBsp~a/2, of the titration

[AA]H’ curve.

=

(II)

[til

Independent study (4) showed that the pK, of N-DSHP value was 4.2 in water. From [H’l _ {[Alo - [AM11 [Ml0 Fig. 1 and equation [5], the formation conK,K, [AMI stant of N-DSHP-Cu(I1) was calculated to = $f [M]o [31 be about 2 X 10’ mol-’ using the pK, of NDSHP as 3.8 in that condition. Kc for HPCu(I1) obtained from this equation agrees where f - -[AM1 denotes the fraction of the well with the value from Fig. 2. Our experLAJo imental results indicate that the Kc value of acid complexed, and can be related to the HP-Cu(I1) is higher than the values previabsorbance of the difference spectra, hA, ously reported by Lages and Stivala (15). ~ for a given [Alo. [AA]+1 can The anomaly can be explained on the basis by f = [AA]f-l of an interaction of Cu(I1) with the Tris be determined by measuring AA with in- and citrate buffer used by them. creasing Cu(I1) concentration. Equation [3] Equations that are the basis of the calcan be written in a linear form culation of stability constants of complexes derived by considering the equilibpH = pK, + pKa [41 were rium between carboxylate and H’, and be- log[M]o - log(1 - f)/f tween carboxylate and Cu(I1). Protonation A plot of pH against log (1 - f)/f would of sulfate group in low pH may disturb the give a straight line and Kc can be calculated equilibrium either by changing ionic from the plot using known values of pK, strength of media or by varying charge and [M]o. Figure 2 shows the plot of equa- density of the polymer. The good linearity tion [4] for the HP-Cu(I1) system. Assumbetween log (1 - f)/f and pH indicates these ing the pK, value of HP as 4.7 (pK, of HP effects are negligible in our experimental in water is 5.1 (14); HP is assumed to have conditions. Cu(I1) also can bind to sulfate the same salt concentration dependence of group. However, such binding, if any, does pK, as DS (4)), the formation constant of not change appreciably the spectral propHP-Cu(I1) is found to be 1 x lo4 mol-‘. erties near 237 nm. Furthermore, experiWhen [AA]f-l is not known, the Kc value ments with high polymer-to-Cu(I1) ratio still can be determined using the following (not shown) suggested that the binding to relationship derived from equations [l] and sulfate group is much weaker than that to

396

MUKHERJEE,

PARK,

carboxylic group. Hence, overall binding of Cu(I1) to a GAG is mainly determined by binding to carboxylate group. The sharp increase in absorption with pH, when pH is above 5, reflects change in the nature of complexes formed by hydroxylation of Cu(I1) and deprotonation of amine group. The apparent plateau in pH in the range of 4.3 to 4.8 in N-DSHP suggests that these reactions are not significant enough in this nH range to produce large error in the determination of the midpoint of the titration curve, and thus in the K, value. Circular

Dichroism

AND

CHAKRABARTI

tion spectra of the N-DSHP-Cu(I1) system at pH 5.2 and 4.5, normalized at 237 nm, and calculated the difference in the absorbance values in higher wavelength regions. The calculated difference spectrum is included in the inset of Fig. 4, showing an absorption maximum at the same wavelength where the new CD band appears. Difference CD spectra of Cu(I1) complexes of HP, HPS, and DS are shown in Fig. 5, where it is seen that the nature of the 0.0

0

of the Complexes

In the presence of Cu(II), HP shows a new CD band at 235 run, the magnitude of which depends on the concentration of the metal ion and on the pH of the solution. Figure 3 shows the CD spectra of HP at a given HP concentration with varying concentrations of Cu(I1). In the inset of Fig. 3, the variation of A[G] at 235 and 210 rnn is shown; A[01 is the difference between the [0] values of the polymer and the polymerCu(I1) system. In the concentration range of Cu(I1) used in the experiment, we could not record the CD spectra below 200 nm because of high noise level. For a given HP and Cu(I1) concentration, lowering the pH from 6 results in a similar set of CD spectra as observed with decreasing concentration of Cu(I1) at fixed pH: the spectrum near pH 2 is virtually identical with that of HP itself, as expected from absorption data. However, a well defined isobestic point (Fig. 3) was not obtained when pH was varied, due to the different CD characteristics of free and protonated carboxylic groups (4,14) of the polymer. These results are in agreement with a previous report (lo), except that we observed a gradual change and detected no red shift of 235 nm band at higher concentration of Cu(I1). N-DSHP in aqueous solution displays a CD band at 210 nm with similar intensity to that of HP at the same pH. No significant change in the CD features was observed for a mixture of N-DSHP (1.4 mg/ml) and Cu(I1) (3 x 10V3~), when pH is below 4.8. However, when the pH was raised to 5.7, the mixture showed a new CD band around 265 nm (Fig. 4). We compared the absorp-

