Infrared spectroscopy of chemically modified heparins

Grant, David M.; Long, William F.; Moffat, Colin F.; Williamson, Frank B.

doi:10.1042/bj2611035

Cited by 23 publications

(20 citation statements)

References 17 publications

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“…[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.…”

supporting

confidence: 64%

“…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.…”

mentioning

confidence: 90%

“…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 higher [cation]/[heparin disaccharide] ratios, destabilization of the phase, generating the non-linear portion of the plot, may occur[2,3].…”

mentioning

confidence: 99%

“…2 shows that the initial Zn2+ addition to low concentrations of heparin is accompanied by the usual decrease in pH, but that, a s the [Zn2+l/[heparin 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 the [Znz+]/[heparin disaccharide] ratio is increased, by dissolution of the complex accompanied by proton removal from solution.…”

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Complexation of Zn2+ ions by heparin

Grant

Long

Williamson

1992

Biochemical Society Transactions

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Fig. 1 shows the binding of Zn2+ to heparin as measured by polarimetry (open symbols) and 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. 1 is 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 higher [cation]/[heparin disaccharide] ratios, destabilization of the phase, generating the non-linear portion of the plot, may occur [2,3].-0 -0 -0. 0 . 0 -0. 0 -8 0 '0 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 I I I I 0 0. 0 4. 0.0 ' I I I I 0 1 2 3 4 [ZnV/lheparindhc&an 'del Fig. 1. Fractions equivalent to isothermal saturation 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 functions of [Zn2+]/[heparin 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. 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&+Ylheparin disa~ha15del: the effect of variation in m -I ZnSO4 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.[heparin]. Such similarity is also seen i n polarimetrically derived plo...

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supporting

confidence: 64%

mentioning

confidence: 90%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Complexation of Zn2+ ions by heparin

Grant

Long

Williamson

1992

Biochemical Society Transactions

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“…This suggests that polarimetric measurements indirectly report on some ginding. In an attempt to assess the importance of the a set Of various anionic grou s of heparin to cation bindin chemically moddiedkeparins was prepared, and t e chaflges in their optical rotations, following the addition of Ca +, measured.The source, preparation and ro erties of the he arin 111 and of the chemically modifiefpoyymers derived from it [2] have been described previously. Polarimetry was carried out as described in the two preceding transactions.…”

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confidence: 99%

Optical rotation changes in chemically modified heparins as a guide to anionic groups involved in Ca2+ binding

Grant

Moffat

Long

et al. 1991

Biochemical Society Transactions

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For a series of cations, the maximal change (increase or decrease) in the optical rotation of heparin, seen when a high concentration of cation is added to the olyanion, is directly related to an independently derivJassociation constant for the interaction of that cation with the polyanion (see the preceding transaction). This suggests that polarimetric measurements indirectly report on some ginding. In an attempt to assess the importance of the a set Of various anionic grou s of heparin to cation bindin chemically moddiedkeparins was prepared, and t e chaflges in their optical rotations, following the addition of Ca +, measured.The source, preparation and ro erties of the he arin 111 and of the chemically modifiefpoyymers derived from it [2] have been described previously. Polarimetry was carried out as described in the two preceding transactions. The water contents of Na+ forms of heparin and of modified forms of heparin, a knowledge of which was necessary for the computation of molecular masses of polymer average disaccharide units, were deduced followm calculation of formula weights from uroNc acid residue 81 or, for carboxy-reduced heparin, glucosamine residue [41 assa .Addition of CaC12 solution to he arin and to the modiied polymers caused an increase in tEe optical rotation of the polymer, except in the case of de-N-sulphonated de-0-sulphated N-acetylated he arin, in which case a decrease in optical rotation occurred. gecause the different degrees of hydration of heparin and of the modified polymers contribute to the polymer mass, but are unlikely to contribute directly to experimentally recur& optical rotations are reported directly rather than after conversion to specific rotation values. Quasi-isothermal saturation fractions of binding sites on he arin and on the chemically modified polymers, were calcuyated from polarimetric measurements as described in the two preceding transactions.Following addition to solutions of Na+ forms of the polymers and measurement of consequent changes in polymer optical rotation, plots of quasi-isothermal saturation fractions of binding sites on heparin and of bindin sit on chemically modified heparins as functions of totahCaeii+l were constructed. These showed that, for each polymer, t change in optical rotation was completed when a high [C%] was reached; no fyther change in optical rotation was detectable at a [Ca +]/[heparin dlsaccharidel ratio of 6. The table shows this maximal change in optical rotation for the polymers studied. In each case, modification decreased the extent of the optical rotation chan e. This is consistent with the idea that the chynical mo8fication lowers the affinity of the heparin for Ca +. In quasi-isothermal saturation plots for heparin itself no shown), a linear to non-linear transition occurs at a ;Cab+] /[heparin disaccharide] ratio of about 0.5; above this ratio, a non-linear portion of the lot indicates that binding of the ion is complete at a ratio orabout 1.0. The same transition can be seen in quasi-isot3rmal saturation plots for the ...

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Structure and Physicochemical Characterisation of Heparin

Mulloy

2011

Heparin - A Century of Progress

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Heparin is a member of the heparan sulphate family of glycosaminoglycans, a linear polysaccharide with a complex sequence resulting from the action of post-polymerisation enzymes on a regular repeating disaccharide background. Its overall conformation is rod-like in solution as well as in the solid state, but the conformational fluctuations of iduronate residues give rise to considerable internal motion and variation in local three-dimensional structure. Structure/function relationships and their relation to sequence are still the subject of argument, but new methodologies to tackle the subject are emerging. Heparin as a therapeutic agent and as the object of research may be characterised by numerous physico-chemical techniques. These include chromatographic methods for measurement of molecular weight; a variety of spectroscopic techniques; separation methods for whole polysaccharides, as well as for oligo- and monosaccharides; and mass spectrometric methods for mapping and sequence analysis. The impetus provided by the discovery of heparin contamination with oversulphated chondroitin sulphate has been influential in bringing combinations of many old and new techniques into use to ensure that heparin is sufficiently consistent and pure to be used safely. Synthetic and semi-synthetic heparins are in development and may become reality in the relatively near future.

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Infrared spectroscopy of chemically modified heparins

Cited by 23 publications

References 17 publications

Complexation of Zn2+ ions by heparin

Complexation of Zn2+ ions by heparin

Optical rotation changes in chemically modified heparins as a guide to anionic groups involved in Ca2+ binding

Structure and Physicochemical Characterisation of Heparin

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