20/4/05 Modified 18/5/08 Reformatted 25/5/09. Plus additional Figure for submission to e.g. N. P.

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

The heparanome and metallome are suggested to interact and modulate the activity of a range of biological processes in animals David Grant (A hypothesis compiled from research carried out at Marischal College, University of Aberdeen* re-copied in modified form from a note written 4/2/05) Summary It is suggested that the heparanome and the metallome cross react in vivo. Heparins (and putatively heparan sulphates) like alginates, unless specially purified, are apparently chemically so constituted to as to retain the full range of multi-inorganic elements which occur in seawater. Of especial interest is the putative requirement for Ca2+ , Mg2+ and possibly also Mn2+ and Cu2+ ions for the linkage of heparan sulphate anionic patterns to target proteins and the requirements for Cu2+ (and Cu+) and Zn2+ ions for the nitrosative generation of heparan sulphate oligosaccharides required for inter- and intracellular signalling. Introduction The metallome, a scientific field which was originally suggested by R.J.P. Williams (cf. 1) but later redefined by H. Haraguchi (2) to emphasize the inter-relationships between the multielement profiles of biological and geological matrices, e.g., between seawater and human blood serum. Human hair and nails (cf. 3) it should be noted, are also common examples of apparent Haraguchi-multielement-matrices. It should be noted that while it is commonly assumed (e.g. as in the recent review of metallomics by Mounicou et al. (1)) that the biological relevance of metallomics is restricted entirely to the provision of the essential metal ions required for the generation of active sites in metallo-proteins, it can rationally be suggested that the concept of the metallome could also be of value to enable a fuller understanding of mechanisms by which polysaccharides interact with other types of bio-molecules. This could especially

apply to those highly anionic polysaccharides (e.g. alginate (4) and heparin (5)) which apparently exist in their native states as well-defined Haraguchi-type multi-inorganic element matrices. This idea could also be applicable to the heparin-like segments which occur in the heparan sulphate side chains which are the basis of the heparanome (6) (cf. 7) which is thought to play major signalling roles throughout animal biochemistry (8-10) including by its ability to behave as a high capacity flexible information holding and processing system. The mechanism by which this heparanome information code functions could, at least partly, it is now argued, be dependent on the provision of inorganic elements by the metallome. This idea is supported by in vitro studies of the binding of metal ions (11, 12) and inorganic anions (13) to heparin and heparan sulphate, the roles of metal ions in heparin and heparan sulphate biochemical signalling (14) and the evidence that high affinity binding between multivalent metal ion labels and heparan sulphate occurs e.g. during in vivo scintigraphic tissue imaging (15). A further role of metal ions seems to be the regulation of nitrosative and enzymic scission processes (16) which can generate messenger heparan sulphate oligosaccharides which are now believed to exert key biochemical signalling actions.

The inorganic constitution of unpurified heparin suggests that heparin/heparan sulphate will exist in vivo as a metallomic matrix The amounts of the numerous kinds of inorganic elements present in several samples of pharmaceutical heparin showed an approximate correlation with the amounts of such elements present in human blood serum or seawater (as illustrated by Fig. 1); this circumstance equally seems to apply to heparins from different manufacturers, and following the conventional partial single counterion enrichment of heparin achieved by the percolation of multi-inorganic element substituted heparin solutions through a selected single counter-ion substituted forms of ion exchange resin columns. .

Since the existence of the above type of seawater-like multi-inorganic-element profile has also been previously fairly well established to occur with the anionic polysaccharides present at the cell walls of for marine algae (4), the origin of the metallomic range of inorganic elements in heparin is therefore probably not, as was formerly supposed, from contamination arising during industrial work-up, but is more likely to have been determined by the in vivo sequestration of inorganic elements from the seawater-like multiinorganic salt solutions present in the originating animal biological fluids in a similar manner to how the marine alginates acquire their bound multi-inorganic ions and inorganic particles by the sequestration of these moieties from seawater. NMR studies suggested that heparin and related polysaccharides possess a complex inorganic ion binding system which allows inorganic ions to be held both in rapid site change locations, at more secure locations and also to occur e.g., in phase separated locations (cf. 12). The relative amounts of the different types of inorganic elements which have become established to

occur in heparin, putatively under equilibrium conditions, (cf. Fig 1) also suggests the existence of a mechanism whereby a preferential sequestration by heparin of the least abundant elements which are present in biological fluids can occur in preference to the sequestration of the more abundant inorganic elements present in the natural multi-element bathing solution ‘mother liquors’. Heparin in addition to having the ability to bind a wide range of counter cations can (paradoxically) also avidly bind anions (13) (including the halides, sulphate, silicate, borate and phosphate) as well as colloidal particles (e.g. sparingly soluble alkaline earth carbonates as well as iron oxides) apparently so as to achieve a heterogeneous seawater-related metallomic- matrix-status putative inorganic element reservoir.

While biological metallomics is currently focussed on the proteome where every third protein is believed to require a metal cofactor [most often Cu, Fe, Zn or Mo (cf., Mounicou et al., (1)] it is now suggested that metallomics could also be of central relevance to the heparanome (6) , the putative animal cell surface and extracellular matrix polysaccharide-based signalling system (7-11) which is believed to depend on the ability of the ultra-anionic heparin-like segments within the heparan sulphate proteoglycan structure to act e.g., during animal organism development and wound healing by processes which may include roles of inorganic cations and anions which are known to bind to heparin (12,13) and which might also contribute to mechanism by which heparan sulphate binds to specific proteins (14) e.g. for the modulation of cell-cell communication and the operation of a ‘smart glue’ trapping mechanism (cf. 145). Roles for inorganic cations as accessory, or even absolute, requirements for such signalling has been indicated (cf., Kan et al. (14-3) cf., Rudd et al. (11) and Guimond et al. (11)). Such inorganic cations may e.g. effect the conformation of the adjacent polysaccharide chains which could also affect the information signal (or ability to trap target molecules including growth factors and their receptors) by which this polysaccharide control system is able to accomplish the regulation of tissue morphogenesis. This hypothesis is consistent with a range of other literature reports (cf. Tables I and II) which confirms that

metal ions may quite often be required for heparan sulphate signalling. Since this phenomenon per se has been comparatively little studied, however, it seems likely that further examples of this effect may come to light if the possibility of such effects is brought to the full attention of the polysaccharide research scientific community, which this article seeks to accomplish.

