Biochemical SocietyTransacttons ( 1 992)20
Complexationof
363s
ions by heparin.
DAVID GRANT, WILLIAM F. LONG and FRANK B. WILLIAMSON Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, ABERDEEN AB9 1AS. Scotland. Fig. 1 shows the binding of Zn2+ to heparin as measured by polarimetry (open symbols) a n d potentiometric titration (continuous plot). The results reinforce the notion that otentiometric titration affords a rapid and simple mettod by which cation-heparin interaction may be indirectly examined [l].The plot in Fig. 1is linear up to a [Zn2+]/[heparin disaccharide] ratio of about one. Linearity in analo ous plots derived from polarimetric examination of C a k and Cu2+ interaction with heparin, and occurrin at low [cationl/[heparin disaccharide] ratios, have i e e n observed [2,3]. The linearity suggests that the interactive process - presumed to be the formation of a species of cation-heparin complex - which gives rise to this part of the plot, occurs by a mechanism that does not conform to simple reversible equilibrium thermodynamics. The plot suggests that, a t higher [cation]/[heparin disaccharide] ratios, further cation interaction occurs. In the case of the Zn2+/heparin interaction this appears, under the conditions explored in Fig. 1, to approach completion at a [cation]/ [heparin disaccharide] ratio of about 3. It has been suggested that such observations may reveal the production, a t low [cation]/[heparin disaccharide] ratios, of a cationpolyanion complex that resembles a discrete, hydrated colloid, mineral-like state, that is therefore not subject to simple aqueous solution equilibrium processes, and that is stabilised by forces additional to electrostatic ones. A t h i g h e r [cation]/[heparin disaccharide] ratios, destabilization of the phase, generating the non-linear portion of the plot, may occur [2,3].
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
[ZG+l/l&eparindismhan 'del
Fig. 2. Fractions equivalent to isothermal saturation fractions of binding sites on heparin occupied by Zn2+, derived from potentiometric measurements, as functions of [Z&+Ylheparindisa~ha15del: the effect of variation in
m-IZnSO4 was added to 20 cm3 of a solution containing the
following concentrations of heparin (mmol.dm-3) in the absence of NaCI: (a) 0.011; (m) 0.042; (0)0.15; ( 0 ) 0.29; ( 0 ) 0.59. Other details are as given in the legend to Fig. 1.
Fig. 2 shows the near identity, a t low [Zn2+]/[heparin disaccharide] ratios, of potentiometrically derived plots obtained from experiments in which Zn2+ and heparin were mixed under conditions of widely differing 1.2
I
1.0
-
I
I
0 0
0.8 -
0
I
I
4.
0.
0 0.6
-
0.4
-
0.2
-
0.0
'
0.
0. 0 0. 0 0
8
'0
0
I
I
I
I
1
2
3
4
[ZnV/lheparindhc&an 'del
Fig. 1. Fractions equivalent to isothermal s a t u r a t i o n fractions of binding sites on heparin occupied by Zn2+, d e r i v e d f r o m p o l a r i m e t r i c and p o t e n t i o m e t r i c measurements, as f u n c t i o n s of [ Z n 2 + ] / [ h e p a r i n disaccharide] A binding site was taken to be an average heparin disaccharide unit. Fractions equivalent to isothermal saturation fractions and derived from polarimetric ( 0 ) o r potentiometric measurements (4 were obtained, in the absence of NaCI, as described in references [l-31.
[heparin]. Such similarity is also seen i n polarimetrically derived plots in which Ca2+- and Cu2+heparin interactions are explored [2,3] and accords with the idea that processes not conforming to reversible equilibrium thermodynamics are occurring. Further, Fig. 2 shows t h a t the initial Zn2+ addition to low concentrations of heparin is accompanied by the usual decrease in pH, but that, a s the [ Z n 2 + l / [ h e p a r i n disaccharide] ratio is increased by further Zn2+ addition, this pH change is reversed. The source of the protons released during metal cation binding to heparin is not known, but may involve direct or indirect replacement of covalently bound protons by metal cations [4]; some of these protons may be present in hydrogen-bonded structures, including those occurring in heparinassociated water molecules [3]. The results suggest that, under the conditions examined, formation of a putative colloid-like ZnZ+-heparin complex with an accompanying release of protons, may be followed, a s t h e [Znz+]/[heparin disaccharide] ratio is increased, by dissolution of the complex accompanied by proton removal from solution. A proton pumping or tranport system based on the formation and dissolution of particular divalent cation-glycosaminoglycan complexes is an intriguing possibility. 1. Grant, D., Long, W.F. & Williamson, F.B. (1992)
Biochem. J., in press 2. Grant. D.. Long, W.F.. Moffat, C.F. & Williamson, F.B. (1992) Bioch
Complexationof
363s
ions by heparin.
