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The Relevance to Metallomics of the Binding of Metal Ions toHeparin/Heparan Sulphate.Heparin may provide a high capacity multilelement binding matrix of especial relevance to biological metallomic research.

The heparanome and metallome are suggested to interact and modulate the activity of a range offundamental biological processes in animals.

David Grant*(A hypothesis compiled from research carried out at Marischal College, University of Aberdeen*re-copied from a note written 4/2/05 at Turriff AB53)

Summary

The metallome, a new scientific field designated by H. Haraguchi which concerns, inter alia therelationships between the multielement profiles of biological and geological matrices, is now suggestedto be highly relevant to the heparanome, the system of heparin/heparan sulphate polysaccharides whichoccur in multicellular animal and which may provide, in addition to the modulation of many proteinactivities, a wideranging system of homeostasis for metal ions and H+ . The heparanome and themetallome may cross react in vivo

IntroductionRecent discussion of the relevance of the large number (ca. 80) of elements routinely detected by massspectroscopic analysis in biological matrices has led H. Haraguchi (1) to elaborate some of the earlierideas of R.J.P. Williams, and to initiate a new scientific field dealing with the occurrence and relevanceofmultielement matrices for which the term metallomics has been suggested. Implicit in the concept ofmetallomics is the possible significance of connections between geology, inorganic chemistry and

biology The metallome was suggested by Haraguchi to apply to biology principally for the provision ofmetal ions required to generate active sites in proteins (1). It is now suggested that a similar fundamentalrequirement of metal ions as essential cofactors for polysaccharides is also of relevance. Metal ions areknown to occur naturally in anionic polysaccharides (1a,b,c) including heparin (1d) which sequestersCu2+ Ca2+ and Zn2+ (1g) in a physiologically relevant manner. This phenomenon could be critically

relevant to the activity of the heparanome** (2) (the heparin/heparan sulphate and (putatively) metalion dependent signalling system in animal biology (2-9)). Heparin may contain a surprisingly largeamounts of the least abundant of the full range of the ultratrace non-physiological elements (1d) whichare also now known to consistently occur in biological fluids including blood serum (1). While it is nowfairly well established that Cu2+, Ca2+ and Zn2+ are essentially required cofactors for various heparanome-related protein control mechanisms (10), (10b) (some examples of which are listed in Table I), thevarious ultratrace inorganic ions which occur in biological fluids (1) but which currently have no known

physiological function, might conceivably also be involved in heparin/heparan sulphate signalling or itsanthropogenic perturbation.

Medical Use of Metal Ion Binding to Heparin/Heparan SulphateBinding of Eu3+ (11) can be used for heparin assay in blood samples.

Pathological lesions can be imaged (12) by an altered binding of metal ion radiolabels such as 67Ga.Similar use of 111In and 99Tc labelled heparin (13), however, seemed to be less effective.67Ga is believed to bind especially strongly to the heparan sulphates which occur at all adherent cellsurfaces but are subject to structural alteration as a consequence of pathological situations e.g. cellularoncogenic transformation (14).

In vitro studies (12a) confirmed that radioimaging by metal ions is facilitated by an alteration in theanionic density of heparan sulphates associated with pathological lesions (rather than, e.g., transferrin),this being confirmed by an in vitro study which confirmed that a wide range of physiologically relevantmetal ions bind to heparan sulphate (11e). These findings also tend support the notion that heparan


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sulphate probably exists in vivo as a form of specific metallomic matrix.

Participation of Metal Ions in Heparin/Heparan Sulphate Signalling.Heparin/heparan sulphate, which contains a linear encoded information system, is produced by an as yetnot fully understood biosynthesis in the Golgi apparatus, which includes epimerisation, deacetylation andsulphation stages (2-9) but also is subjected to a postsynthetic modification by both enzymic (15) and

non-enzymic (16) (16a) (16b) pathways which are apparently used for the creation and transmission ofencoded information in the form of anionic polysaccharides. These anionic polysaccharides are nowsuggested always to occurin vivo in the form of (multiple) metal ion complexes. This information

processing system apparently also includes possibly complex metal ion-dependent inputs from enzymicand non-enzymic scission by redox metal ion generated radicals as well as by specific redox metal iondependent deaminative cleavage reactions (17).

The postsynthetic coding alteration of heparin/heparan is not restricted, as is DNA, by a requirement topreserve genetic information, so that while the entire signal in heparan sulphate chains is believed to beof physiological significance (e.g. as a sort of biological postcode for defining particular cellular typesandlocations the details of which have not yet been established owing to the lack of sequencing methods asgood as those available for nucleic acids) the heparan sulphate chains are normally utilised after beingspecifically modified to generate fragments containing information packets designed to be read at distantsites (18-20).

Metal Ions Can Link Heparin/Heparan Sulphate Domains to ProteinsA effect of the binding to heparin/heparan of (non-redox) metal ions may be to assist heparan sulphate-

protein binding which at least in some cases, has an absolute requirement for specific divalent metal ions,which are apparently needed to create a correct linkage between the two types of polymers to facilitatenormal heparin/heparan sulphate biochemical control processes.

Cf. the anticoagulant heparin/heparan sulphate antithrombin (III) binding sequence which is the major mammalian bloodanticoagulant mechanism which operates in conjunction with the action of divalent metal ions which similarly can affect growthfactor receptor activation by a process which could further be relevant to the toxic actions ofe.g., barium ions in the aetiology ofdegenerative diseases in which abnormal growth factor activities are apparent.

Table I collects some of the reported evidence for an absolute requirement for the presence of specificdivalent metal ions (e.g. Ca2+ or Zn2+) putatively required to generate the required polysaccharideconformations in order to achieve the correct interactions with their target proteins.These actions, it may be supposed, may be perturbable by such toxic ions as Pb2+, Cd2+ and Ba2+.

It must be emphasised that although metal ions are known (Table I) to have critical roles in themodulation of heparin/heparan sulphate - protein interactions, this may only be discovered fortuitously,as exemplified by how the requirement for the presence of Zn2+ ions to permit endostatin - heparansulphate binding was discovered (10b). Whilst binding to Zn2+ will normally occurin vivo, this bindingwas found to be it is abolished in vitro unless Zn2+ ions, evidently removed during column polysaccharidegel purification processes, were reintroduced. Required metal ions can evidently also bind strongly to the

polysaccharides used for usual chromatographic separations which may therefore be the unexpectedsource of experimental errors.

In vivo, sufficient metal ions for facilitation of the correct protein-heparan sulphate interactions willnormally be present in common physiological fluids but may be absent in sufficient amounts after

column gel fractionations or in physiological saline solutions prepared by use of chemically pure sodiumchloride.

A direct observation of Ca2+ ions linking annexin-V and heparin is shown by the X-ray structure of aheparin oligosaccharide annexin-V complex (10c). Heparin/heparan sulphate and Ca2+ are required forinvivo structure building of functionally active annexin-V aggregates at the plasma membrane.The structure of fibroblast growth factor receptor dimer (10e) shows a similar divalent cation dependent


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interlinking of heparan sulphate to protein.

The Role of Heparin/Heparan in Controlled Assembly/Disassemblyof Solid Phases

The assembly, disassembly and inhibition of crystalline and semi-amorphous substance formation isstrongly affected by the presence of anionic polysaccharides, which includes the important function ofheparin/heparan sulphate for the inhibition of pathological calcification (21) and also for an apparent

antioxidant mechanism involving the removal of Cu2+ (22), Fe2+ (23) and Fe3+ (24) ions from the solutionphase including by the binding of these ions to heparin/heparan sulphate, and their assembly into redox-inert aggregates (25) similarly to how chitosan and hyaluronan have recently been reported (26) toachieve a similar antioxidant protection. Heparin may bind paramagnetic ions in aggregated insolubleforms (26a).

DiscussionPolysaccharides, occurring abundantly throughout biota, characteristically bind to and may, in vivo,naturally contain a wide range of both nutrient and toxic elements present in amounts which shows anapproximate correlation with the amounts of such elements in blood serum and seawater (1).This circumstance seems relevant, inter alia, to a full definition of the scope of metallomics (1) a new

branch of biometal science, which aims to compare and evaluate the relevance of various multielementprofiles throughout biology and geology.

Electrostatic theory (e.g. that of Manning) predicts that polyelectrolytes will bind oppositely chargedcounterions equally for equally charged counterions. In depth studies of the mechanism of binding ofcounterions to heparin (26a) dis not, however, support the Manning electrostatic binding theory, despitethe similarity of the strength of binding found to be achieved of many similarly charged cations toheparin which had been predicted by this theory. The Manning electrostatic attraction theory cannotaccount for the well-established the ability of the ultraanionic heparin anions to strongly to associatedwith SO4

2- anions (27). The non-electrostatic binding activity of polyol groups within allpolysaccharides may, however, be responsible for this. Such a general mode of binding by allpolysaccharides evidently become modulated by additional electrostatic fields present in anionicpolysaccharides exemplified by heparin/heparan sulphate. These formally ultra-anionic polysacchairdesalso show great structural diversity (to which inorganic ions it is now suggested also contribute). Thisflexible diversity apparently enables these molecules to perform a wide range of signalling and control

functions in animal biochemistry. It is therefore predicted that the precise physical chemical andultrastructural details of the counterion profile (and its ability to engage in variable rapid site exchange(cf. 28)) and the possible perturbation of this by anthropogenic input, could be highly relevant to the invivo molecular (/supramolecular) structure forming and physiological activities of these polysaccharides.

Specific metal ions (e.g. Ca2+) have previously been reported to be absolutely required for severalhepain/heparan sulphate protein binding and regulation functions, some examples of these effects beinglisted in Table I. Inorganic cations and anions have also been reported to modulate heparan sulphate

primarly biosynthesis as exemplified in Table II and specific cations may be are required forpostsynthetic structural modification (cf. Table I).

Table I

Examples of facilitation by divalent cations of the binding of heparin/heparanto proteins.