Table I Examples of facilitation by divalent cations of the binding of heparin/heparan to proteins. Cation Process Involving Metal Ion Determined Heparin/Heparan Sulphate Protein Linkage References or Analogous Crosslinking | __________________________________________________________________________ M2+ ions Assembly of Annexin-V Capila et al. 2001, ref. (14-3) at Phospholipid membranes ------------------------------------------------------------------------------------------------------------------------------------Mn2+ Serum lipid (LDL) aggregate formation Heparin -M2+ -Phospholipid Lindahl & Hook 1978, ref. (4) ----------------------------------------------------------------------------------------------------------------------------------M2+ ion Modulation of FGF-2 activity Kan et al. 1996, ref. (14-1) Ca2+, 2+ divalent cation required for dimerisation of Mg FGF receptor for activation or 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) ----------------------------------------------------------------------------------------------------------------------------------2+ Ca Inhibition of heparin binding to Thrombin Spreight & Griffith 1983, ref. (29-3) -----------------------------------------------------------------------------------------------------------------------------------Ca2+ Fibronectin Hayashi & Yamada 1982, ref. (29-4) ------------------------------------------------------------------------------------------------------------------------------------Ca2+ β 2 and β3 Integrins, Discussed by Kan et al. 1996, ref. (14-1) Ca2+ Platelet/Endothelial Cell Adhesion Molecule-1 (PECAN-1) Discussed by Kan et al. 1996, ref . (14-1) ) ------------------------------------------------------------------------------------------------------------------------------------Ca2+ Cadhedrin, Discussed by Kan et al. 1996 ref. (14-1) ------------------------------------------------------------------------------------------------------------------------------------2+ Ca L-Selectin Koenig et al. 1998, ref. (29-5) Norgard-Sumnich et al. 1993, ref (29-6) -------------------------------------------------------------------------------------------------------------------------------Ca2+ Serum Amyloid P (SAP) Nielson et al. 1994, ref. (29-7) ----------------------------------------------------------------------------------------------------------------------------------Ca2+ Porcine Brain Synaptosome Shinjo et al. 2004, ref (29-8) Ca-ATPase inhibition Zhao & Zhang 2003, ref. (29-9) ----------------------------------------------------------------------------------------------------------------------------------Ca2+ Recycling of heparan sulphate proteoglycans by parathyroid Takeuchi et al. 1990, ref. (29-10) cell lines Ca2+ Dependent -Heparan Sulphate-Neurological Activity Ca2+ Signaling in neurons induced by S100A4 Kiryushko et al. 2006, ref. (29-11) and heparan sulphate May act as co-receptors of S100 proteins in neurons ------------------------------------------------------------------------------------------------------------------------------------Ca2+/Na+ Smooth muscle Na+/Ca2+ exchanger inhibition heparin fragments bearing C4-5 unsaturation Schinjo et al. 2004, ref. (29-8) at the non-reducing end, produced by bacterial lyase activity Heparin binds with high affinity to Knaus et al. 1990, ref. (29-12) voltage-dependent L-type Ca2+ channels Vascular flow sensor potentiated via Ca2+/Na+ induced heparan conformation alteration Siegel et al. 1998, ref (29-13) Zn2+ (and Ca2+ ) Modulation of heparan sulphate binding 2+ Zn Inhibition of Fibroblast growth factor-2 (FGF-2) activity Ricard Blum S et al. 2004 ref. (10b)

Endostatin --------------------------------------------------------------------------------------------------------------------------------Zn2+ Modulation of MRP-8/14 S100 activity Robinson et al. 2002, ref. (29-14) --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Zn2+ H-Kininogen. Stanley et al. 1995, ref. (29-16) ------------------------------------------------------------------------------------------------------------------------------------Zn2+ Histidine Rich Glycoprotein Jones et al. 2006, ref. (29-17) ------------------------------------------------------------------------------------------------------------------------------------Zn2+ Histamine Kerp 1963, ref (29-18) ------------------------------------------------------------------------------------------------------------------------------------2+ Cu Fibrinogen Lages & Stivala 1973, ref. (29-19) ------------------------------------------------------------------------------------------------------------------------------------Cu2+ Prion Proteins Gonzalez-Inglesias et al. 2002, ref. (29-20) ---------------------------------------------------------------------------------------------------------------------------------Ca2+ ( or Mg2+) Inhibition of heparin binding to Antithrombin(III) Yamane et al. 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 sulphate chains by NO metabolites via Cu+ and Cu2+ redox recycling (putatively involving ascorbate) which enables heparan sulphate oligosaccharide generation e.g. , Ding et al.., 2002, ref. (16a) A possibly related process is the inhibition by 2+ sulphated polysaccharides of a Mn - dependent soluble guanylate cyclase Liebel et al. 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 but not an imidazole buffer (Vilar et al., 1997, ref. (16a-1)). This might suggest that trace amounts of redox metals such as Cu and Fe, known to occur in phosphate buffers, promote this reaction. ------------------------------------------------------------------------------------------------------------------------------------------------------------------Amyloid Formation Zn2+ 50nM Zn2+ promotion of binding of heparin to Inhibition of APP proteolysis by heparin is abolished by Zn2+ --------------------------------------------------------------------------------------------------------------------------------------------Inorganic Crystal Formation Calcium Oxalate Crystals (or precursor high oxalate concentration) Borges et al., 2005, ref. (29-24) Inhibition of formation by heparan sulphate

Table II Examples of Reports of The Effects of Inorganic Cations and Anions on Heparan Sulphate Biosynthesis Mn2+ Promotion of heparan sulphate biosynthesis ----------------------------------------α-GlcNAc transferase I can use both Mn2+ and Ca2+ but α-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 the metabolism of glycosaminoglycans, including heparan sulphate P. Jaya and P.A. Kurup,J. Biosci., 1986, 10, 487-493 (ref. 29-27) Ca2+ -----------------------------------------2+ Pb Suppression of proteoglycan including heparan sulphate synthesis by calcium ionophore A23187 in cultured vascular endothelial cells --------------------------------------Implication of intracellular calcium accumulation in Pb2+ inhibition of endothelial proteoglycan including heparan sulphate biosynthesis Y. Fujiwara and T. Kaji , J. Health Sci., 2002, 48, 460-466 (ref. 29-28) Cd2+ Inhibition of the incorporation of [35-S] sulphate into glomerular membranes of rats chronically exposed to Cd2+ and its relation with urinary glycosaminoglycans and proteinuria A. Cardenas et al.,Toxicology 1992, 76, 219-231 (ref.29-29) Hg2+, Ni2+ Inhibition of glomerular heparan sulphate biosynthesis. Metal-proteoglycan interactions in the regulation of renal mesangial cells: implication for metal induced nephropathy. D.M. Templeton, Proc. Trace Element Health Disease, IUPAC Int. Symp.,1990, p. 209-219 (ref. 29-30) SiO2 Putative promotion of heparan sulphate biosynthesis Si, putatively as SiO2 nanoparticles, may always occur in association with heparin/heparan sulphate M.F. McCarty Med Hypoth.,1997, 49, 177-179 Cf., R.M. Iler The Chemistry of Silica , Wiley, 1979, cf. p. 762 (ref. 29-31) and D. Grant et al. (1992, Med Hypoth 38 46-8 Additional References) FF- 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)

