Biochemical SocietyTransactions ( 1 9 9 2 ) 20
ComplexationofF& ions by heparin.
1.2 I
DAVID GRANT, WILLIAM F. LONG and FRANK B. WILLIAMSON
1 .o
Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, ABERDEEN AB9 lAS, Scotland.
0.8
A knowledge of the binding of Fez+ to heparins and heparans is relevant in several biochemical and clinical situations. These include inhibition of tumour growth by Fe chelators [l], neural function [2], effects on symptoms of Alzheimer's disease of heparin-like preparations [3] t h a t perhaps affect the Fe-rich areas of the brain implicated in memory function [4], antioxidant effects of glycosaminoglycans [ 5 ] and of Fe-chelating sugars [6], the cause and possible therapy of rheumatoid arthritis 171, a n d modulation of nucleation events i n biocrystallization [8]. F u r t h e r , altered heparan microstructure is a characteristic feature of transformed cells (references cited in 9 and Kojima et al. [lo] showed that the Fe2+-like isotope Ga bound to tumours via their heparan chains, allowing detection of such lesions by nuclear medical techniques. The effectiveness against replication of human immunodeficiency virus of heparin and related substances [ll] may, in part, be related to polyanion-Fe2+ binding and suppression of superoxide formation activity, since superoxide anions are able to activate replication of the virus [12]. Results of experiments involving polarimetry and otentiometric titration suggest that partly irreversible ginding may occur between heparin and Ca2+ or Cu2+ [13,14]. Kinetic effects, influenced by the order and vigour of mixing of the reagents, appear to influence the strength and stoichiometry of binding [15]. A probably comparable observation is the finding that particular glycosaminoglycans apparently occur in a phase, separate from that of their aqueous medium, which allows them to bind Ca2+ [16,17]. Plots analogous to adsorption isotherms derived from potentiometric titrations and olarimeteic measurements, and most readily obtainef when solution components 'are gently mixed, suggest linear increases in the amount of binding with increasing [divalent metal cationl/[heparin disaccharide] ratio, for ratios less than a critical value. This critical value may be calculated from a sum of factors representing amounts of anionic and N-acetyl groups resent in the polymer [13,14], suggesting that thermo&namic equilibria are not involved in the binding rocess under such conditions. In this transaction, ginding of Fez+ to heparin is investigated by potentiometric titration and polarimetric studies. The source and degree of hydration of the heparin have been previously described [18]. For polarimetry, 10 mm3 aliquots of AnalaR FeNH4S04 (from BDH, Poole, U.K.) solution (600 mmol.dm-3) were added to 6 cm3 of a 4.78 mmol.dm-3 solution of Na+-heparin. Optical rotation a t 541 nm increased from 0.1470 degrees arc in the absence of Fez+ to a maximum of 0.1545 a t a [Fe2+l/[heparin disaccharide] r a t i o g r e a t e r t h a n unity. For potentiometric titration, aliquots of a 14.8 mmo1.dm-3 FeNH4S04 solution were added to a solution of Na+heparin (1.2 mmol.dm-3). The maximum pH decrease observed on Fez+ addition was 1.56 pH units, and occurred at a [Fe2+]/[heparin disaccharide] ratio of 2. Quoted heparin concentrations are on a hydrated disaccharide basis. Results were expressed in terms of quasi-isothermal saturation fractions, as described by Grant et al. [13,14]. The results of polarimetric and potentiometric titrations are shown in the figure (graphs indicated by open and closed circles, respectively). The former graph resembles quasi-isotherms obtained under conditions of gentle mixing of components during the binding of other bivalent cations to heparin [13,14]. In the present case, linearity persisted up to a [FeZ+]/[heparin disaccharide] ratio of about 0.5. Possible inflections in the latter graph
d?
361 S
I
I
I
I
0.4
0.8
1.2
1.6
t
0.6 0.4
0.2
nn 0.0
Fe(II)/heparindisaccharide Fig. Polarimetric ( 0 ) and potentiometric ( 0 ) titration of Na+.heparin(4.7 and 1.3moLdm4 solutions respectively) withFeNH@O4 Curve (01 is curve (o)minus curve ( 0 ) . which somewhat resembles similarly obtained results seen when Cu2+ was added to heparin under more vigorous mixing conditions [14,15], may be due to the formation of discrete metal-ion-heparin complexes [ 191. The graph indicated by open squares is constructed by subtraction of the potentiometric titration curve from that obtained polarimetrically. We attribute its form to the effect of separate proton uptake and release processes induced by heparin-Fe2+ interaction. The quantity of protons released is likely to reflect the strength of cationpolyanion binding [20]. Our results indicate that, for Fe2+ binding, this is of a similar order of magnitude to that found for Cu2+ binding [ E l . These results accord with those in which binding to heparin of other bivalent cations has been studied, in suggesting t h a t such interactions cannot readily be described in terms of simple single-phase reversible equilibrium thermodynamics. 1.