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Cation Process Involving Heparin/Heparan Sulphate | References_____________________________________________________________________________________

M2+ ions Assembly of Annexin-V Capila et al. 2001, ref. (10c)at phospholipid membranes

-------------------------------------------------------------------------------------------------------------------------------------

Mn2+ Serum lipid aggregate formationlow density lipoprotein (LDL) Lindahl & Hook 1978, ref. (4)phospholipid linking M2+ ions

-----------------------------------------------------------------------------------------------------------------------------------M2+ ion Modulation of FGF-2 activity Kan et al. 1996, ref. (10e)Ca2+, divalent cation required for dimerisation ofMg2+ FGF receptor for activationor Mn2+-------------------------------------------------------------------------------------------------------------------------------------Ca2+ Heparin-Mn+-LDL linking Keskes et al. 1983, ref. (29-1)-------------------------------------------------------------------------------------------------------------------------------------Ca2+ Factor X - prothrombin complex Ofosu et al. 1982, ref. (29-2)-----------------------------------------------------------------------------------------------------------------------------------Ca2+ Inhibition of heparin binding to thrombin Spreight & Griffith 1983, ref. (29-3)------------------------------------------------------------------------------------------------------------------------------------Ca2+ Fibronectin Hayashi & Yamada 1982, ref. (29-4)-------------------------------------------------------------------------------------------------------------------------------------

Ca

2+

2 and 3 integrins, Discussed by Kan etal. 1996, ref. (10e)-------------------------------------------------------------------------------------------------------------------------------------Ca2+ Platelet/endothelial

cell adhesion molecule-1 (PECAN-1) Discussed by Kan etal. 1996, ref. (10e)-------------------------------------------------------------------------------------------------------------------------------------Ca2+ Cadhedrin, Discussed by Kan etal. 1996 ref. (10e)-------------------------------------------------------------------------------------------------------------------------------------Ca2+ L-selectin, Koenig et al. 1998, ref. (29-5)

Norgard-Sumnich et al. 1993, ref (29-6)--------------------------------------------------------------------------------------------------------------------------------Ca2+ Serum amyloid P (SAP) Nielson etal. 1994, ref. (29-7)-----------------------------------------------------------------------------------------------------------------------------------Ca2+ Porcine brain synaptosome Shinjo et al. 2004, ref (29-8)

Zhao & Zhang 2003, ref. (29-9)Ca-ATPase inhibition

-----------------------------------------------------------------------------------------------------------------------------------Ca2+ Recycling of heparan sulphate proteoglycans by parathyroid cell lines

is dependent on extracellular Ca2+concentration Takeuchi etal. 1990, ref. (29-10)

-----------------------------------------------------------------------------------------------------------------------------------Ca2+ - NEUROLOGICAL ACTIVITY -Heparan Sulphate

Ca2+ Signaling in neurons induced by S100A4 Kiryushko et al. 2006, ref. (29-11)Glycosaminogycans (especially heparan sulphate)may act as co-receptors of S100 proteins in neurons

-------------------------------------------------------------------------------------------------------------------------------------Ca2+/Na+ Smooth muscle Na+/Ca2+ exchanger

heparin fragments bearing C4-5 unsaturation Schinjo et al. 2004, ref. (29-8)at the non-reducing end, produced by

bacterial lyase activity

Table I, cont.Heparin binds with high affinity to Knaus etal. 1990, ref. (29-12)

voltage-dependent L-type Ca2+ channels

cf. putative Ca2+/Na+ vascular flow sensor via induced heparan conformation alteration Siegel et al. 1998, ref (29-13)

Zn2+ (and Ca2+ ) Modulation of heparan sulphate binding

Zn2+ Inhibition of FGF-2 activity Ricard Blum S etal. 2004 ref. (10b)---------------------------------------------------------------------------------------------------------------------------------Zn2+ Modulation of MRP-8/14 S100 activity Robinson etal. 2002, ref. (29-14)-----------------------------------------------------------------------------------------------------------------------------------


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Zn2+ Fibrillin-1, Tidemann et al. 2001, ref. (29-15)-----------------------------------------------------------------------------------------------------------------------------------Zn2+ H-kininogen. Stanley etal. 1995, ref. (29-16)-------------------------------------------------------------------------------------------------------------------------------------Zn2+ Histidine rich glycoprotein Jones etal. 2006, ref. (29-17)-------------------------------------------------------------------------------------------------------------------------------------Zn2+ Histamine Kerp 1963, ref (29-18)-------------------------------------------------------------------------------------------------------------------------------------

Cu2+ Fibrinogen Lages & Stivala 1973, ref. (29-19)-------------------------------------------------------------------------------------------------------------------------------------Cu2+ Prions Gonzalez-Inglesias etal. 2002, ref. (29-20)----------------------------------------------------------------------------------------------------------------------------------Ca2+ ( or Mg2+) Inhibition of heparin binding to Antithrombin(III) Yamane etal. 1983, ref. (29-21)

-----------------------------------------------------------------------------------------------------------------------------------------------------------------Deaminative cleavage of heparan sulphate via heparan sulphate (e.g. via core protein storage of nitric oxide)

Metal ion modulation promotes scission of syndecan-1 heparan sulphatechains by NO metabolites viaCu+ and Cu2+ redox recycling (putatively involving ascorbate)

enables heparan sulphate oligosaccharide generation e.g. , Ding etal.., 2002, ref. (16a)A possibly related process is theMn2+ dependent soluble guanylate cyclase inhibition by sulphated polysaccharides Liebel etal. 1982, ref. (29-22)

The nitrosative scission of heparin/heparan sulphate at physiological pH was found to occur in the presence of a phosphate buffer butnot an imidazole buffer (Vilaret al., 1997, ref. (16a-1)). This might suggest that trace amounts of recox metals such as Cu and Fe,known to occur in phosphate buffers, might promote this reaction.-------------------------------------------------------------------------------------------------------------------------------------------------------------------Amyloid FormationZn2+ 50nM Zn2+ promotion of binding of heparin to

Amyloid Precursor Protein (APP)

Inhibition of APP proteolysis by heparin abolished by Zn2+ Masters etal. 1993, ref. (29-23)---------------------------------------------------------------------------------------------------------------------------------------------

Inorganic Crystal FormationCalcium Oxalate Crystals or precursor high oxalate concentration Borges et al., 2005, ref. (29-24)

Table IIExamples of Reports of The Effects of Inorganic Cations and Anions on Heparan Sulphate BiosynthesisMn2+ Promotion of heparan sulphate

biosynthesis

------------------------------------------GlcNAc transferase I can use both Mn2+

and Ca2+

but-GlcAc transferase II can use only Mn2+

A.Z. Kalea et al., Biometals 2006, 19,535-546 (ref. 29-25)

------------------------------------T.A. Fritz et al.,J. Biol. Chem., 1994, 269,

28809-28814 (ref. 29-26)

Mg2+ Effect of Mg2+ deficiency on themetabolism of glycosaminoglycans,including heparan sulphate

P. Jaya and P.A. Kurup,J. Biosci., 1986,10, 487-493 (ref. 29-27)

Ca2+

------------------------------------------

Suppression of proteoglycan includingheparan sulphate synthesis by calciumionophore A23187 in culturedvascular endothelial cells--------------------------------------- Y. Fujiwara and T. Kaji , J. Health Sci.,


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Pb2+ Implication of intracellular calciumaccumulation in Pb2+ inhibition ofendothelial proteoglycan includingheparan sulphate biosynthesis

2002, 48, 460-466(ref. 29-28)

Cd2+ Inhibition of the incorporation of [35-S]sulphate into glomerular membranes ofrats chronically exposed to Cd2+ and itsrelation with urinary glycosaminoglycans

and proteinuria

A. Cardenas et al.,Toxicology1992, 76, 219-231 (ref.29-29)

Hg2+, Ni2+ Inhibition of glomerular heparan sulphatebiosynthesis.Metal-proteoglycan interactions in theregulation of renal mesangial cells:implication for metal inducednephropathy.

D.M. Templeton, Proc. Trace ElementHealth Disease, IUPAC Int. Symp.,1990,

p. 209-219 (ref. 29-30)

SiO2 Putative promotion of heparan sulphatebiosynthesis

Si may always occur in heparin/heparansulphate

M.F. McCartyMed Hypoth.,1997, 49, 177-179Cf., R.M. Iler The Chemistry of Silica ,Wiley, 1979, cf. p. 762 (ref. 29-31)

F- F- ions inhibit heparan sulphate sulphation M. Pawalowska Goral et al., Fluoride,1998, 31, 193-201 (ref. 29-32)

SeO42- Inhibits heparan sulphate biosynthesis C.P. Dietrich et al., FASEB J., 1988, 2, 56-

59 (ref. 29-33)

The reports listed in Table I and Table II, taken together, suggest that heparanome associated metal ionscould normally be essential components of the modus operadi of this complex information system inwhich inorganic ions may perhaps act as part of a servo feedback messenger system acting in response toachieve alterations in extracellular metal ion concentrations (similar to the known dependence onextracellular Ca2+ concentration of heparan sulphate biosynthsis in rat parathyroid cells (29-10)).

The X-ray crystal structure of a heparin oligosaccharide annexin-V adduct indicates how interlinking Ca2+

ions might influence the aggregation of annexin V at plasma membranes and how this activity might beperturbed by Ba2+ which is reported to disturb Ca2+ binding to annexin-V (2) this being of relevance forannexin ion channel building, anticoagulant and cellular apoptosis activity.

While Ba2+

occurs in pharmaceutical porcine heparin and also in human scalp hair, there is a largervariation in the reported values for Ba2+ in human hair consistent with an environmental intoxicationsource of this metal (30).

The ability to bind a wide range of elements by heparin/heparan sulphates is found to be shared withother anionic polysaccharides such as those (e.g. alginates and carrageenans) occurring extracellularly inmarine algae. This is the basis of the use of the algal biomass of kelp for plant and animal nutrition (3-4)and for its use for the removal toxic heavy metal ions from polluted waters (5,6) .

Heparin, however is believed to be the most highly anionic polysaccharide, is also readily available sinceit is manufactured as a largely protein-free pharmaceutical agent for blood anticoagulation, mainlyattributable to its content of the antithrombin (III) binding sequence which is also present inanticoagulant-type-HSPG present at vascular walls from which it can be released by proteolytic ornitrosative cleavage (processes which are also subject to perturbation by inappropriate multielements

constituents of blood serum).

Heparin-like structures are known to also to occur in segments of similar structure present in the highlysulphated domains of heparan sulphate (e.g. at endothelial surface and, in liver (31) and in glial cell

progenitors (32).

The physiological role of multi-element binding to heparin may suggest that heparin and relatedpolysaccharides also function as a nutrient sequesters able rapidly to release when required bound Fe2+,Cu2+, Mn2+, Mg2+ and Ca2+ by ion exchange.


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A possible role as a endogenous heavy metal detoxifier can also be suggested from the lower observedefficiency of release of some toxic ions (e.g. Pb2+, Ce4+).The phase change process in which redox iron ions are made unavailable by the promotion by heparin ofthe oxidation of Fe(II) to Fe(III) and formation of stable colloidal particle formation is a process whichalso has been reported to occur with with hyaluronate for the elimination of excess Fe(III) in livingorganisms has been proposed (26), may be an original function of such polyanionic polysaccharidessince this will also protect against free radical damage to DNA (by e.g. Fenton reactions of Fe, Cu, V,

Mn, Cr and Ti ion promoted free radical induced damage).Similar activity may also be relevant to the reported in vivo and in vitro inhibition of lipid peroxidation

by heparin (23a,b).The gathering and safe handling of major and trace nutrients, it might further be suggested, could have

provided the driving force for the early evolution of heparin-like polysaccharides having highly evolvedmetal ion dependent functions.