Discussion
The roles of metal ions in heparin/heparan sulphate biochemistry summarized in Table I indicate that metal ions can be required to link (of segments of) heparin/heparan sulphate to phospholipids and proteins and also are required for the generation of the oligosaccharides which are believed to act as messenger molecules which enable cell-cell communication. The role of metal ions in this process includes the Cu2+ Cu+ redox cycling which is required for the release of nitric oxide from conserved cysteines present in heparan sulphate protoeglycans core protein stores, a process which is believed (16-2) to contribute to the regulation of oligosaccharide generation via the deaminative cleavage of heparan sulphate chains by nitric oxide metabolites (for which reaction there also seems to be a a less well-defined catalytic role of Zn2+ ions, which also indirectly affect the alternative mechanism of oligosaccharide production via the action of heparanase which has recently been recently indicated (by Zcharia et al. 16-1) to occur in conjunction with metalloproteinase activity). Other indications of the importance of metal ions in glycosaminoglycan biochemistry are that the physico-chemical behaviour of cartilage is believed to be modulated e.g. for calcification and swelling (18-1) by an association of metal ions with anionic polysaccharides and their aggregates. In related studies, heparin was found to interact more strongly with monovalent and metal divalent cations than did the other glycosaminoglycans which are the main components of cartilage (182). The ability of glycosaminoglycans to inhibit calcification, a phenomenon which has been suggested to be of critical importance to the prevention of blood vessel wall calcification (32,33), is also (putatively) affected by the secondary inorganic elements (which may be augmented by anthropogenic input) which are likely to become enriched in cell wall heparan sulphates since anionic extracellular polysaccharides seem generally to have the ability to simultaneously bind and selectively become

enriched in the least abundant ultratrace elements which occur in natural waters and biological fluids. This multi-element sequestration property is also the basis of the use of the algal biomass of kelp for plant and animal nutrition (cf. 4) and for its use for the removal toxic heavy metal ions from polluted waters (cf. Davis et al. Supplem. refs). This suggests that toxic metal ion trapping by animal polysaccharides may serve similar functions.

A further potential function of metal and other small inorganic ions in the heparanome may be related to an apparent servo feedback system which enables the presence of specific inorganic ions and inorganic surfaces (summarised in Table II) to signal for the de novo synthesis of heparan sulphate microstructures which are putatively generated in response to the extracellular presence of such inorganic entities via alteration of heparan sulphate in the Golgi apparatus. This seems to allow for the animal organism to

protect itself from potential threats posed by the presence of potentially threatening extracellular environments (such as calcium oxalate crystals cf. refs. (29-24) 33 and 34). The provision of inorganic cofactors which may assist in general tissue protection processes afforded by the heparan sulphate polysaccharide system may be achieved by an inbuilt reservoir system consists of a metallomic array of inorganic elements attached to heparin/heparan sulphate protoeglycans. Heparin seems to be chemically constituted so as to simultaneously bind the full range of seawater elements in both cationic, anionic and non-ionic forms. By inference heparin-like segments within heparan sulphate polysaccharide chains are predicted to behave similarly. While Ca2+ and Zn2+ are the most common currently known inorganic cofactors for heparanome actions (cf. Table I) it is suggested that an in depth study of the inorganic elements silicon titanium, the rare earths, manganese, phosphorus, vanadium, arsenic and gallium which consistently occur in heparin could be of possible relevance for a fuller understanding of heparan sulphate signalling and should be evaluated as possible cofactors for heparanome activities.. Heparan sulphates were reported by Nader et al., to occur in greater abundance in those marine invertebrates with habitats having greater salinities; this relationship seems to be a mathematically exact one for sulphated glycosaminoglycan contents (7-2). This phenomenon, and it’s apparent continuation in

more complex animals (exemplified by the ionic filtration roles of heparan sulphate in glomerular basement membranes (34-1)) agrees with the concept that the original biological function of heparan sulphate polysaccharides may have been for the regulation of the ionic balance at the cell surface and perhaps also to sequester essential nutrient elements from the bathing solutions. The need for an improved readily-reversible sequestration seems to have arisen at the time of the first evolution of multicellular animals in the sea which was coincident with the evolution of an isomerase enzyme to convert the glucuronic acid residues of the precursor bacterial-like polysaccharides in order to yield the improved metal ion chelation abilities of iduronate groups which can flexibly bind and release metal ions (cf., e.g., ref (15-4)). This process can be suggested to have further evolved into a complex morphogenic mechanisms involving crosstalk between the metallome and the heparanome sulphates with feedback processes allowing postsynthetic modification involving nitric oxide which is putatively used for control of morphogenesis in multicellular animals (cf. 16-2). This complex postsynthetic modification process (which enable a rational alteration of the signalling code and produces oligosaccharides) by both enzymic (16-1) and non-enzymic (16-2) pathways achieves direct and indirect sensitivity to the presence of inorganic ions and organic (e.g. ascorbate, retinoic acid, fatty acids, etc.). The reports listed in Table I and Table II, taken together, might suggest that heparanome associated metal ions could normally be important components of the modus operadi of a complex information system in which inorganic ions and particles apparently can act (via servo feedback modes?) to change the heparan sulphate microstructure in response to external stresses. This phenomenon is most apparent for the altered synthesis of heparan sulphates in parathyroid cells in response to changes in extracellular Ca2+ concentrations (29-10) and for the alteration of synthesis of heparan sulphates towards a form more able to effectively combat calcium oxalate crystal stone formation (29-24)). Metal Ions & Heparan Sulphate Cooperate for Control of Fibroblast Growth Factor Activity Attempts to correlate fibroblast growth factor variant activities (a phenomenon which is likely to be under a strict temporal and positional control) with the binding behaviour of separated-out and purified specific heparan sulphate-core-anion pattern oligosacchrides, failed (as reported by Kreuger et al. (28)) to show any of the expected discriminated binding. This had been assumed by these authors to occur by a mechanism which recognised only the naked anionic patterns present in the individual oligosaccharides. But, if it is correct as is now suggested that specific counterions may also be needed to complete the signal it is conceivable that these were removed during standard column purification of oligosaccharides. Ricard-Blum et al. (14-2) reported an example of this effect. Zn2+ ions were found to be essentially required to allow endostatin to bind to heparan sulphate only after the inadvertently removal of Zn2+ can caused failure of this binding to occur. Rudd et al. (11) and Guimond et al. (11) have more recently reported in depth studies of the specific binding of a range of metal ions to heparin/heparan sulphate in a manner able to greatly influence basic fibroblast growth factor receptor signalling activity, which supports the idea that inorganic elements, including K+ and Cu2+ could be essential components of a heparan sulphate microstructure “signal”.

The following ‘metallome-heparanome’ sub-hypotheses can be suggested. 1) Heparan sulphate proteoglycans are so structured as to facilitate the initial uptake, rapid transport and release of nutrient elements and promote heavy metal detoxification processes. 2) Anionic polysaccharides may generate specific conformations as a consequence of the binding of specific types of metal ions. These conformations may play direct roles in heparan sulphate signalling 3) The heparan sulphate nutrient element sequestration action contains a feedback loop to altered heparan sulphate synthesis for the generation of altered anionic patterns which potentially can signal for an altered tissue and organ assembly process (principally via the regulation of the provision and the activation of growth factors and their receptors). This may have enabled the evolution of animal species to occur in response to inorganic nutrient stress oxidative stress or nitrosative stress.