DAVID GRANT, WILLIAM F. LONG and FRANK B. WILLIAMSON Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, ABERDEEN AB9 1AS. Scotland. Fig. 1 shows the binding of Zn2+ to heparin as measured by polarimetry (open symbols) a n d potentiometric titration (continuous plot). The results reinforce the notion that otentiometric titration affords a rapid and simple mettod by which cation-heparin interaction may be indirectly examined [l].The plot in Fig. 1is linear up to a [Zn2+]/[heparin disaccharide] ratio of about one. Linearity in analo ous plots derived from polarimetric examination of C a k and Cu2+ interaction with heparin, and occurrin at low [cationl/[heparin disaccharide] ratios, have i e e n observed [2,3]. The linearity suggests that the interactive process - presumed to be the formation of a species of cation-heparin complex - which gives rise to this part of the plot, occurs by a mechanism that does not conform to simple reversible equilibrium thermodynamics. The plot suggests that, a t higher [cation]/[heparin disaccharide] ratios, further cation interaction occurs. In the case of the Zn2+/heparin interaction this appears, under the conditions explored in Fig. 1, to approach completion at a [cation]/ [heparin disaccharide] ratio of about 3. It has been suggested that such observations may reveal the production, a t low [cation]/[heparin disaccharide] ratios, of a cationpolyanion complex that resembles a discrete, hydrated colloid, mineral-like state, that is therefore not subject to simple aqueous solution equilibrium processes, and that is stabilised by forces additional to electrostatic ones. A t h i g h e r [cation]/[heparin disaccharide] ratios, destabilization of the phase, generating the non-linear portion of the plot, may occur [2,3].
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
[ZG+l/l&eparindismhan 'del
Fig. 2. Fractions equivalent to isothermal saturation fractions of binding sites on heparin occupied by Zn2+, derived from potentiometric measurements, as functions of [Z&+Ylheparindisa~ha15del: the effect of variation in
m-IZnSO4 was added to 20 cm3 of a solution containing the
following concentrations of heparin (mmol.dm-3) in the absence of NaCI: (a) 0.011; (m) 0.042; (0)0.15; ( 0 ) 0.29; ( 0 ) 0.59. Other details are as given in the legend to Fig. 1.
Fig. 2 shows the near identity, a t low [Zn2+]/[heparin disaccharide] ratios, of potentiometrically derived plots obtained from experiments in which Zn2+ and heparin were mixed under conditions of widely differing 1.2
I
1.0
-
I
I
0 0
0.8 -
0
I
I
4.
0.
0 0.6
-
0.4
-
0.2
-
0.0
'
0.
0. 0 0. 0 0
8
'0
0
I
I
I
I
1
2
3
4
[ZnV/lheparindhc&an 'del
Fig. 1. Fractions equivalent to isothermal s a t u r a t i o n fractions of binding sites on heparin occupied by Zn2+, d e r i v e d f r o m p o l a r i m e t r i c and p o t e n t i o m e t r i c measurements, as f u n c t i o n s of [ Z n 2 + ] / [ h e p a r i n disaccharide] A binding site was taken to be an average heparin disaccharide unit. Fractions equivalent to isothermal saturation fractions and derived from polarimetric ( 0 ) o r potentiometric measurements (4 were obtained, in the absence of NaCI, as described in references [l-31.
[heparin]. Such similarity is also seen i n polarimetrically derived plots in which Ca2+- and Cu2+heparin interactions are explored [2,3] and accords with the idea that processes not conforming to reversible equilibrium thermodynamics are occurring. Further, Fig. 2 shows t h a t the initial Zn2+ addition to low concentrations of heparin is accompanied by the usual decrease in pH, but that, a s the [ Z n 2 + l / [ h e p a r i n disaccharide] ratio is increased by further Zn2+ addition, this pH change is reversed. The source of the protons released during metal cation binding to heparin is not known, but may involve direct or indirect replacement of covalently bound protons by metal cations [4]; some of these protons may be present in hydrogen-bonded structures, including those occurring in heparinassociated water molecules [3]. The results suggest that, under the conditions examined, formation of a putative colloid-like ZnZ+-heparin complex with an accompanying release of protons, may be followed, a s t h e [Znz+]/[heparin disaccharide] ratio is increased, by dissolution of the complex accompanied by proton removal from solution. A proton pumping or tranport system based on the formation and dissolution of particular divalent cation-glycosaminoglycan complexes is an intriguing possibility. 1. Grant, D., Long, W.F. & Williamson, F.B. (1992)
Biochem. J., in press 2. Grant. D.. Long, W.F.. Moffat, C.F. & Williamson, F.B. (1992) Bioch