Heparin has been subjected to numerous in vitro metal ions binding studies (e.g. by Grant et al. (1f) andfurther listed in the Appendix, which confirm the existence of complex binding mechanisms for metalions).In cartilage, the association of metal ions and their aggregates with anionic glycosaminoglycan

proteoglycans is thought to be involved in the modulation of calcification and swelling (33). A study ofsuch effects by NMR relaxation seemed to indicate they arose simply from a physico-chemicalassociation between divalent counterions and chondroitin or dermatan sulphate polysaccharide chains butthe NMR detected effect of metal ions on heparin showed a much greater alteration consistent with amuch greater both monovalent and divalent cation metal ion association (34).Such enhanced polyanionic attraction exerted by heparin seems to be augmented by non-electrostaticforces, since although heparin is the most highly anionic of mammalian biopolymers, it can evidentlysequester elements present in both cationic and anionic forms (27).

The multi-elemental character of heparin is correlated to modern seawater suggests that the requirementfor sequestration of elements from seawater at the time of the early evolution of multicellular animals

prompted the evolution of heparan sulphate proteoglycans (including the evolution of the isomeraseenzyme to convert the glucuronic acid residues of precursor bacterial-like polysaccharides into theiduronate moieties which are better able to bind metal ions further evolved into the complexmorphogenic mechanism

for the development of animals. The amounts of glycosaminoglycans most especially of heparansulphate present in marine invertebrates was found to increase with the salinity of aquatic habitats (24a)The ionic filtration activity of glomerular basement membranes is also believed also to be dependent ontheir heparan sulphate containing anionic sites (24b).

The heparan sulphate nutrient element sequestration function could have encouraged the evolution ofvarious additional mechanisms by which evolutionary pressure could be transmitted via heparan sulphatemicrostructure e.g. by modulation of this by the Hofmeister effects of ionic environments, oxidative(16a,b) and/or nitrosative (16,17) stress or cation-induced alteration in enzymic biosynthesis (this isknown to depend on extracellular Ca2+ (29-10), Mg2+ (29-27) and Mn2+ (29- 25) which can be altered bythe presence of inappropriate levels of heavy such metal ions as Pb2+ (29-28) and Cd2+ (29-29) whichinhibit, respectively the biosynthesis of the core protein and reduce the sulphation of the polysaccharidechains).

Marine algal polysaccharides are known to bind a large variety of counter ions and although this seems tooccur rather non-specifically and at only moderate affinity, this type of binding seems to be that mostsuited to create a potential reservoir and buffer system for major and trace metal ion nutrient binding and

pH control, a situation which also seems to pertain to heparin/heparan sulphate in animals.

The following polysaccharide metallomic derived hypotheses can then be suggested:

1) anionic extracellular polysaccharides which contain highly conserved chemicallycomplex interfaces (such as present in the antithrombin binding site of heparin)generate specific conformations as a consequence of the binding of specific types


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of metal ions.Such stabilisation of anionic polysaccharide shapes by metal ions could have evolved to deal with metalion related processing both being a stimulus for the evolution of the polysaccharides themselves and forthe evolution of adducts between polysaccharides and other biological molecules;

2) heparan sulphate proteoglycans may be so structured as to facilitate the initial uptake, rapid transportand release of nutrient elements and promote heavy metal detoxification processes.

References

(1)H. HaraguchiMetallomics as integrated biometal scienceJ. Anal. At. Spectrom., 2004, 19, 5-14

[The use of modern mass spectroscopic techniques suggests the common occurrence of 50+ multi-inorganicelements in a range of biological matrices (where these often occur with a similar abundance and type to thosewhich occur in the sea) seems to have prompted Haraguchi to suggest that such studies should form the basis of anew branch of science: metallomics.The above review is largely concerned with the relevance of metallomics to proteomics.Earlier reports had, however, also suggested that natural anionic polysaccharide-based matrices occur asmulti-element salts,cf.

(1a)W.A.P. Black and R.L. MitchellTrace elements in the common brown algae and in sea waterJ. Mar. Biol. Assoc. U.K., 1952, 30 (3) 575-584 and

(1b)A. WassermannCation adsorption by brown algae. The mode of occurrence of alginic acidAnn. Bot. N.S., 1949; 13 (49): 81-88.

The studies reported in refs 1a and 1b clearly suggest that anionic polysaccharides in the cell walls of marinealga


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are in the form ofmulti-inorganic metal salts.

More recent studies further confirm this idea. The multi-element contents, in e.g., kelp, has been indicated toderive

largely from the in vivo binding by the extracellular anionic polysaccharides present in the algal cell walls of theionic and particulate multi-inorganic elements present in seawater,Cf.,

(1c)K. Truus, M. Vaher and I.Taure,Proc. Estonian Acad. Sci. Chem., 2001, 50, 95-103,andData for the multi-element analysis ofAscophylum nodosum which is cited in several internet sites (e.g. the

report ofthe multi-elements inEcklonia maxima given in http://www.gairesearch.co.za/kelp.html, and kelp elements

given inhttp://www/alginure.co.uk/ascophylum-nodosum.html which were apparently provided by the Norwegian

Instituteof Seaweed Research.

Cf. also T.A. Davis et al. (Supplementary references)

(1d)Multi-ion content data available to the author for heparin suggests that this material tends always to occur as aextremely well-defined metallomic matrix, cf., the multi elemental composition data briefly reported by D.

Grant etal., in Biochem. J., 1987, 244, p. 143 and more fully discussed by D. Grant in Chemistry Preprint Archive ,

2000,October, 2000, (10), 94-103, showed that a sodium porcine mucosal heparin sample (prior to the final ion

exchangereduction of multi-elements needed to achieve pharmaceutical grade heavy metal ion contents; this provided by

apharmaceutical industry source as being suitable for this type of academic research) was found by massspectrometric analysis to contain, (amounts in g/g)Ca (30000), Si (5900), Cl (5600), K (2000) Fe (1100), F (890) Cu (730), P (440) Ti (


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I. Rodushkin and M.D. AxelssonApplication of double focussing sector field ICP-MS for multielemental characterization of human hair and

nails.Part I. Analytical methodologySci. Tot. Environ., 2000, 250, 83-100]

(1f) The results ofin vitro studies of ion binding by heparin by D. Grant et al. also indicated that a range

of inorganic cations and anions will likely to bind simultaneously to heparin. Cf., D. Grant, W.F. Long and F.B. Williamson:

Infrared spectroscopy of heparin-cation complexesBiochem. J., 1987, 244, 143-149

Similarity and dissimilarity in aspects of the binding to heparin of Ca2+ and Zn2+as revealed by potentiometric titrationBiochem. Soc. Trans., 1996, 24, 203S;

Zn2+-heparin interaction studied by potentiometric titration Ibid., 1992, 287, 849-853;

A potentiometric titration study of the interaction of heparin with metal cations Ibid., 1992, 285, 477-480;

A study of Ca2+ heparin complex formation by polarimetry Ibid., 1992 282, 601-604 Biochem J., 1991, 277, 569-571;

N.m.r spectroscopy of Ca2+-heparin suggests delocalized binding of the cationAbstracts of the 641st Meeting of the Biochemical Society, Issued with The Biochemist,Royal Holloway and Bedford New College 17-20 December 1991,P56 Abstract No 157;

(1g) The normal occurrence of a wide range of metal ions in heparin may also be concluded from the sporadic reports in theliterature of the presence of a variety of individual metal ions, e.g.,G.F. Harrsion and A Sutton, Nature, 1963 (4869) 809, noted the presence of calcium, strontium and barium inheparin and H.J.M. Bowen in Trace Elements in Biochemistry Academic press, London, 1966, p. 63,noted the ubiquitous presence of barium, calcium, copper, manganese, strontium and zinc in heparin (this author also statedthat such multi-inorganic element presence in heparin was normally sufficiently great to disallow the use of procedures usingthis anticoagulant for the evaluation of trace metals (especially manganese) in blood fractions; a similar situation evidentlystill existed in 2005 when the full metallomic range of inorganic elements was confirmed (ref. 1d) to occur in heparinized

blood collection tubes, a circumstance which requires to be addressed for the determination of particular metal ions inblood fractions (it should also be noted that a similar range of inorganic elements may also occur in EDTA, also used as ananticoagulant, but the amounts of inorganic elements present with this reagent were reported (ALS, ref., 1d) however, to be, ona molar anionic site basis, some two orders of magnitude less than was the case with heparin); other possible sources of errorsin inorganic element determination in blood samples evidently can evidently arise from needles etc. and from antimonycatalyst residues leached from PET tube walls (cf. ALS, ref. 1d)).N.W. Alcock had also previously reported (Elem. Metab. Man Anim. Proc. Int. Symp., 4th., 1981 (Pub. 1982) Eds. J.M.

Gawthorme, J.M.M.M.C. Howell, C.L. White p. 678-680, Springer, Berlin; Chem. Abs., 96, 213646) the presence ofpotentially toxic amounts of manganese and chromium in some heparin samples and S.A. Katz (1984) Amer. BiotechnologyLab., 2, 24-30 had reviewed reports of the presence of calcium, copper, manganese, strontium and zinc in heparin (seeming,however, to suggest that the source of such elements was laboratory dust); G. Heinemann and W. Vogt (J. Biol.Trace Elem.Res., 2000, 75, 227-234) had noted the occurrence of variable amounts of vanadium in heparin, and D. Bohreret al. hadreported the occurrence of variable amounts of arsenic (Parenteral Enteral Nutrition, 2004, 29 (1) 1-7) and aluminium (RBAC

(Brasil), 2005, 36 (2) 99-103) in heparin; the aluminium ions in heparin were found to be effectively removed by cationexchange resin percolation; this procedure seemed warranted in some instance prior to the use of the heparin batchesevaluated by these authors as anticoagulants for kidney dialysis patients).

It is likely that individual heparin preparations will show differences in multi-element contents due tooriginal in vivo multi-element contents and work-up procedure (which of course also applies to humanhair samples etc.) and further work is warranted to more fully study this.