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

References
(1)
S. Mounicou, J Szpunar and R. Lobinski Metallomics: the concept and methodology Chem. Soc. Rev., 2009, 38, 1119-1138 doi:10.1039/b713633c

(2) H. Haraguchi Metallomics as integrated biometal science J. Anal. At. Spectrom., 2004, 19, 5-14 Cf. http://www.apchem.nagoya-u.ac.jp/06-III-3/index-e.html
[The use of modern mass spectroscopic techniques suggests the common occurrence of 80+ multi-inorganic elements in a range of biological matrices (where these often occur with a similar abundance and type to those which occur in the sea). This information seems to have prompted Haraguchi to suggest that such studies should form the basis of a new branch of science: “metallomics”. (3) I. Rodushkin and M.D. Axelsson Application of double focussing sector field ICP-MS for multielemental characterization of human hair and nails. Part I. Analytical methodology Sci. Tot. Environ., 2000, 250, 83-100; cf., ibid., 2000, 262, 21-6 Cf., also V.N. Senofonte and S. Caroli, J. Trace Elem. Med. Biol., 2000, 14, 6-13; D.A. Bass et al., Altern. Med. Rev., 2001, 6, 472-481 A.R. Bleise et al., Analytical Quality Control Services (Hari Elements Reference Sheet) Interntional Atomic Energy Agency, A1499, Vienna , Austria, and http://www.analytica.se/hem2001/human/research.asp) (4) A. Wassermann Cation adsorption by brown algae. The mode of occurrence of alginic acid Ann. Bot. N.S., 1949; 13 (49): 80-88; cf., W.A.P. Black and R.L. Mitchell Trace elements in the common brown algae and in sea water J. Mar. Biol. Assoc. U.K., 1952, 30 (3) 575-584, [These authors noted that “Seawater probably contains all the chemical elements, although a number of them have not yet been detected”] The above studies clearly suggest that anionic polysaccharides in the cell walls of marine algae occur in the form of multi-inorganic metal salts. More recent studies also further confirm this idea. The multi-element contents, in e.g., kelp, has been indicated to derive largely from the in vivo binding by the extracellular anionic polysaccharides present in the algal cell walls of the ionic and particulate multi-inorganic elements present in seawater, E.g., K. Truus, M. Vaher and I.Taure, Proc. Estonian Acad. Sci. Chem., 2001, 50, 95-103, and The multi-element analysis of Ascophylum nodosum reported at http://www/alginure.co.uk/ascophylum-nodosum.html contain data were apparently provided by the Norwegian Institute of Seaweed Research.

Cf. also T.A. Davis et al. (Supplementary references) ; the multi-elements in Ecklonia maxima given in http://www.gairesearch.co.za/kelp.html.

(5)
A sodium porcine mucosal heparin sample (prior to the final ion exchange reduction of multi-elements needed to achieve pharmaceutical grade heavy metal ion contents was analysed by spark source mass spectrometry (further details are given in D. Grant et al., in Biochem. J., 1987, 244 p. 143 and C.F. Moffat Synthesis, “Characterisation and applications of chemically modified heparins”, Ph.D. Thesis, University of Aberdeen Department of Biochemistry, 1987, and D. Grant, Chemistry Preprint Archive , 2000, October, 2000, (10), 94-103) where unmodified, unfractionated heparin was shown to contain a seawater-like distribution of inorganic elements; a lesser amount (but also a seawater-like distribution) was obtained after modification of the starting (‘natural’) heparin-multi-inorganicion- complex routine ion exchange resin purification procedure detailed by D. Grant (1987) loc.cit.
This replacement is highly effective for most elements by less so for Pb2+, Ce4+ and other rare earths.

The elements present in heparin are exemplified below which shows the amounts of such element which were present in amounts greater than 1µg/g are listed thus: ( )[ ]{ }; the values as µg/g given in [ ] and { } brackets are respectively the elemental composition in µg/g following single counterion [Tl} enrichment and compared { } with ALS (Sweden)literature-reported (ref. (51)) values (available on the internet) for of a typical ‘industrial’ sodium heparin: Na ca. 9% of dry weight, Ca (30000) [30]{171}, Si (5900)[100]{116}, Cl (5600)[1000]{n.d.}, K (2000)[40]{23}, Mg(1300)[10]{?}, Fe (1100)[10]{5}, F (890)[4](n.d.}, Cu (730)[5]{16}, P (440)[30]{61}, Ti (<390)[4]{0.2}, Ni (<170)[10]{8}, Ba (140)[1]{1}, Br (130[0.9]{?}, Zn (80[7]{16}, Sr (65)[1.4]{2}, Co (<80)[2]{<0.01}, B (<25)[1]{10}, Ga (20)[1]{0.005}, As (15)[1]{<78}, Pb (16)[4]{0.8}, I (10)[<1]{1.3}, Cs (9)[0.7]{0.01}, Tl(8) [principal counterion]{0.2}, Mo (7)[<1]{0.01}, La (7)[4]{0.005}, Ce (7)[3.5]{0.02}, Ag (4)[0.5]{0.002}, Sn (5)[0.3]{<0.3}, Nd (5)[1]{0.01},W (5)[<1]{<0.02}, Zr (5)[0.3]{0.05}, Rb (3)[0.3]{0.1},V(3)[<1]{<0.3}, Mn (1) [<0.1]{0.4}, Cd(1)[1.5]{0.3}), confiming previous reports (cf. ref. 1c) that Ca, Si, Sr, Ba, Cu, Zn, As, Al, Mn, Cr and V normal occur in heparin. The multi-inorganic element contents of heparin as a state of matter were indirectly fully documented by the major international analytical laboratory ALS, (1b-1) vide infra for two commercial end-users evidently employing at least two different brands of sodium and lithium, which contained a similar multi-element presence to the (Aberdeen University experimental) Tl heparin. (N.b. these multi- element contents, although greatly reduced in amounts for most elements are still considered sufficiently great to define such ‘purified’ heparins as metallomic matrices which also putatively allows these heparins to provide a range of metal ions required for possible biochemical cofactors . Problems of reproducibility of inorganic residues in heparin can however arise from differences in work-up procedures between manufacturers (this may be a problem when industrially prepared heparin is used as laboratory model for heparan sulphate and also for the occurrence of potentially toxic amounts of Al in some industrial heparins as discussed by D. Bohrer et al., RBAc (Brasil) 2004, 36 (2) 99-103)). (5-1) <http://www.analytica.se/hem20045/pdf/blood_collection_tubes.eng.pdf> . (These data were apparently published to allow for a greater awareness of sources of error in the determination of the inorganic element contents of blood sample fractions; the ICP-MS elemental data include that of inorganic sulphur from sulphate half ester moieties, allowing the calculation of the multelemental contents of heparins (which had been leached from several types of blood collection tubes {which may have caused a diminution of some elements (perhaps Co, Mo, Lam Ce, Ag, Nd and W) associated with less leachable fractions n.b.,there is no attempt in this report to discuss any wider relevance of these data). While brine or untreated municipal water used for work up of polysaccharide extracts using during industrial scale manufacture of pharmaceutical heparin may, in part, be a multielement source (the possibility of which requires further research) the in vivo equilibration of heparin with the multielement composition of biological fluids having multi-elemental compositions similar to blood serum seems to offer the most rational explanation of