2. Garcia-Se ra, L.M. (--. . , 3. Santani, $1(1989) Mod. Pro1 1M
4. H&well, B. (1989) Acta Neurol. Scand. 126, 23-33 5. Ross M.A., Long, W.F. & Williamson, F.B. (1992) Biochem. SOC. Trans. 20, 216s 6. Burkitt, M.J. & Gilbert, B.C. (1991) Free Rad. Res. Cnmmiin. - -______ __.. -14. - 107-122 --7
7. Tud e, C. (1982) New Scient. 93,23 8. Tavkr, M.G.. Greaves. G.N. & Simkiss. K. (1990) Eur. J. Biochem. 192,783-789 9. Woodhead N.E., Long, W.F. & Williamson, F.13. (1986) IRdS J. Med. Sci. (Libr. Com end.) 14,427-428 10. Kojima S., Hama, Y. Sasaki, & Kubodera, A. (1983) dur. J. Nucl. Med. 8,52-59 11. de Clercq E. (1990) Microbiologica 13, 165-178 12. Schreck, k., Rieber, P. & Baeuerle, P.A. (1991)EMBO J. 10,2247-2258 13. Grant, D., Lon , Moffat, C.F. & Williamson, F.R. (1992)Biochem. 282,601-604 14. Grant, D., Lon Moffat, C.F. & Williamson, F.R. (1992)Biochem. f:283,243-246 15. Grant, D., Long, W.F. & Williamson, F.13.(1992) Trans. [third in this series] Biochem. SOC. 16. Dunstone, J.R. (1962)Biochem. J. 85,336-351 17. Dziewiatkowski, D.D. (1987) Calcif. Tissue I n t . 40. 265-269 18. Grant, D., Long, W.F. & Williamson, F.B. (1987) Biochem. J. 244, 143-149 19. Burger, K. Gaizer, F., Pekli,, M., Takacsi Na G. & Siemroth, (1984) Inorg. Chim. Acta 92, 173-7?6 20. Van Wazer, J.R. & Campanella, D.A. (1950) J . A m . Chem. SOC. 72,655-663
3
f.
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1
ComplexationofF& ions by heparin.
1.2 I
DAVID GRANT, WILLIAM F. LONG and FRANK B. WILLIAMSON
1 .o
Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, ABERDEEN AB9 lAS, Scotland.
0.8
A knowledge of the binding of Fez+ to heparins and heparans is relevant in several biochemical and clinical situations. These include inhibition of tumour growth by Fe chelators [l], neural function [2], effects on symptoms of Alzheimer's disease of heparin-like preparations [3] t h a t perhaps affect the Fe-rich areas of the brain implicated in memory function [4], antioxidant effects of glycosaminoglycans [ 5 ] and of Fe-chelating sugars [6], the cause and possible therapy of rheumatoid arthritis 171, a n d modulation of nucleation events i n biocrystallization [8]. F u r t h e r , altered heparan microstructure is a characteristic feature of transformed cells (references cited in 9 and Kojima et al. [lo] showed that the Fe2+-like isotope Ga bound to tumours via their heparan chains, allowing detection of such lesions by nuclear medical techniques. The effectiveness against replication of human immunodeficiency virus of heparin and related substances [ll] may, in part, be related to polyanion-Fe2+ binding and suppression of superoxide formation activity, since superoxide anions are able to activate replication of the virus [12]. Results of experiments involving polarimetry and otentiometric titration suggest that partly irreversible ginding may occur between heparin and Ca2+ or Cu2+ [13,14]. Kinetic effects, influenced by the order and vigour of mixing of the reagents, appear to influence the strength and stoichiometry of binding [15]. A probably comparable observation is the finding that particular glycosaminoglycans apparently occur in a phase, separate from that of their aqueous medium, which allows them to bind Ca2+ [16,17]. Plots analogous to adsorption isotherms derived from potentiometric titrations and olarimeteic measurements, and most readily obtainef when solution components 'are gently mixed, suggest linear increases in the amount of binding with increasing [divalent metal cationl/[heparin disaccharide] ratio, for ratios less than a critical value. This critical value may be calculated from a sum of factors representing amounts of anionic and N-acetyl groups resent in the polymer [13,14], suggesting that thermo&namic equilibria are not involved in the binding rocess under such conditions. In this transaction, ginding of Fez+ to heparin is investigated by potentiometric titration and polarimetric studies. The source and degree of hydration of the heparin have been previously described [18]. For polarimetry, 10 mm3 aliquots of AnalaR FeNH4S04 (from BDH, Poole, U.K.) solution (600 mmol.dm-3) were added to 6 cm3 of a 4.78 mmol.dm-3 solution of Na+-heparin. Optical rotation a t 541 nm increased from 0.1470 degrees arc in the absence of Fez+ to a maximum of 0.1545 a t a [Fe2+l/[heparin disaccharide] r a t i o g r e a t e r t h a n unity. For potentiometric titration, aliquots of a 14.8 mmo1.dm-3 FeNH4S04 solution were added to a solution of Na+heparin (1.2 mmol.dm-3). The maximum pH decrease observed on Fez+ addition was 1.56 pH units, and occurred at a [Fe2+]/[heparin disaccharide] ratio of 2. Quoted heparin concentrations are on a hydrated disaccharide basis. Results were expressed in terms of quasi-isothermal saturation fractions, as described by Grant et al. [13,14]. The results of polarimetric and potentiometric titrations are shown in the figure (graphs indicated by open and closed circles, respectively). The former graph resembles quasi-isotherms obtained under conditions of gentle mixing of components during the binding of other bivalent cations to heparin [13,14]. In the present case, linearity persisted up to a [FeZ+]/[heparin disaccharide] ratio of about 0.5. Possible inflections in the latter graph
d?