(2)J. Turnbull, A. Powell and S. GuimondTrends Cell Biol., 2001, 11, 75-82

[Heparan sulphate protoglycans (HSPGs), glypicans, syndecans, agrin etc.) constitute a majormulticellular animal cellular control system which is believed to be functionally dependent on the

presence of conserved sugar sequences required for specific interactions with proteins]

(3)H.B. Nader, T.M.P.C. Ferreira, L. Toma,S.F. Chavanto, C.P. Dietrich, B. Casu and G. Torri


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Maintenance of heparan sulphate through evolutionCarbohydr. Res., 1988, 184, 292-300

(4)U. Lindahl and M. HookGlycosaminoglycans and their binding to biological macromoleculesAnn. Rev. Biochem., 1978, 47, 385-417

(5)L. Kjellen and U. LindahlProteoglycans: structures and interactionsAnnu. Rev. Biochem., 1991, 60, 443-465

(6)M. Lyon and J.T. GallagherBiospecific sequence and domains of heparan sulphate and the regulation of cellgrowth and adhesionMatrix Biol., 1998, 17 (7) 485-493

Lyon M. and J.T. GallagherCf.J.T. GallagherHeparan sulfate: growth control with a restricted sequence menuJ. Clin. Invest., 2001, 108 (3) 357-361

(7)M. Bernfield, M. Gotte, P.W. Park, O. Reizes, M.I. Fitzgerald, J. Lincecum, and M ZakoFunctions of cell surface heparan sulfate proteoglycansAnn. Rev. Biochem., 1999, 68, 729-777

(8)N. Perrimon and M. BernfieldSpecificities of heparan sulphate proteoglycans in developmental processesNature, 2000, 404, 725-728J. Biol. Chem., 2000, 275, 29923-29926

(9)P.W. Park, O. Reizes and M. BernfieldSpecificities of heparan sulphate proteoglycans : selective regulators ofligand-receptor encountersJ. Biol. Chem., 2000, 275, 29923-29926

(10)W.F. Long and F.B. WilliamsonGlycosaminolycans, calcium ions and the control of cell proliferation

IRCSJ. Med. Sci., 1979, 7, 429-434;[Cf., related studies , of theinorganic biochemistry of heparin/heparan sulphate by the Aberdeen polysaccharide group, initiated

bythe above review, include those described in ref. (10a) , refs (1f) and (21- 23) and other publications

ofD. Grant et al., listed in the Supplementary References}].

(10a)Boyd J., F.B. Williamson and J. Gettins


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Physico-chemical study of heparin. Evidence for a calcium-inducedco-operative conformational transitionJ. Mol. Biol., 1980, 137, 175-190

(10b)S. Ricard-Blum, O. Feraud, H. Lortat-Jacob and M. van der RestCharacterization of endostatin binding to heparin and heparan sulfate by

surface plasmon resonance and molecular modelling: role of divalent cationsJ. Biol. Chem., 2004, 279, 2927-2936

(10c)I. Capila, M.J. Hernaiz, T.R. Mealy, B. Campos, J.R. Dedman, R.J. Linhardt and B.A. SeatonAnnexin V-heparin oligosaccharide complex suggests heparan sulfate-mediatedassembly on cell surfacesStructure (Camb.) 2001, 9, 57-64

(10d)A. Lewit-Bentley, S. Morera, R. Huber and G. BodoThe effect of metal binding on the structure of annexin V and implications formembrane bindingEur. J. Biochem., 1992, 210, 73-77

(10e)M. Kan, F. Wang, M. Kan, T. Bao, J.L Gabriel and W.L. McKeehanDivalent cations and heparin/heparan sulphate cooperate to control assembly andactivity of the fibroblast growth factor receptor complexJ. Biol. Chem., 1996, 271, 26143-26148[These authors reviewed the other cell surface recognition molecules whose structuresand activities are modulated by divalent cations and heparan sulphate or otherglycosaminoglycans in the pericellular matrix, including

2 and 3 integrins, platelet/endothelial cell adhesion molecule-1 (PECAM-1)cadhedrin and L-selectin]

(10f)M. Kan, X. Wu, F. Wang and W.L. McKeeganSpecificity of factors determined by heparan sulfate in a binary complex with receptorkinase

J. Biol. Chem., 1999, 274, 15946-15952[Anticoagulant heparan sulphate is required for FGF receptor divalent cationdependent association between heparan sulphate and the exodomain]

(11)M. Rizk , Y. El-Shabrawy , N.A. Zakhari and S.S. ToubarSpectroscopic determination of heparin sodium using Eu(III) as a probe ionSpectroscop. Lett., 1995, 28 (8) 1235

[Eu3+

formed an adduct with heparin with log10K=5.079]

(12)Y. Hama, T. Sasaki, S. Kojima and A. Kuberoda67-Ga accumulation and heparan sulfate metabolismEur. J. Nucl. Med., 1984, 9, 51-56; Chem. Abs., 100, 188079t

Cf., S. Kojima, Y. Hama, T. Sasaki and A. KuberodaElevated uptake of 67-Ga and increased heparan sulphate contentin liver-damaged rats


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Eur. J. Nucl. Med., 1983, 8, 52-59,and Y. Hama, S. Kojima, and A. KuberodaRelation of heparan sulphate content and67-Ga uptake in various tissues of ratsKagaku Igaku, 1982, 19 (6) 855-861; Chem Abs., 97, 211645e[67-Ga forms stronger complexes with heparan sulphatethan other GAGs allowing tissue distribution of heparan sulphate to be visualised]

S. KojimaUptake of 67-Ga citrate in tissue and heparan sulphateRadioisotopes, 1986, 35 (8) 437-445; Chem. Abs., 191815j

(13)E.g. D.S. Millbraith et. al.,

Eur. Pat. Appl., EP 55028; Chem. Abs., 97, 168966;A. Mostbecket. al.,

Nuklearmedizin Suppt. (Stuttgart), 1981, 18, 343

(14)E.g. N.E. Woodhead, W.F. Long and F.B. WilliamsonThe heparan sulphates of a normal and virus-transformed hamster fibroblastsBiochem. Soc. Trans., 1981, 9, 555-556N.E. Woodhead, W.F. Long WF, F.B. Williamson and W.J. Harrisibid., 1984, 12, 300-301; and (1986)Heparan sulphates from fibroblasts exhibiting a temperature-dependent transformed growth traitIRCS J. Med. Sci. (Lib. Comp.) 14, 427-428W.F. Long and F.B. WilliamsonHeparan structure and the modulation of angiogenesisMed. Hypotheses, 1984, 13, 385-394D. Grant, W.F. Long and F.B. WilliamsonDifferences in the properties of heparin from BHK and PyY cellsEur. J. Cell Biol., 1985, 36, 14Infrared and proton nuclear magnetic resonance spectroscopy of carbohydrates from BHK andPyY cells

ibid., 36,14

A role for glycosaminoglycans in cellular adhesion of relevance to the cancer stateBiochem. Soc. Trans.,1985, 13, 389

D. Grant, W.F. Long, G. Mackintosh and F.B. WilliamsonRoles of heparins and heparans in inflammatory aspects of cancer and the potential ofheparinoids as anti-cancer drugsProc. Int. Congr. Inflamm., Vienna, 10-15 Oct. 1993

(14a)P. Tapanadechopone, S. Tumova, X. Jiang and J.R. CouchmanEpidermal transformation leads to increased perlecan synthesis with heparin-binding growthfactor affinityBiochem. J., 2001, 355 (2) 517-527

(15) Heparanase degradationE.g.C.F. Moffat, W.F. Long, M.W. McLean and F.B. WilliamsonHeparanase-(II)-catalysed degradation of N-propionylated heparinBiochem. Soc. Trans., 1997, 25, S654Heparanase II fromFlavobacterium heparinum. Action on chemically modified heparinsEur. J. Biochem., 1991, 197, 449-459Heparanase II fromFlavobacterium heparinum. HPLC analysis of the saccharides generated fromchemically modified heparin


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Ibid., 1991, 202, 531-541

(16)Nitrous acid degradation of heparin/heparan sulphate - metal ion effectsK. Ding, K. Mani, F. Cheng, M. Belting and L.-A. FranssonCopper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxidederived from intrinsic nitrosothiols

J. Biol. Chem., 2002, 277 (36) 33353-33360cf., K. Mani, M. Jonsson, G. Edgren, M. Belting and L.-A. FranssonGlycobiology, 2000, 10, 577-586[Endogenous internal degradation of heparan sulphate during recycling ofglypican-1 in vascular endothelial cells]

Cf., M. Belting, S. Persson and L.-A. FranssonProteoglycan involvement in polyamine uptakeBiochem. J., 1999, 338, 317-323

(16a)

B. Lahiri, P.S. Lau, M. Pousada, D. Stanton, and I. DanishefskyDepolymerization of heparin by complexed ferrous ionsArch. Biochem. Biophys., 1992, 293, 54-68

Cf.,J.P. LahievBio Metals, 1996, 9 (1) 10; Chem. Abs., 124, 109969t[Fe(II) selectively degrades heparin]

(16b)Z. Liu and A.S. PerlinEvidence of a selective free radical degradation of heparin mediated by cupric ionCarbohydr. Res., 1994, 255, 183-191

(17)Cf., R.E. Vilar, D. Ghael, M. Li, D.D. Bhagat, L.M. Arrigo, M.K. Cowman, H.S. Dweck

and L.Rosenfeld Nitric oxide degradation of heparin and heparan sulphateBiochem. J., 1997, 324, 473-497Cf., D. Ghael et al., Biochem. Mol. Biol. Int., 1997, 43, 183-188[The reason why different buffers support different degrees of reactivity of nitritefor deaminative cleavage of heparin/heparan sulphate can be deduced to arise fromthe presence of trace amounts of copper and iron in phosphate buffers; n.b. thispossibility was not discussed by these authors but arises from unpublished workcarried out in the field by the Author at Aberdeen University]

{Rapid movement of bound ions along anionic polysaccharide chains apparentlypromotes formation of nanoparticles of Fe3+ oxides and thereby protects againstoxidative damage (a possible normal function of hyaluronan in vivo, cf. ref. (26) ) .It is possible that the formation of such Fe(III) nanoparticles is reversed by the action of nitric oxide.Toxic metal ions may therefore play an potentially important role in the

perturbation of such antioxidative activities (suggesting a further hypothesies for how excessivenitric oxide production may promote of degenerative diseases including cancer and multiple sclerosis(to be the subject another communication)}.