why all commercial heparin contain a blood serum/seawater-like inorganic element profile. (5-2) The normal occurrence of a wide range of metal ions in heparin may be concluded from sporadic reports in the literature of the co-occurrence of part of the metal ion profile now suggested always to occur with heparin , e.g., G.F. Harrsion and A Sutton, Nature, 1963 (4869) 809, noted the presence of calcium, strontium and barium in heparin 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 stated that such multi-inorganic element presence in heparin was normally sufficiently great to disallow the use of procedures using this anticoagulant for the evaluation of trace metals (especially manganese) in blood fractions; a similar situation evidently still 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 in blood fractions (it should also be noted that a similar range of inorganic elements may also occur in EDTA, also used as an anticoagulant, but the amounts of inorganic elements present with this reagent were reported (ALS, ref., (5-1)) however, to be, on a molar anionic site basis, some two orders of magnitude less than was the case with heparin); other possible sources of errors in inorganic element determination in blood samples evidently can evidently arise from needles etc. and from antimony catalyst residues leached from PET tube walls (cf. ALS, ref. (5-1)). 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 of potentially toxic amounts of manganese and chromium in some heparin samples and S.A. Katz (1984) Amer. Biotechnology Lab., 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. Bohrer et al. had reported 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 cation exchange resin percolation; this procedure seemed warranted in some instance prior to the use of the heparin batches evaluated by these authors as anticoagulants for kidney dialysis patients.

In depth in vitro studies of the binding of a range of types of metal ions to heparin (D. Grant et al., Supplementary References collected in an Appendix, vide infra) suggested that

binding was sufficiently similar for a range of multivalent cation to discourage the formation of any single counterion substituted complex in accord with the simultaneous uptake of a range of different counterions when heparin-like molecules are bathed in multi-inorganic ion containing bathing solutions. (6) J. Turnbull, A. Powell and S. Guimond Trends Cell Biol., 2001, 11, 75-82 [Heparan sulphate protoglycans (HSPGs), glypicans, syndecans, agrin etc.) constitute a major multicellular animal cellular control system which is believed to be functionally dependent on the presence of conserved sugar sequences required for specific interactions with proteins] Of especial relevance to the post human geneome era which seeks to understand how the unexpectedly small size of the human genome can serve such highly complex species as humans. This may Be due to the ability of heparan sulphate to hold and process information Cf., D. Fernig, The University of Liverpool <http://www/newlightsource.org/events/presentations/NLS %20life%20science%20Fernif>. Pdf Decoding the matrix: dynamics of the key regulatior of cell-cell communication [E.g. this presentation discussed why the information encoded in heparan sulphate may be more pertinent to medical research than is the information stored in the geneome; it was noted that while the original estimate of the number of protein encoding genes in humans was ca. 1,000,000 it is now belived that this number is similar to that of Caenorhabditis elegans 20,000. {Although Fernig mentions 35,000 for the protein encoding genes present in Homo sapiens more recent work (Clamp et al.) suggests that this value is ca. 20,500] (7-1) H.B. Nader, Te, C.P. Dietrich, B. Casu and G. Torri

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

(7-2) H.B. Nader, M.G.L. Medeiros. J.F. Paiva, V.M.P. Paiva, S.M.B. Jerônimo, T.M.P.C. Ferreira and C.P. Dietrich A correlation between the sulphated glycosaminoglycan concentration and degree of salinity of the habitiat in fifteen species of the classes Crustacea, Pelecypoda and Gastropoda. Comp. Biochem. Physiol., 1983, 76, 433-436. [The mathematically exact nature of the sulphated polysaccharide especially for heparan sulphate requirements for aquatic organisms might point to a primitive osmoregulatory role; that heparan sulphate shows the greatest interspecies variation which is related to aquatic salinity suggests that this polysaccharide may have had a primitive evolutionary function, retained to a major extent in modern organisms, of the provision of multiple inorganic ion binding sites]. (8-1) U. Lindahl and M. Höök Glycosaminoglycans and their binding to biological macromolecules Ann. Rev. Biochem., 1978, 47, 385-417 (8-2) L. Kjellén and U. Lindahl Proteoglycans: structures and interactions Annu. Rev. Biochem., 1991, 60, 443-465 (9) M. Lyon and J.T. Gallagher Biospecific sequence and domains of heparan sulphate and the regulation of cell growth and adhesion Matrix Biol., 1998, 17 (7) 485-493 Lyon M. and J.T. Gallagher Cf . J.T. Gallagher Heparan sulfate: growth control with a restricted sequence menu J. Clin. Invest., 2001, 108 (3) 357-361 (10-1) M. Bernfield, M. Götte, P.W. Park, O. Reizes, M.L. Fitzgerald, J. Lincecum, and M Zako Functions of cell surface heparan sulfate proteoglycans Ann. Rev. Biochem., 1999, 68, 729-777 (10-2) N. Perrimon and M. Bernfield Specificities of heparan sulphate proteoglycans in developmental processes Nature, 2000, 404, 725-728 J. Biol. Chem., 2000, 275, 29923-29926 (10-3) P.W. Park, O. Reizes and M. Bernfield Specificities of heparan sulphate proteoglycans : selective regulators of ligand-receptor encounters J. Biol. Chem., 2000, 275, 29923-29926 (11) T.R. Rudd, S.E. Guimond, M.A. Skidmore, L. Duchesne, M. Guerrini, G. Torri, C. Cosentino,

A. Brown, D.T. Clarke, J.E. Turnbull, D.G. Fernig and E.A. Yates Influence of substitution patterns and cation binding in conformation and activity of heparin derivatives Glycobiology, 2007, 17 (9) 983-993 [The traditional view that signalling by heparin/heparan sulphate depends only on the polysaccharide anionic sequence is indicated to be incorrect, since the binding of individual cations including K+ and Cu2+ seems dramatically to alter the activities of such polysaccharide anionic sequences (e.g. those required for regulation of fibroblast growth factor and receptor activity); this work strengthens the notion that the heparanome-metallome interaction concept is a valid hypothesis for how heparin/heparan sulphate signalling occurs in animal biochemistry] Cf., T.R. Rudd, M.A. Skidmore, S.E. Guimond, M. Guerrini, C. Cosentino, R. Edge, A. Brown, D.T. Clarke, G. Torri, J.E. Turnbull, R.J. Nichols, D.G. Fernig and E.A. Yates Site-specific interactions of copper(II) ions with heparin revealed with complementary (SRCD, NMR, FTIR and EPR) spectroscopic techniques Carbohydr. Res. , 2008, 343, 2184-2193 Cf. S.E. Guimond, T.R. Rudd, M.A. Skinner, A. Ori, D. Gaudesi, C. Cosentino, M. Guerrini, R. Edge, D. Collinson, E. McInnes, G. Torri, J.E. Turnbul, D.G. Fernig and E.A. Yates Cations modulate polysaccharide structure to determine FGF-FGFR signaling: a comparison of signaling and inhibitory polysaccharide interactions with FGF-1 in solution Biochemistry. 2009, 48. 4772-4779

Cf .also Supplementary References Ayotte et al., Burger et al., Casu et al., Dais et al , Dunstone, Grushka et al., Liang et al., Panov et al.