361 S
I
I
I
I
0.4
0.8
1.2
1.6
t
0.6 0.4
0.2
nn 0.0
Fe(II)/heparindisaccharide Fig. Polarimetric ( 0 ) and potentiometric ( 0 ) titration of Na+.heparin(4.7 and 1.3moLdm4 solutions respectively) withFeNH@O4 Curve (01 is curve (o)minus curve ( 0 ) . which somewhat resembles similarly obtained results seen when Cu2+ was added to heparin under more vigorous mixing conditions [14,15], may be due to the formation of discrete metal-ion-heparin complexes [ 191. The graph indicated by open squares is constructed by subtraction of the potentiometric titration curve from that obtained polarimetrically. We attribute its form to the effect of separate proton uptake and release processes induced by heparin-Fe2+ interaction. The quantity of protons released is likely to reflect the strength of cationpolyanion binding [20]. Our results indicate that, for Fe2+ binding, this is of a similar order of magnitude to that found for Cu2+ binding [ E l . These results accord with those in which binding to heparin of other bivalent cations has been studied, in suggesting t h a t such interactions cannot readily be described in terms of simple single-phase reversible equilibrium thermodynamics. 1.
2. Garcia-Se ra, L.M. (--. . , 3. Santani, $1(1989) Mod. Pro1 1M
4. H&well, B. (1989) Acta Neurol. Scand. 126, 23-33 5. Ross M.A., Long, W.F. & Williamson, F.B. (1992) Biochem. SOC. Trans. 20, 216s 6. Burkitt, M.J. & Gilbert, B.C. (1991) Free Rad. Res. Cnmmiin. - -______ __.. -14. - 107-122 --7
7. Tud e, C. (1982) New Scient. 93,23 8. Tavkr, M.G.. Greaves. G.N. & Simkiss. K. (1990) Eur. J. Biochem. 192,783-789 9. Woodhead N.E., Long, W.F. & Williamson, F.13. (1986) IRdS J. Med. Sci. (Libr. Com end.) 14,427-428 10. Kojima S., Hama, Y. Sasaki, & Kubodera, A. (1983) dur. J. Nucl. Med. 8,52-59 11. de Clercq E. (1990) Microbiologica 13, 165-178 12. Schreck, k., Rieber, P. & Baeuerle, P.A. (1991)EMBO J. 10,2247-2258 13. Grant, D., Lon , Moffat, C.F. & Williamson, F.R. (1992)Biochem. 282,601-604 14. Grant, D., Lon Moffat, C.F. & Williamson, F.R. (1992)Biochem. f:283,243-246 15. Grant, D., Long, W.F. & Williamson, F.13.(1992) Trans. [third in this series] Biochem. SOC. 16. Dunstone, J.R. (1962)Biochem. J. 85,336-351 17. Dziewiatkowski, D.D. (1987) Calcif. Tissue I n t . 40. 265-269 18. Grant, D., Long, W.F. & Williamson, F.B. (1987) Biochem. J. 244, 143-149 19. Burger, K. Gaizer, F., Pekli,, M., Takacsi Na G. & Siemroth, (1984) Inorg. Chim. Acta 92, 173-7?6 20. Van Wazer, J.R. & Campanella, D.A. (1950) J . A m . Chem. SOC. 72,655-663
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