(18)M. Herbert and J.P. MaffrandJ. Cell Physiol., 1989, 138, 424-432[Oligosaccharides following internalization show antiproliferative effects onvascular endothelial and smooth muscle cells]


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(19)R. Hahnenberger, A.M. Jakobson, A. Ansari, T. Wehler, C.M. Svahn and U. LindahlLow-sulphated oligosaccharides derived from heparan sulphate inhibit normal angiogenesis .Glycobiology, 1993, 3 (6), 567-573[Inhibition of angiogenesis by low sulphated oligosaccharides from heparan sulphatefor which endothelial cell surface-bound heparan sulphate proteoglycans may

constitute a pool of precursors for anti-angiogenic polysaccharides]

(20)S. Ihrcho, L.E. Wrenshall, B.J. Lindman and J.L. PlattImmunol. Today, 1993, 14, 500-501; Chem. Abs., 120, 51877v[Review on the release of heparan sulphate from endothelial cells duringinflammation and possible regulation by soluble heparan sulphate fragmentsof the functioning of lymphocytes at sites of inflammation]

(21)D. Grant, W.F. Long and F.B. WilliamsonInhibition by glycosaminoglycans of CaCO3 (calcite) crystallizationBiochem. J., 1989, 259, 41-45

Cf., Degenerative and inflammatory diseases may result from defects inantimineralization mechanism afforded by glycosaminglycansMed. Hypotheses, 1992, 38, 49-55

(22)D. Grant, W.F. Long, C.F. Moffat and F.B. WilliamsonCu2+-heparin interaction studied by polarimetryBiochem. J., 1992, 283, 243-246;

Cf., There is a lack of paramagnetic broadening of NMRabsorbances in heparin containing 1100ppm Fe and 730ppm Cu [these values were obtained fromspark source mass spectroscopic analysis (Moffat, Colin F, Ph.D. ThesisSynthesis Characterisation and Applications of Chemically Modified HeparinUniversity of Aberdeen 1987);Cf. alsoD. Grant., W.F. Long and F.B. Williamson

Complexation of Fe2+ ions by heparinBiochem. Soc. Trans., 1992, 20, 361s[This is not a simple thermodynamic process]

(23a)

M.A. Ross, W.F. Long and F.B. WilliamsonInhibition by heparin of Fe(II)-catalysed free-radical peroxidation of linolenic acid

Biochem. J., 1992, 286, 717-720 Cf. M.A. Ross et al.,

Heparin inhibits potentiation of thiobarbituric acid reactive substances inthe presence of linoleic acid and Fe2+ ionsBiochem. Soc. Trans., 1992, 20(4) 364sCf. D. Grant et al. (1996) ref. (23b(

(23b)D. Grant, W.F. Long and F.B. WilliamsonPericellular heparans may contribute to the protection of cells from free radicals


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Med. Hypotheses, 1987, 23, 67-71Cf. also Ross, Marion A Heparin as an Antioxidant M.Sc. Thesis University of Aberdeen,1992D. Grant, W.F. Long, G. Mackintosh and F.B. WilliamsonThe antioxidant activity of heparinBiochem. Soc. Trans., 1996, 24, 194S[Cf. R. Albertini, A. Passi , P.M. Abjuja and G. De LucaThe effect of glycosaminoglycans on lipid peroxidation

Int. J. Mol. Med. 2000, 6,129-136R. Albertini, S. Rindi, A. Passi, G. Palladini, G., Pallavicini and G. De LucaThe effect of heparin on Cu2+-mediated oxidation of human low-density lipoproteinsFEBS Lett., 1995, 377, 240-242], cf. also http://electra.chemistry.upatras.gr/fects/final/w-shops.htm]

(24a)H.B. Nader, M.G.L. Medeiros. J.F. Paiva, V.M.P. Paiva, S.M.B. Jeronimo, T.M.P.C. Ferreira

T.M.P.C.and C.P. DietrichA correlation between the sulphated glycosaminoglycan concentration and degree of salinity of thehabitiat in fifteen species of the classes Cructacea, Pelecypoda and GastropodaComp. Biochem. Physiol., 1983, 76, 433-436[The mathematically exact nature of the sulphated polysaccharide especially for heparan sulphaterequirements for aquatic organisms might point to a primitive osmoeregulatory role; that heparansulphate shows the greatest interspecies variation which is related to aquatic salinity suggests that

thispolysaccharide may have had a primitive evolutionary function, retained to a major extent in modernorganisms, of the provision of multiple inorganic ion binding sites]

(24b)H. Morita, A. Yoshimura, K. Inui, T. Ideura, H. Watanake, L. Wang, R. Soininen and K.

TryggvasonHeparan sulfate of perlecan is involved in glomerullar filtrationJ. Am.Soc. Nephrol., 2005, 16, 1703-1710

(25)M.F. Williamson, W.F. Long and F.B. WilliamsonThe effect of heparin on the u.v. absorption properties of Fe(II) and Fe(III)Biochem. Soc. Trans., 1992, 20, 360S

(26)A.L. Merce, L.C. Marques Carrera, L.K. Santos Romanholi and M.A. Lobo RecioAqueous and solid complexes of iron (III) with hyaluronic acid.Potentiometric titrations and infrared spectroscopy studiesJ. Inorg. Biochem., 2002, 89, 212-218

[Hyaluronan promoted Fe3+

nanoparticle formation was described in this paper] Cf., P. Sipos, O. Berkesi, H, Tombacz, T.G. St Pierre and J. WebbFormation of spheroidal iron(III) nanoparticles sterically stabilized by chitosan in aqueous solutionsJ. Inorg. Biochem., 2003, 95, 55-63[Iron-containing nanoparticles also form at chitosan surfaces]

(26a) 13C n.m.r. spectroscopy of Cu2+- heparin suggests phase separation of the complex from Cu2+ in

aqueous solution Ibid. 1992; 20: 214S and

Cation interaction with heparin at antithrombin-binding sites may differ


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from that occurring elsewhere in the polymerCell Biology International Reports, 1987, 11, 220;

Cation interactions with heparins/heparans are not explicable in terms of simple electrostatic effectsCell Biology International Reports, 1987,11, 221;

Cation complexation with heparin studied by 13C-NMR spectroscopyIRCS Med. Sci., 1986, 14, 903-4;

Binding of copper to heparin. Physiochemical studies suggest interaction ofpharmacological significanceBrit. Soc. Cell. Biol., 1985 36, Suppl. 8 (Abstr. 26), 13

andM.A. Ross, W.F. Long WF , F.B. Williamson and C.F. Moffat CF

Effect of chemically modified heparins , and of heparin fragments onFe(II)-catalysed peroxidation of linoleic acidBiochem Soc Trans., 1992, 20, 216s[cf. Abstracts of the 641st Meeting of the Biochemical SocietyRoyal Holloway and Bedford New College London,17-20 Dec 1991 p. 56 Abstract No. 159]

M.F. Williamson, W.F. Long and F.B. WilliamsonEffect of heparin on the UV absorption properties of Fe(II) and Fe(III)

Biochem. Soc. Trans. 1992, 20, 360s,and

N.E. Woodhead, W.F. Long and F.B. WilliamsonZinc ion binding by heparin

Biochem. Soc. Trans., 1983, 11, 96-97;Biochem. J., 1985, 237, 281-284 and

(26b)D. Grant (2000) Seminar presentation and discussion University of Glasgow(Discussions Relating to Ascorbate/Heparan Sulphate /Upper Stomach Cancer etc.)http://web.ukonline.co.uk/dgrant/dg2/ alsohttp://web.ukonline.co.uk/dgrant/dg1/also http://web.ukonline.co.uk/dgrant/dg4/ and http://web/ukonline.co.uk/dgrant/dg5/cf.http://web.ukonline.co.uk/dgrant/dg8

These notes suggested that the anti-cancer activity of ascorbate is more likely arise from the roles of redox plus non recox metals inasocrbate/nitric oxide determined heparan sulphate fuzzy logic control systems than via collagenor DNA-damage dependentmechanisms (cf., Linus Pauling had suggested that the anti-cancer activity of ascorbate was due to a general promotion of collagencrosslinking and Albert Szent Gyorgi had suggested that anti-cancer actions of ascorbate were due to a DNA protection antioxidant/DNA quantum dot physics mechanism [cf. also the DNA semiconductor hypothesis of B Marczynski, Med. Hypotheses, 1988, 26 (4)239-249 (Carcinogensis as the result of the deficiency of some trace elements)].

(27)Binding of anions to heparin

J.R. Helbert and M.A. MariniStructural studies of heparin II. Exchangeable anionsBiochim. Biophys. Acta, 1964, 83, 120-122[Heparin binds SO4

2- so that the normal presence of inorganic sulphate in heparin can increases thetotal amount of sulphur in heparin by a factor of 2+ greater than that due to the presence of sulphatehalf ester and N-sulphonate groups]Cf.,K.H. SimonNaturwiss Rudschau, 1982. 11, 452-455andL.B. JaquesHeparin: an old drug with a new paradigmScience, 1978, 206, 528-533Cf also., Fo-We (Forschungs und Verweltungs Anstalt)

Brit. Pat. Appl. 890,622 (1962)[Formation of complexes between heparin and inorganic salts]

(28)Z. Liu and A.S. PerlinEvidence of a selective free radical degradation of heparin mediated by cupric ionCarbohydr. Res., 1994, 255,183-191Cf.R.N. Rej, K.R. Holme and A.S. PerlinMarked stereoselectivity in the binding of copper ions by heparin.
http://web.ukonline.co.uk/dgrant/dg1/http://web.ukonline.co.uk/dgrant/dg1/http://web.ukonline.co.uk/dgrant/dg1/http://web.ukonline.co.uk/dgrant/dg8http://web.ukonline.co.uk/dgrant/dg8http://web.ukonline.co.uk/dgrant/dg1/http://web.ukonline.co.uk/dgrant/dg8


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Contrasts with the binding of gadolinium and calcium ionsCarbohydr. Res., 1990, 207, 143-152[Trace amounts (ca 1/185 mol ratio with respect to heparin) of Cu2+ together witha slight molar excess of H2O2 + ascorbate reduced (e.g., by 30% over 30 min at 40

oC)the anti-FactorXa activity of heparin without causing any detectable alteration in the

NMR spectrum of the heparin; Fe2+ caused a less selective alteration in the heparin.Evidently Cu2+ engages in more site specific binding to heparin than does Fe2+]

Tables I &II (Additional References)

(29-1)E. Kecskes, K.G. Bucki, P.I. Bauer., R. Machovich., and I. HorvathThromb. Haemost., 1983, 49, 138-141; Chem. Abs., 99, 3504x[Heparin forms a complex with LDL in the presence of Ca 2+; this complex retains the anticoagulantactivity of the heparin]

(29-2)F.A. Ofosu, G. Modi, A.L. Cerskus, J. Hirsh and M.A. BlajchmanCa binding to heparin inhibits phospholipid dependent assembly of Factor X and

prothrombin activated complexThromb Res., 1982, 28 (4) 487-497; Chem. Abs., 98, 69550