12 Some Aberdeen University research results on the binding of metal ions to heparin
D. Grant, W.F. Long, C.F. Moffat and F.B. Williamson Cu2+-heparin interaction studied by polarimetry Biochem. J., 1992, 283, 243-246; Cf., There is a lack of paramagnetic broadening of NMR absorbances in heparin containing 1100ppm Fe and 730ppm Cu [these values were obtained from spark source mass spectroscopic analysis (Moffat, Colin F, Ph.D. Thesis “Synthesis Characterisation and Applications of Chemically Modified Heparin” University of Aberdeen 1987); Cf. also D. Grant., W.F. Long and F.B. Williamson Complexation of Fe2+ ions by heparin Biochem. Soc. Trans., 1992, 20, 361s [This is not a simple thermodynamic process]
13C n.m.r. spectroscopy of Cu2+- heparin suggests phase separation of the complex from Cu2+ in aqueous solution Ibid. 1992; 20: 214S and
Pharmaceutical

heparin was also suggested, from NMR spectroscopic evidence, to bind paramagnetic ions in aggregated insoluble forms (26a)). Sulphated polysaccharides may provide antioxidant protection by sequestering Cu2+ (11), Fe2+ and Fe3+ (24,26) ions from the solution phase (e.g., by the binding of these

ions to heparin/heparan sulphate, and enabling their assembly into redox-inert aggregates (26). Chitosan and hyaluronan are also believed to similarly act as antioxidants cf., Albertini et al. (24-2)
Cation interaction with heparin at antithrombin-binding sites may differ from that occurring elsewhere in the polymer Cell Biology International Reports, 1987, 11, 220; Cation interactions with heparins/heparans are not explicable in terms of simple electrostatic effects Cell Biology International Reports, 1987,11, 221; Cation complexation with heparin studied by 13C-NMR spectroscopy IRCS J Med. Sci., 1986, 14, 903-904; Binding of copper to heparin. Physiochemical studies suggest interaction of pharmacological significance Brit. Soc. Cell. Biol., 1985 36, Suppl. 8 (Abstr. 26), 13 and M.A. Ross, W.F. Long WF , F.B. Williamson and C.F. Moffat CF Effect of chemically modified heparins , and of heparin fragments on Fe(II)-catalysed peroxidation of linoleic acid Biochem Soc Trans., 1992, 20, 216s [cf . Abstracts of the 641st Meeting of the Biochemical Society Royal Holloway and Bedford New College London, 17-20 Dec 1991 p. 56 Abstract No. 159] M.F. Williamson, W.F. Long and F.B. Williamson Effect 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. Williamson Zinc ion binding by heparin Biochem. Soc. Trans., 1983, 11, 96-97; Biochem. J., 1985, 237, 281-284

Boyd J., F.B. Williamson and J. Gettins Physico-chemical study of heparin. Evidence for a calcium-induced co-operative conformational transition J. Mol. Biol., 1980, 137, 175-190 Cf., W.F. Long and F.B. Williamson Glycosaminolycans, calcium ions and the control of cell proliferation IRCS J. Med. Sci., 1979, 7, 429-434; [Cf., related studies, of the inorganic biochemistry of heparin/heparan sulphate by the Aberdeen polysaccharide group listed in the Supplementary References}].

(13) Binding of anions to heparin (13-1) J.R. Helbert and M.A. Marini Structural studies of heparin II. Exchangeable anions Biochim. Biophys. Acta, 1964, 83, 120-122 [Heparin binds SO42- so that the normal presence of inorganic sulphate in heparin can increases the total amount of sulphur ‘in heparin’ by a factor of 2+ greater than that due to the presence of sulphate half ester and N-sulphonate groups] Cf., K.H. Simon Naturwiss Rundschau, 1982.,11, 452-455 and L.B. Jaques Heparin: an old drug with a new paradigm

Science, 1979, 206, 528-533 Cf also., Fo-We (Forschungs und Verweltungs Anstalt) Brit. Pat. Appl. 890,622 (1962) [Formation of complexes between heparin and inorganic salts] (13-2) T. Takeuchi , Anon. Safni, T. Miwa, Y. Hashimoto and H Moriyama Ion chromatography using anion exchangers modified with heparin Analusis, 1998, 26, 61-64 [A form of heparin- based ion exchange chromatography can separate cations and anions on a single column] (14-1) M. Kan, F. Wang, M. Kan, B. To, J.L Gabriel and W.L. McKeehan Divalent cations and heparin/heparan sulphate cooperate to control assembly and activity of the fibroblast growth factor receptor complex J. Biol. Chem., 1996, 271, 26143-26148 [These authors reviewed the other cell surface recognition molecules whose structures and activities are modulated by divalent cations and heparan sulphate or other glycosaminoglycans in the pericellular matrix, including β 2 and β3 integrins, platelet/endothelial cell adhesion molecule-1 (PECAM-1) cadhedrin and L-selectin] M. Kan, X. Wu, F. Wang and W.L. McKeegan Specificity of fibroblast growth factors determined by heparan sulfate in a binary complex with receptor kinase J. Biol. Chem., 1999, 274, 15947-15952 [Anticoagulant heparan sulphate is required for FGF receptor divalent cation dependent association between heparan sulphate and the exodomain] (14-2) S. Ricard-Blum, O. Féraud, H. Lortat-Jacob, A. Rencurosi, N. Fukai, F. Dkhissi, D. Vittet, A. Imberty, B.R. Olsen and M. van der Rest Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modelling: role of divalent cations J. Biol. Chem., 2004, 279, 2927-2936 (14-3) I. Capila, M.J. Hernáiz, T.R. Mealy, B. Campos, J.R. Dedman, R.J. Linhardt and B.A. Seaton Annexin V-heparin oligosaccharide complex suggests heparan sulfate-mediated assembly on cell surfaces Structure (Camb.) 2001, 9, 57-64 (14-4) A. Lewit-Bentley, S. Morera, R. Huber and G. Bodo The effect of metal binding on the structure of annexin V and implications for membrane binding Eur. J. Biochem., 1992, 210, 73-77 (14-5) e.g. Medical new Today 22 may 2006 press release from Uppsala Univerity discussed a glue like (“entirely new)” heparan sulphate mechanism by which communication can occur between cells (15-1) Y. Hama, T. Sasaki, S. Kojima and A. Kuberoda 67-Ga accumulation and heparan sulfate metabolism Eur. J. Nucl. Med., 1984, 9, 51-56; Chem. Abs., 100, 188079t