..I FIG. 3. CD spectra of 1.5 mg/mI HP-Cu (II) at pH 5.8. Cu(I1) concentration is 0.0, 0.25, 0.50, 1.0, 1.5 X 10m3m. Inset is variation of A[e]*, vs A[@]zLo ,,,,,.

I

0.08: 0.040.0

200

243

280

A.nm

FIG. 4. CD spectra of 1.6 mghnl N-DSHP (I) and 1.6 mg/ml N-DSHP-3 x 10m3~ Cu(I1) at pH 5.8. Inset is calculated A[A] of N-DSHP-Cu(II) at pH 5.2.

OPTICAL

PROPERTIES

spectrum of DS-Cu(I1) is different from those of HP-Cu(I1) and HPS-Cu(I1) systems. Upon addition of Cu(II), we could not detect any significant change in the CD spectra of chondroitin, ChS, and KS over a wide range of pH, 2-6. DISCUSSION

Since glucuronic acid, in the presence of Cu(II), shows an absorption band at 235 nm (12), the observed band at 237 nm for all uranic acid-containing GAG must involve the carboxyl group as one of the ligands. The pH dependence of the absorption band supports this view. Studies (16-18) have shown that n- VT*transition of glucuronic acid is located near 225 nm and this magnetic dipole-allowed transition is optically active, displaying two CD bands in the wavelength region of 206-230 nm for two rotational isomers. Iduronic acid (17) also shows a strong CD band near 215 nm, which is presumably due to n- YT* carboxyl transition. The intensity of the absorption band at 237 nm of Cu(II)-GAG complexes is high enough to rule out the possibility of assigning it as a shifted n- r* carboxyl transition; rather, we attribute it to a chargetransfer transition involving ligand to metal ion (12). Since the ligand in this case does not possess any acceptor orbital, metal to ligand charge-transfer can be safely excluded.

knm

FIG. 5. Difference w,ymer, of (I) HP-Cu(II), Cu(I1) at pH 5.8.

CD spectra, [O]cu.po~ymer - [e](II) HPS-Cu(II), and (III) DS-

OF

Cu(I1)

COMPLEXES

397

The stability constant of Cu(II)-HP complex is much higher than that of the complex between N-DSHP and Cu(I1). The difference in stability constants of HPCu(I1) and N-DSHP-Cu(I1) can be explained largely by the charge distribution near the carboxyl group; the negative charge in the N-sulfate group of HP, compared to the positive one in the amine group of N-DSHP, stabilizes the complex. This is supported by the difference in pK, value between these polymers. However, the stability of a complex can not be ascribed solely to electrostatic forces. The effects of chelation may also appear as increased stability constants of complexes. The marked difference in behaviors of CD between these complexes below pH 5 (see following section) suggests chelation of the N-sulfate group in addition to the carboxyl group in the HP-Cu(I1). The Cotton effect near 235 and 265 nm for the Cu(I1) complexes with HP and with N-DSHP, respectively, originates from the stereoselective properties of the complex, since the polymers alone do not show any band in those regions. The accepted explanation (19) of such effect rests on the puckering of chelate rings, where the ligands are bonded to metal ions. Alternatively, the optical activity of the complex might arise from the binding of the chromophoric molecule to the asymmetric site of the polymer. Despite the fact that Cu(I1) binds (as evident from absorption data) to the asymmetric site of the molecules, in the case of hyaluronate and N-DSHP at pH