(29-3)M.O. Spreight and M.J. GriffithCalcium inhibits the heparin-catalysed antithrombin III/thrombin reaction bydecreasing the apparent binding of heparin for thrombinArch. Biochem. Biophys., 1983, 225 (2) 958-963

(29-4)M. Hayashi, and K.M. Yamada

Divalent cation modulation of fibronectin binding to heparin and to DNAJ. Biol. Chem., 1982, 257, 5263-7

(29-5)A. Koenig, K. Norgard-Sumnicht, R. Linhardt, and R. VarkiDifferential interaction of heparin and heparan sulfate glycosaminoglycanswith the selectins. Implications for the use of unfractionated and low molecularweight heparin as therapeutic agentsJ. Clin. Invest., 1998, 101 (4) 877-889

(29-6)K.E. Norgard-Sumnich, N.M. Varki, and A. VarkiCalcium-dependent heparin-like ligands for L-selectin in nonlymphoidendothelial cells

Science, 1993, 261, 480-483

(29-7)E.H. Nielson, I.J. Sorensen, K. Vilsgaard, O. Andersen and S.E. SvehagCalcium enhanced aggregation of serum amyloid P and its inhibition by the ligandsheparin and heparan sulphate. An electron microscope and immunoelectrophoretic studyAPMIS, 1994, 102, (6) 420-426; Chem. Abs., 122,181976a

(29-8)S.K. Schinjo, L.L.S. Tersario, V. Olivera, C.R. Nakaie, M.E.M. Ochiro, A.T. Ferreora,


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I.A. Santos, C.P. Dietrich and H.B. NaderHeparin and heparan sulfate disaccharides bind to the exchanger inhibitor

peptide region of the Na+/Ca2+ exchanger and reduce the cytosolic calciumof smooth muscle cell linesJ. Biol. Chem., 2004, 277 (50) 48227-48233

(29-9)

Y. Zhao and X. ZhangHeparin inhibits the reconstituted plasma membrane Ca-ATPase fromPorcine brain synaptosomeGlycoconjugate J., 2003, 19, 373-378

(29-10)Y. Takeuchi, K. Sasaguchi, M. Yanagishita, G.D. Aurbach and V.C. HascallExtracellular calcium regulates distribution and transport of heparan sulfate

proteoglycans in a rat parathyroid cell lineJ. Biol. Chem., 1990, 265 (23) 13661-13668Cf. Y. Takeuchi, M. Yanagashita and V.C. HascallRecycling of transferrin receptors and heparan sulfate proteoglycans in arat parathyroid cell lineJ. Biol. Chem., 1992, 267 (21) 14685-14690

(29-11)D. Kiryushko, V. Novitskaya, V. Soroka, J. Klingelhogfer, E. Ludkanidin, V. Berezin, and E. BockMolecular mechanism of Ca2+ signaling by the S100A4 proteinMol. Cell. Biol., 2006, 26 (9) 3625-3638(29-12)H.-G. Knaus, F. Scheffauer, C. Romanin, H.-G. Schindler and H. GlossmannHeparin binds with high affinity to voltage-dependent L-type Ca2+ channels.Evidence for an agonistic actionJ. Biol. Chem., 1990, 265, 11156-11166

(29-13)

G. Siegel, M. Malmsten and B. LindmanFlow sensing at the endothelium-blood interfaceColloids and Surfaces A: Physiochemical and Engineering Aspects, 1998, 138, 384-351[Heparan sulphate may serve as a vascular flow sensorvia conformation changes elicited mechanicallyand electrostatically by Na + and Ca2+ cation binding]

(29-14)M.J. Robinson, P. Tessier, R. Poulson and N. HoggThe S100 family heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfateglycosaminoglycans on endothelial cellsJ. Biol. Chem., 2002, 277 (5) 3658-3665

(29-15)Tiedemann K.., Batge B., Muller P.K. and Reinhardt D.P. (2001)

Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assemblyJ. Biol. Chem., 276 (380) 36035-36042[Ca2+ dependence of extracellular microfibrils fibrillin-1 binding toheparan sulphate proteoglycan]

(29-16)M.J. Stanley, B.F. Liebersbach, W. Liu, D.J. Anhalt and R.D. SandersonHeparan sulfate-mediated cell aggregationJ. Biol. Chem., 1995, 270 (10) 5077-5083[Aggregation of syndecan-1-transfected cells mediation by divalent cations]


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(29-17)A.L. Jones, M.D. Hulett and C.R. ParishHistidine rich glycoprotein HRG- a novel adapter protein in plasma thatmodulates the immune vascular and coagulation systemsImmunol. Cell Biol., 2006, 83 (2) 106-118

(29-18)L. KerpImportance of zinc for histamine storage in mast cellsIntern. Arch. Allergy Apppl. Immunol., 1963, 22, 112-123Cf. L. Kerp and G. SteinhauserOn a ternary heparin-metalhistamine-complexKlin. Wochschr., 1961, 39, 762-764

(29-19)B. Lages and S.S. StivalaCopper ion binding and heparin interactions of human fibrinogenBiopolymers, 1973, 12, 961-974

(29-20)R. Gonzales-Iglesias, M.A. Pajares, C. Ocal, J.C. Espoinosa, B. Oescg and M. GassetPrion protein interaction with glycosaminoglycan occurs with the formation ofoligomeric complexes stabilized by Cu(II) bridgesJ. Mol. Biol., 2002, 319, 527-540

(29-21)Y. Yamane, S. Saito and T. KoizumiEffects of calcium and magnesium on the anticoagulant action of heparinChem. Pharm. Bull (Tokyo) 1983, 31, (9) 3214-3221; Chem. Abs., 99, 205875e

(29-22)M.A. Liebel and A.A. WhiteInhibition of the soluble guanylate cyclase from rat lung by sulphated polyanions

Biochem. Biophys. Res. Commun., 1982, 104 (3) 957-964; Chem. Abs., 96,138749q

(29-23)C.L. Masters et al.PCT Int. Appl., WO 9310459 (1993); Chem. Abs., 119, 136893bAlzheimers disease is treated by modulation of metal ion/ heparin/amyloid precursor protein (APP);Zn2+ at 50nM promoted heparin binding to APP;Zn2+ abolished a protective effect afforded by heparin of proteolysis of APP

(29-24)F.T. Borges, Y.M. Michelacci, J.A.C. Aguiar, M.A. Dalboni, A.S. Garofalo and N. SchorCharacterization of glycosaminoglycans in tubular epithelial cells: Calcium oxalate and oxalate ionseffects

Kidney Int., 2005, 68, 1630-1642[Kidney tubular cells apparently can upregulate the synthesis of {specifically microstructured?}glycosaminoglycans when cultured in the presence of calcium oxalate crystals or high concentration ofoxalate which can induce the formation of (harmful) calcium oxalate crystals. A servo feedback systemcould be suggested to create {specifically microstructured heparan sulphate molecules} designed to

protect kidney cells from such calcium oxalate crystals or the precursors of such crystals. {N.b.,heparin/heparan sulphate is well known to be an effective inhibitor of calcium oxalate crystallization}].

(29-25)A.Z. Kalea, F.N. Lamari, A.D. Theocharis, D.A. Schuster, N.K. Karamanis and D.J. Klimis-Zacas


21/32

Dietary manganese affects the concentration , composition and sulfation pattern of heparan sulfateglycosaminoglycans in Sprague-Dawley rat aortaBiometals, 2006, 19 (5) 535-546

(29-26)T.A. Fritz, M.M. Gabb, G. Wei and J.D. EskoTwo N-acetylgluocosaminyltransferases catalyse the biosynthesis of heparan sulfate

J. Biol. Chem., 1994, 269 (46) 28809-28814[-GlcNAc transferase I can use both Mn2+ and Ca2+ while-GlcNAc transferase II can only use Mn2+. Mn2+ status may thereforeaffect heparan sulphate proteoglycan biosynthesis by this mechanism]

(29-27)P. Jaya and P.A. KurupEffect of magnesium deficiency on the metabolism of glycosaminoglycans in ratsJ. Biosci., 1986, 10, 487-493

(29-28)Y. Fujiwara and T. KajiSuppression of proteoglycan synthesis by calcium ionophore A23187 in culturedvascular endothelial cells; implication of intracellular calcium accumulation in lead inhibition ofendothelial proteglycan synthesisJ. Health Sci., 2002, 48, 460-466Cf., Y. Fujiwara, and J. KajiLead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan

by proliferating vascular endothelial cells in cultureToxicology, 1999, 133 (2,3) 159-169; Chem. Abs., 131, 154569Cf. T. Kaji, C. Yamamoto and M. SakamotoEffect of lead on the glycosaminoglycan metabolism of bovine aortic endothelial cellsin culture.

Ibid., 1991, 68, 249-257

(29-29)A. Cardenas, A. Bernard and R. Lauwerys

Incorporation of [35-S] sulfate into glomerular membranes of rats chronicallyexposed to cadmium and its relation with urinary glycosaminoglycans and proteinuriaToxicology, 1992, 76, 219-231

(29-30)D.M. TempletonMetal-proteoglycan interactions in the regulation of renal mesangial cells:Implication for metal induced nephropathyProc. Trace Element Health Disease IUPAC Int. Symp.,1990, p. 209-219; Ed., Aito A.