Cf., S. Kojima, Y. Hama, T. Sasaki and A. Kuberoda Elevated uptake of 67-Ga and increased heparan sulphate content in liver-damaged rats Eur. J. Nucl. Med., 1983, 8, 52-59, and Y. Hama, S. Kojima, and A. Kuberoda Relation of heparan sulphate content and 67-Ga uptake in various tissues of rats Kagaku Igaku, 1982, 19 (6) 855-861; Chem Abs., 97, 211645e [67-Ga forms stronger complexes with heparan sulphate than other GAGs allowing tissue distribution of heparan sulphate to be visualised] S. Kojima Uptake of 67-Ga citrate in tissue and heparan sulphate Radioisotopes, 1986, 35 (8) 437-445; Chem. Abs., 191815j Cf. also ref. 13 (15-2) E.g. D.S. Millbraith et. al., Eur. Pat. Appl., EP 55028; Chem. Abs., 97, 168966; A. Mostbeck et. al., Nuklearmedizin Suppt. (Stuttgart), 1981, 18, 343 [Heparin complexes of 111In and 99Tc may also be used for medical imaging] Cf. (15-3) M. Rizk et al. Spectroscopic determination of heparin sodium using Eu(III) as a probe ion Spectroscop. Lett., 1995, 28 (8) 1235 [Eu3+ formed an adduct with heparin with log10K=5.079] (15-4) D.M. Whitfield., J. Choay and B. Sarkar Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site Biopolymers, 1992, 32, 585-596 Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation Ibid., 1992, 32, 597-619 (16) Oligosaccharide Generation (16-1) Heparanase degradation Bacterial heparanase activity is metal ion dependent E.g. C.F. Moffat, W.F. Long, M.W. McLean and F.B. Williamson Heparanase-(II)-catalysed degradation of N-propionylated heparin Biochem. Soc. Trans., 1997, 25, S654 Heparanase II from Flavobacterium heparinum. Action on chemically modified heparins Eur. J. Biochem., 1991, 197, 449-459 Heparanase II from Flavobacterium heparinum. HPLC analysis of the saccharides generated from chemically modified heparin Ibid., 1991, 202, 531-541 Cf., E. Zcharia, J. Jia, X. Zhang, L. Baraz, U. Lindahl, T. Peretz, I. Vlodavsky, J.-P. Li Newly generated heparanase knock-out mice unravel co-regulation of heparanase and matrix metalloproteinases PLoS ONE 2009, 4 (4): e5181Epub 2009 Apr.10 [This suggests that the generation of key oligosaccharides messenger molecules is achieved by a ‘smart’ system in which Zn2+-dependent metalloproteinsases act in concert with heparanase}

(16-2) Nitrous acid degradation of heparin/heparan sulphate - metal ion effects K. Ding, K. Mani, F. Cheng, M. Belting and L.-Å. Fransson Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols J. Biol. Chem., 2002, 277 (36) 33353-33360 cf., K. Mani, M. Jonsson, G. Edgren, M. Belting and L.-A. Fransson Glycobiology, 2000, 10, 577-586 [Endogenous internal degradation of heparan sulphate during recycling of glypican-1 in vascular endothelial cells] Cf., M. Belting, S. Persson and L.-Å. Fransson Proteoglycan involvement in polyamine uptake (16-3) Nitrous acid degradation of heparin/heparan sulphate under physirolgical conditions 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 sulphate Biochem. J., 1997, 324, 473-497 Cf., D. Ghael, M. Mileva, H.S. Dweck and L. Rosenfeld Biochem. Mol. Biol. Int., 1997, 43, 183-188 [The reason why different buffers support different degrees of reactivity of nitrite for deaminative cleavage of heparin/heparan sulphate can be deduced to arise from the presence of trace amounts of copper and iron in phosphate buffers; n.b. this possibility was not discussed by these authors but arises from unpublished work carried out in the field by the Author at Aberdeen University]. Cf. Possible Nitric Oxide Metal Ion Heparan Sulphate Related Phenomenona {Rapid movement of bound ions along anionic polysaccharide chains apparently promotes formation of nanoparticles of Fe3+ oxides and thereby protects against oxidative 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 excessive nitric oxide production may promote of degenerative diseases including cancer and multiple sclerosis (the subject of a communication in preparation)}. (16-4) B. Lahiri, P.S. Lau, M. Pousada, D. Stanton, and I. Danishefsky Depolymerization of heparin by complexed ferrous ions Arch. Biochem. Biophys., 1992, 293, 54-60 Cf., J.P. Lahiev Bio Metals, 1996, 9 (1) 10; Chem. Abs., 124, 109969t [Fe(II) selectively degrades heparin] (17) Z. Liu and A.S. Perlin Evidence of a selective free radical degradation of heparin mediated by cupric ion Carbohydr. Res., 1994, 255,183-191 Cf. R.N. Rej, K.R. Holme and A.S. Perlin Marked stereoselectivity in the binding of copper ions by heparin. Contrasts with the binding of gadolinium and calcium ions Carbohydr. Res., 1990, 207, 143-152 [Trace amounts (ca 1/185 mol ratio with respect to heparin) of Cu2+ together with

a slight molar excess of H2O2 + ascorbate reduced (e.g., by 30% over 30 min at 40oC) 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+] 18 Effects of metal ions on cartilage (18-1) R.D. Campo Effects of cations on cartilage structure: swelling of growth plate and degradation of proteoglycans induced by chelators of divalent cations Calcif. Tissue Int., 1988, 43 (2) 108-121 (18-2) L. Lerner and D.A. Torchia A multinuclear NMR study of the interactions of cations with proteoglycans heparin and Ficoll. J. Biol. Chem., 1986, 261, 12706-12714. [23-Na, 39-K, 25-Mg and 43-Ca NMR relaxation assessment of cation binding to heparin]. Possible relevance of perturbation of heparan sulphate signalling to pathology (19) M. Purdey Chronic barium intoxication disrupts sulphated proteoglycan synthesis: A hypothesis for the origin of multiple sclerosis Med. Hypotheses., 2004, 62, 746-754 [Cf., Anthropogenic perturbation of heparin/heparan sulphate signalling: Some of the reported requirements for the presence of specific divalent metal ions (e.g. Ca2+ or Zn2+) for heparin/heparan sulphate binding to proteins which is listed in Table I can also be rationally suggested to be subject to anthropogenic perturbation (e.g. by Ba, Pb, Cd and Hg )].
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 but this activity might be perturbed by Ba2+ which is reported to disturb Ca2+ binding to annexin-V this being of relevance for annexin ion channel building, anticoagulant and cellular apoptosis activity. While Ba2+ occurs in pharmaceutical porcine heparin and also in human scalp hair, there is a larger variation in the reported values for Ba2+ in human hair consistent with an environmental intoxication source of this metal].