(29-31)M.F. McCartyReported anti atherosclerotic activity of silicon may reflect increasedendothelial synthesis of heparan sulfate proteoglycans

Med. Hypotheses, 1997, 49, 175-176Cf., R.M. Iler, The Chemistry of Silica , Wiley, 1979, cf. p. 762

(29-32)K. Pawalowska-Goral, M. Wardas, W. Wardas and U. MajnuszThe role of fluoride ions in glycosaminoglycan sulphation in cultured fibroblastsFluoride, 1998, 31, 193-202

(29-33)C.P. Dietrich, H.B. Nader, V. Buonassisi and P. Colburn


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Inhibition of synthesis of heparan sulfate by selenate: possible dependence on sulfation for chainpolymerizationFASEB J., 1988, 2, 56-59

(30)M. PurdeyChronic barium intoxication disrupts sulphated proteoglycan synthesis:

A hypothesis for the origin of multiple sclerosisMed. Hypotheses., 2004, 62, 746-754

(31)M. Lyon, J.A. Deakin and J.T. GallagherLiver heparan sulphate structure. A novel molecular designJ. Biol. Chem., 1994, 269 (15) 11208-112115

(32)S.E. Stringer, M. Mayer-Proschel, A. Kalyani, M. Rao and J.T. GallagherHeparin is a unique marker of progenitors of the glial cell lineageJ. Biol. Chem., 1999, 274 (36) 25455-25460

(33)R.D. CampoEffects of cations on cartilage structure: swelling of growth plate and degradation of proteoglycansinduced by chelators of divalent cationsCalcif. Tissue Int., 1988, 43 (2) 108-121

(34)L. Lerner and D.A. TorchiaA multinuclear NMR study of the interactions of cations with proteoglycansheparin and FicollJ. Biol. Chem., 1986, 261, 12706-12714[23-Na, 39-K, 25-Mg and 43-Ca NMR relaxation assessment of cation binding to

heparin]

Supplementary ReferencesOf relevance to the hypothesis that heparin/heparan sulphate function as metallomic matrices

Listed Alphabetically According to First Named Author

Ayotte L., and A.S. PerlinNmr spectrosocpy observations related to the function of sulfategroups in heparin. Calcium binding vs. biological activityCarbohydr. Res., 1986, 145 (2) 267-277

Bodini M.E., and D.T. SawyerElectrochemical and spectroscopic studies of manganese(II), (III) and (IV) gluconateComplexes 2. Reactivity and equilbria with molecular oxygen and hydrogen peroxide

J. Amer. Chem. Soc., 1976, 98 (26) 8366-8371[Formation of hydroxyl radicals by Fenton type reactions from Mn(II) alsosimilar reactions from V(IV), Cr(II), Ti(III) and Fe(II) discussed]

Burger, K., F. Gaizer, M. Pekli, G.Takacsi Nagy and J. SiemrothThe effect of cations on the calcium ion coordination of heparinInorganica Chimica Acta, 1984 92, 173-176[Ca and Zn selective electrodes show complex effects of Li, Na, K and Mg onCa binding; Zn more strongly bound than Ca and Zn binding stronglyreduces K binding]


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Fransson L.-A., I. Carlstedt, L. Coster and A. MalmstromBinding of transferrin to the core protein of fibroblast protoheparan sulfateProc. Natl. Acad. Sci., USA, 1984, 81 (18) 5657-5661(however, cf., A. Schmidtchen et al.Biochem. J., 1990, 265 (1) 289-300, did not confirm the original findings)

Grant D. et al. , Additional References

Grant D., W.F. Long and F.B. WilliamsonAnalysis by infrared spectroscopy of the association of water and metal ions withheparinBiochem. Soc. Trans., 1983, 11, 96

Grant D., C.F. Moffat, W.F. Long and F..B. WilliamsonAltered water structure in mixtures of heparin and metal ions

Ibid.,, 1984, 12, 302

Grant D., W.F., Long and F.B. WilliamsonA role for glycosaminoglycans in cellular adhesion of relevance to the cancer stateBiochem. Soc. Trans., 1985, 13, 389

Grant D., W.F., Long and F.B. WilliamsonPericellular heparans may contribute to the protection of cells from free radicalsMed. Hypotheses, 1987, 23, 67-72

Grant D., W.F. Long and F.B. WilliamsonA model of two conformational forms of heparins/heparans suggested by infraredspectroscopyMed. Hypotheses, 1987, 24, 131-137

Grant D., W.F. Long and F.B. WilliamsonEffect of heparin on dismutation of superoxide anionBiochem. Soc. Trans., 1988,, 16, 1030-1031

Grant D., W.F. Long, C.F. Moffat and F.B. WilliamsonInfrared spectroscopy of chemically modified heparinsBiochem. J., 1989, 261, 1035-1038

Grant D., W.F. Long and F..B. WilliamsonBiochem. J., 1989, 259, 41-45;Heparin-polypeptide interaction. Near-i.r spectroscopy in an anhydrousdispersant allows the involvement of polymer-associated water to be assessed

Grant D., W.F. Long. C.F. Moffat and F.B. WilliamsonInfrared spectra of chemically modified heparinBiochem. J., 1989, 261,1035-1038;

Grant, D., W.F. Long., C.F. Moffat and F.B. WilliamsonInfrared spectroscopy as a method of investigating the conformationof iduronate saccharide residues in glycosaminoglycansBiochem. Soc. Trans., 1990, 18, 1277-1279

Grant D., W.F. Long and F. B. WilliamsonInfrared spectroscopy of heparin suggests that the region 750-950cm-1


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is sensitive to changes in iduronate ring conformation;Biochem. J., 1991, 275, 193-197

Grant D., W. F. Long and F.B. WilliamsonExamination of cation-heparin interaction by potentiometric titrationAbstracts of the 641st Meeting of the Biochemical Society Issued with The Biochemist,Royal Holloway and Bedford New College 17-20 December 1991, P. 56 Abstract No 158;

Grant D., W.F. Long and F.B. WilliamsonA possible ring conformational change of iduronate residues detected by irspectroscopy of aqueous solutions of lithium heparinBiochem. Soc. Trans.,1991, 18 (6) 1281-1282;

Grant D., W.F. Long and F.B. WilliamsonThe dependence on counter-cation of the degree of hydration of heparinBiochem. Soc. Trans., 1991, 18 (6) 1283-1284

Grant D., C.F. Moffat., W.F. Long W.F. and F.B. WilliamsonCa2+ -heparin interaction investigated polarimetricallyBiochem. Soc. Trans., 1991, 19 (4) 391S

Grant D., C.F. Moffat., W.F. Long and F.B. WillliamsonA relationship between cation-induced changes in heparin optical rotation andheparin-cation associated constantsBiochem. Soc. Trans., 1991, 19 (4) 392S

Grant D., C.F. Moffat, W.F. Long and F.B. WilliamsonOptical rotation changes in chemically modified heparins as a guide toanionic groups involved in Ca2+ bindingBiochem. Soc. Trans., 1991, 19, 393S

Grant D., C.F. Moffat, W.F. Long and F.B. WilliamsonCarboxylate symmetric stretching frequencies and optical rotation shifts ofheparin cation complexes

Biochem. Soc. Trans., 1991, 19 (4) 394S

Grant D., W.F. Long and F.B. WilliamsonI.r. spectrosocpic analysis of heparin-polypeptide interactionBiochem. Soc. Trans., 1991, 19 (4) 395S

Grant D., W.F. Long, C.F. Moffat and F.B. WilliamsonPolarimetry of mixtures of Cu(II) ions and chemically modified heparinsBiochem. Soc. Trans., 1991, 20, 2S

Grant D., W.F. Long and F.B. WilliamsonIR spectroscopy of heparan sulphates isolated from the surfaces of normally and

virally transformed fibroblastsNIR News,1992,19-21Cf., Grant D., W.F. Long and F.B. Williamson

NIR spectroscopy shows that animal cell adhesion to non-biological solid surfacesmay require surface water structuring

NIR News, 1992, 22-24(Proc. Int. Conf., Aberdeen, Near Infrared Spectroscopy, 1991 pub. 1992Ed. Murray I,, and Cowe I.A.VCA Weinheim Germany; Chem. Abs., 118 164498z)


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Grant D., W.F. Long and F.B. WilliamsonMultiple-specular-reflectance i.r. spectrocospy ofglycosaminoglycan-cetylpyridinium complexesBiochem. Soc. Trans., 1992, 20(1) 4S

Grant D., W.F. Long and F.B. WilliamsonA putative role for colloidal silicates in primitive evolution deduced in part form their

relevance to modern pathological afflictionsMed. Hypotheses., 1992, 38, 46-48(Later discussions took this hypothesis further. The original background to this hypothesis was theobservations partly reported in Brit Pats 1143014 and 1136016 of biological-like self assembly of silicasols. Different types of silica sols underwent self seeding of new particle growth and therefore a systemof different silica sols (generated by slight difference in initial conditions) seemed to be potentiallycapable of competing with each other for the acquisition of low molecular weight (water soluble) silicicacid and thereby suggested a hypothesis for the generation of high levels of molecular complexity andcellular structures such as the precursors of modern organisms. The natural ability form structures ofincreasing complex structures over geological timescales (akin to a process of Darwinian evolution) can

be suggested to have been driven by the unique abilities of silica sols in this regard; such advantages forobtaining nutrient for growth (initially silicic acid) might have been achieved by the evolution of motilitythis needing energy generation e.g. via mechanisms using sequestered inorganic energy-rich phosphatestructures in the pores of the silica sols; primitive pro-biological polysilicates would have been capablefrom the start of multi-ion adsorption from seawater; evolution of polysaccharides proteins and nucleicacids would have followed from advantages produced by these systems for gaining nutrients.Cf., Grant D., W.F. Long and F.B. WilliamsonDegenerative and inflammatory diseases may result from defects inantimineralization mechanisms afforded by glycosaminoglycansMed Hypotheses, 1992, 38, 49-55This article argued that polyanions similar to polyphosphates and glycosaminoglycansmay have influenced early biological evolution by modulating the morphology,surface chemistry and activity of polyoxyanionic minerals such as silica and apatite

Grant D., W.F. Long and F.B. WilliamsonThe binding of Pt(II) to heparinBiochem. Soc. Trans., 1996, 24, 204S

Grant D., W.F. Long, G. Macintosh and F.B. WilliamsonAntioxidant activity of heparinBiochem. Soc. Trans., 1996, 24 (2) 194S

Grant D., W.F. Long and F.B. WilliamsonIncompletely published (but displayed as conference posters) of studies conducted by procedures similarto these described in ref. (21) showed that the carrageenans and other anionic polysaccharides as wellnatural polyanions such as humic materials, by binding to nascent crystallization nuclei also inhibitCaCO3 crystallization; the highly efficient action of humic polymers is probably of majorrelevance to global CO2 balance in the sea since this seems to potentially be subject to anthropogenic

perturbation by land-derived humic matter e.g. produced following deforestations and intensiveagriculture]

Grant D. (2000)http://web.ukonline.com.uk/dgrant/dg5

Grant D. (2000)Ascorbate and nitric oxide in redox control of heparan sulphatehttp//www.ukonline.co.uk/dgrant/dg4[Are the roles of inorganic cofactors intrinsically different between heparan sulphate and DNA?It may be intrinsically imperative to strongly hinder the access of redox metal ions to DNA. Heparan sulphate occurs abundantly atuniquely accessible extracellular sites in contrast with the DNA location. The suggested normal physiolgical heparan sulphatemultielement sequestration behaviour seems intrinsically to contrast strongly with the perceived situation with DNA which it might be


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anticipated, must be shielded from potential disruption of its encoded information e.g. by Fenton reactions inducible by contact withdamaging metal ions which is prevented in eukaryotes by holding DNA intracellularly, shielded by the cytoskeleton and histones andsubject to the protective actions of antioxidants, high affinity metal ion binding proteins and specific damage correction repairmechanisms. Hence although both heparin/heparan sulphate and DNA contain conceptually similar linearly encoded informationsystems, quite different regulation of metal ion interactions may be required for proper function of these two linearly information

encoded biopolymer systems].

Grant D. (2000)

Metallome-Heparanome Crosstalk HypothesisAlthough there are a large number of possible in vivo relevant multielement biological ligands additionalto specific metal ion ligands such as calmodulin, transferrin and caeruloplasmin (e.g. nucleic acids,

proteins, polysaccharides and thousands of types of lower molecular weight biomolecules) of which theextracellular polysaccharides seem uniquely placed to associate with metal ions, (at often modest but

physiologically relevant affinities) with metal ions present in multielement solutions such as blood serum(1) by several mechanisms (e.g., electrostatic, hydrogen-bonding and phase change engulfment).Multi-metal-ion and multielement binding may determine the behaviour of the unusually ultra-anionicbiological polysaccharide systems including the heparan oligosaccharides are apparently involved in as

yet poorly understood servo-feedback intracelluar signalling involving inorganic ions and particles.Metal ion binding studies of heparin, and mass spectroscopic multielement analysis (by SSMS and ICP-MS) show that heparin acts as an efficient multielement matrix, this was formerly thought to be of trivial

significance arising from contamination during extraction and work-up procedures. However, suchpolysaccharide inorganic complexes may normally arise from an equilibration with physiological media

which normally contain a similar large range of dissolved ions to those present in seawater (or bloodserum). The multielement character of heparin and heparan sulphate is of obvious relevance to themechanism of heparanome protein interactions designed for subsequent easy release of a wide variety ofmetal ions required, e.g., for specific nucleic acid, protein or polysaccharide structure building andrelated functions. an absolute requirement for

specific divalent metal ions can potentiate signalling by growth factors and could be critically relevantfor fundamental studies of the biological roles of heparin/heparan sulphate--metal ion--protein andheparan sulphate--metal ion--nucleic acid interactions and wider mechanisms.{The multielement contents of biological samples, whole cells and complex multi molecular protein,organic and inorganic component solutions such as blood serum and geological matrices such as seawater are now thought to be of fundamental interest to the fuller understanding of the roles of metal ionsin biology. Highly anionic extracellular polysaccharides however could provide suitable metallomicligands.Heparin, it is now suggested is perhaps the single most relevant such ligand since this is the most ultraanionic polysaccharide in biology. The uniquely high multielement binding capacity heparin canuniquely provide insight into mammalian metal ion presence. Further study of the mass spectrosocpicmultielement evaluation of polysaccharides derived from human and animal tissues is warranted}.

Grushka E. and A.S. CohenThe binding of Cu(II) and Zn(II) ions by heparinAnal. Lett., 1982, 15 (B16), 1277-1288

Hamazaki H.Ca2+-mediated association of human serum amyloid P component withheparan sulfate and dermatan sulfateJ. Biol. Chem., 1987, 262,1456-1460(No binding was observed in the absence of added Ca2+ but other

M

2+

ions studied (M: Ba, Cu, Mg, Mn and Sr) did not promote binding)

Herwats L., P. Laszlo and P. GenardHow heparin binds sodium: a sodium-23 NMR study

Noveau J de Chemie, 1977, 1 (2) 173-176

Hu W.-L., and R. ReogoecziHepatic heparan sulphate proteoglycan and the recycling of transferrinBiochem. Cell Biol., 1992, 70, 535-538


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Iler R.K.The Chemistry of SilicaWiley, New York, 1979(Cf., p. 762[silica is bound in tissues with glycosaminoglycans and polyuronides.About 800 ppm SiO2 was bound to purified hyaluronic acid, chondroitin 4-sulfateand heparan sulfate. Silica is also reported to be bound to pectin and alginic acid

Some association of polysaccharide with silica has probably existed since life began]

Jorpes J.E.Heparin: A mucopolysaccharide and an active antithrombotic drugCirculation, 1959, 19, 87-91[A historical review of the discovery and early use of heparin as an anticoagulant]{The high ash content of heparin had noted from the start of scientific interest in heparin. Previously thiswas regarded as impurities}

N.b.,heparin extracted from (mast cells) in mammalan tissue is a pharmaceutical agent and a convenientmodel for elucidating the behaviour of the structurally related heparan sulphate proteoglycan systemmanagement system which apparently most often depends for its activity on the information encodedheparin-like segments.

Karlinsky J.B. and R.H. GoldsteinRegulation of sulfated glycosaminoglycan production by

prostaglandin E2 in cultured lung fibroblastsJ. Lab. Clin. Med., 1989, 114, 170-184

Kazama Y. and Koide T. (1992)Role of Zn and Ca ions in the heparin neutralizing ability of histidine rich glycoproteinThromb Haemostasis, 67 (1) 50 Chem. Abs., 114,187724t

Kjellen L. and U. LindahlProteoglycans: structures and interactionsAnnu. Rev. Biochem., 1991, 60, 443-465

Lewit-Bentley A., S. Morera, R. Huber and G. Bodo

The effect of metal binding on the structure of annexin V and implications formembrane bindingEur. J. Biochem., FEBS., 1992, 210, 73-77

Liang J.N., B. Chakrabarti, L. Ayotte and A.S. PerlinAn essential role for the 2-sulfamino group in the interaction ofcalcium ion with heparinCarbohydr. Res., 1982, 106,101-109

Luck W.Ber Bunsenesgesel Phys, Chem., 1965, 69(1) 69(Cf. also ibid., 826)[Salt effects on the association of water: the explains the Hofmeister effect which may in turn explainthe role of water clusters associated with sulphated polyanions as modulators of protein folding]

Lyon M.E.Specific heparin properties interfere with simultaneous measurement ofionized Mg and ionized CaClin. Biochem., 1995, 28 (1) 79[Time dependent bias was observed in ionized Mg and Caconcentrations with Zn heparin but not with Li or electrolyte balanced heparin]

McKeehan W.L., X. Wu and M. KanRequirement for anticoagulant heparan sulfate in the fibroblast growth factor


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Tajmir-Riahi H.-A.D-Glucose adducts with zinc-group metal ions. Synthesis and spectroscopic and structuralcharacterization of Zn(II), Cd(II) and Hg(II) complexes with D-glucose, and the effects of metal-ion

binding on the sugar anomeric structuresCarbohydr. Res., 1989, 190, 29-37

Templeton D.M.Acceleration of the mercury-induced aquation of bromopentammine Co(III)

by naturally occurring glycosaminoglycansCan. J. Chem., 1987, 65, 2411-2420Timpl R.Structure and biological activity of basement membrane proteinsEur. J. Biochem., 1989, 180, 487-502

Toida T.E. with R.J. Linhardt, et al.Detection of GAGs Cu(II) complex in capillary electrophoresisElectrophoresis, 1996, 17, 341 346; J. Chromatog., 1997, 787 (1-2) 266-270

Vandewalle B., F. Revillon, L. Hornez and J. LefebvreCalcium regulation of heparan sulphate proteoglycans in breast cancer cellsJ. Cancer Res. Clin. Oncol., 1994, 120(7) 389-392 ; Chem. Abs., 121,105487j

Whitfield D.M., J. Choay and B. SarkarHeavy metal binding to heparin disaccharides. I.Iduronic acid is the main binding siteBiopolymers, 1992, 32, 585-596Heavy metal binding to heparin disaccharides. II.First evidence for zinc chelation

Ibid., 1992, 32, 597-619

Williams R.J.P.The biochemistry of sodium, potassium, magnesium and calciumQuart. Rev., 1970, 24 (3) 331-365

Yamaguchi S., T. Yoshioka, M. Utsunomiya, T. Koide, M. Osafune, A. Okuyana and T. SonadaHeparan sulfate in the stone matrix and its inhibitory effect on calcium oxalate crystallizationUrol. Res., 1993, 21 (3) 187-192; Chem. Abs., 119, 243946t[Heparan sulphate is a potent inhibitor of calcium oxalate crystallization in vivo]

Zou S., C.E. Magura and W.L. HurleyHeparin-binding properties of lactoferrin and lysozymeComp. Biochem. Physiol., 1992, 103B (4) 889-895[Biotinylated heparin binding to lactoferrin was dependent on Na, Ca, Cu, Zn and Fe cations]

_____________________________________________________________________________________

Postscript. The following papers which were published after the initial drafting of this document arerelevant to the hypothesis that the metallome and the heparanome cross react.

Rudd T.R. et al., Influence of substitution patterns and cation binding in conformation and activity ofheparin derivatives[The traditional view that signalling by heparin/heparan sulphate depends on the polysaccharide anioncsequence is suggested to be incorrect; the binding of individual cations including K+ and Cu2+

dramatically alters activities (e.g. for regulation of fibroblast growth factor regulation); this indicates thatthe heparanome-metallome interaction concept provides a plausible hypothesis for how heparan sulphate


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exerts critical control behaviour in animal biochemistry]

ZchariaE et al., Newly generated heparanase knock-out mice unravel co-regulation of heparanase andmatrix metalloproteinasesPLoSD ONE 2009, 4 (4): e5181Epub 2009 Apr.10

**The heparanomeThe heparanome is the name recently suggested for the system of animal polysaccharides whichcontain evolutionary conserved (3) domains of mineral-like anionic arrays of sequences of highlysulphated polyiduronate/glucuronate glucosamine N-sulphonate residues.Studies of HSPG biochemistry suggest an especially important role for such informationencoded sequences occurring in side chain polysaccharide structures which act like biological postcodesto facilitate the interaction with conserved HSPG binding sites in proteins.They have well established roles in morphogenesis (providing a reservoir and control system for basicfibroblast growth factor and its receptors, regulation of embryo assembly) mediation of adhesion andmorphogenesis, the provision of links betwen cytoskeleton and extracellular matrix, modulation ofantioxidant activity including the anchoring of endothelial antioxidant enzymes, the assembly of matrix

phosphatidyl-inositol linkages, regulation of blood coagulation and apoptosis, modulation ofsynaptic and neurological activity, and are implciated in the mechanisms of memory, cognition andageing.Although such activities are more pronounced for HSPGs than for other glycosaminoglycansthe latter however share various primitive functions with heparan sulphates especially in the provision

of ion and pH balance, water activity, mechanical support, modulation of collagen fibrillogenesisincluding the transparency of the cornea.Glycosaminoglycans generally provide a regulation of calcification, cell migration, aggregation anddevelopment, a filtration barrier and stabilization of basement membrane, synaptic structures,endothelial surfaces, mediate of tranferrin uptake and have roles in antigen presentation.

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*Home based research continued from former institutional affiliated research at the University ofAberdeen (Marischal College) and discussions with F.B. Williamson and other former AberdeenPolysaccharide Group members and others including R.J.P. Williams (Oxford University) and K.E.L.McColl (Glasgow University).


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