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

ibid., 36,14 A role for glycosaminoglycans in cellular adhesion of relevance to the cancer state Biochem. Soc. Trans.,1985, 13, 388 D. Grant, W.F. Long, G. Mackintosh and F.B. Williamson Roles of heparins and heparans in inflammatory aspects of cancer and the potential of heparinoids as anti-cancer drugs Proc. Int. Congr. Inflamm., Vienna, 10-15 Oct. 1993 While numerous other laboratories have studies the pathological alteration of heparan sulphate cf., P. Tapanadechopone, S. Tumova, X. Jiang and J.R. Couchman Epidermal transformation leads to increased perlecan synthesis with heparin-binding growth factor affinity Biochem. J., 2001, 355 (2) 517-527, a full review is outwith the scope of this article

(21) M. Herbert and J.P. Maffrand J. Cell Physiol., 1989, 138, 424-432 [Oligosaccharides following internalization show antiproliferative effects on vascular endothelial and smooth muscle cells] (22) R. Hahnenberger, A.M. Jakobson, A. Ansari, T. Wehler, C.M. Svahn and U. Lindahl Low-sulphated oligosaccharides derived from heparan sulphate inhibit normal angiogenesis. Glycobiology, 1993, 3 (6), 567-573 [Inhibition of angiogenesis by low sulphated oligosaccharides from heparan sulphate for which endothelial cell surface-bound heparan sulphate proteoglycans may constitute a pool of precursors for anti-angiogenic polysaccharides] (23) S. Ihrcho, L.E. Wrenshall, B.J. Lindman and J.L. Platt Immunol. Today, 1993, 14, 500-501; Chem. Abs., 120, 51877v [Review on the release of heparan sulphate from endothelial cells during inflammation and possible regulation by soluble heparan sulphate fragments of the functioning of lymphocytes at sites of inflammation] Heparin-like molecules as redox metal de-activating antioxidants (24-1) M.A. Ross, W.F. Long and F.B. Williamson Inhibition 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 in the presence of linoleic acid and Fe2+ ions Biochem. Soc. Trans., 1992, 20(4) 364s Cf. D. Grant et al. (1996) ibid., 24 194S and ref. 24-2 (24-2) D. Grant, W.F. Long and F.B. Williamson Pericellular heparans may contribute to the protection of cells from free radicals Med. Hypotheses, 1987, 23, 67-71 Cf. also Ross, Marion A “Heparin as an Antioxidant” M.Sc. Thesis University of Aberdeen,1992

D. Grant, W.F. Long, G. Mackintosh and F.B. Williamson The antioxidant activity of heparin Biochem. Soc. Trans., 1996, 24, 194S [Cf. R. Albertini, A. Passi , P.M. Abjuja and G. De Luca The effect of glycosaminoglycans on lipid peroxidation Int. J. Mol. Med. 2000, 6,129-136 R. Albertini, S. Rindi, A. Passi, G. Palladini, G., Pallavicini and G. De Luca The effect of heparin on Cu2+-mediated oxidation of human low-density lipoproteins FEBS Lett., 1995, 377, 240-242; ibid., 1996, 383,155-158], cf. also http://electra.chemistry.upatras.gr/fects/final/w-shops.htm] (25) M.F. Williamson, W.F. Long and F.B. Williamson The 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 Recio Aqueous and solid complexes of iron (III) with hyaluronic acid. Potentiometric titrations and infrared spectroscopy studies J. 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. Webb Formation of spheroidal iron(III) nanoparticles sterically stabilized by chitosan in aqueous solutions J. Inorg. Biochem., 2003, 95, 55-63 [Iron-containing nanoparticles also form at chitosan surfaces]
(27) 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/ also <http://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 in asocrbate/nitric oxide determined heparan sulphate fuzzy logic control systems than via collagen or DNA-damage dependent mechanisms (cf., Linus Pauling had suggested that the anti-cancer activity of ascorbate was due to a general promotion of collagen crosslinking 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)].

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J. Kreuger, P. Jemth, E. Sanders-Lindberg, L. Eliahu, D. Ron, C. Basilico, M. Salmivirta and U. Lindahl Fibroblast growth factors share binding sites in heparan sulphate Biochem. J., 2005, 389, 145-150

Tables I &II (Specific References)

(29-1) E. Kecskes, K.G. Bucki, P.I. Bauer., R. Machovich., and I. Horvath Thromb. Haemost., 1983, 49, 138-141; Chem. Abs., 99, 3504x [Heparin forms a complex with LDL in the presence of Ca2+; this complex retains the anticoagulant activity of the heparin] (29-2) F.A. Ofosu, G. Modi, A.L. Cerskus, J. Hirsh and M.A. Blajchman

Ca binding to heparin inhibits phospholipid dependent assembly of Factor X and prothrombin activated complex Thromb Res., 1982, 28 (4) 487-497; Chem. Abs., 98, 69550 (29-3) M.O. Speight and M.J. Griffith Calcium inhibits the heparin-catalysed antithrombin III/thrombin reaction by decreasing the apparent binding of heparin for thrombin Arch. 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 DNA J. Biol. Chem., 1982, 257, 5263-7 (29-5) A. Koenig, K. Norgard-Sumnicht, R. Linhardt, and R. Varki Differential interaction of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparin as therapeutic agents J. Clin. Invest., 1998, 101 (4) 877-889 (29-6) K.E. Norgard-Sumnicht, N.M. Varki, and A. Varki Calcium-dependent heparin-like ligands for L-selectin in nonlymphoid endothelial cells Science, 1993, 261, 480-483 (29-7) E.H. Nielson, I.J. Sørensen, K. Vilsgaard, O. Andersen and S.E. Svehag Calcium enhanced aggregation of serum amyloid P and its inhibition by the ligands heparin and heparan sulphate. An electron microscope and immunoelectrophoretic study APMIS, 1994, 102, (6) 420-426; Chem. Abs., 122, 181976a (29-8) S.K. Shinjo, I.L. Tersariol, V. Oliveira, C.R. Nakaie, M.E.. Oshiro, A.T. Ferreiora, I.A. Santos, C.P. Dietrich and H.B. Nader Heparin and heparan sulfate disaccharides bind to the exchanger inhibitor peptide region of the Na+/Ca2+ exchanger and reduce the cytosolic calcium of smooth muscle cell lines J. Biol. Chem., 2004, 277 (50) 48227-48233 (29-9) Y. Zhao and X. Zhang Heparin inhibits the reconstituted plasma membrane Ca-ATPase from porcine brain synaptosome Glycoconjugate J., 2002, 19, 373-378 (29-10) Y. Takeuchi, K. Sakaguchi, M. Yanagishita, G.D. Aurbach and V.C. Hascall Extracellular calcium regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line J. Biol. Chem., 1990, 265 (23) 13661-13668 Cf. Y. Takeuchi, M. Yanagashita and V.C. Hascall Recycling of transferrin receptors and heparan sulfate proteoglycans in a rat parathyroid cell line J. Biol. Chem., 1992, 267 (21) 14685-14690

(29-11) D. Kiryushko, V. Novitskaya, V. Soroka, J. Klingelhofer, E. Lukanidin, V. Berezin, and E. Bock Molecular mechanism of Ca2+ signaling in neurons induced by the S100A4 protein Mol. Cell. Biol., 2006, 26 (9) 3625-3638 (29-12) H.-G. Knaus, F. Scheffauer, C. Romanin, H.-G. Schindler and H. Glossmann Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. Evidence for an agonistic action J. Biol. Chem., 1990, 265, 11156-11166 (29-13) G. Siegel, M. Malmsten and B. Lindman Flow sensing at the endothelium-blood interface Colloids and Surfaces A: Physiochemical and Engineering Aspects, 1998, 138, 384-351 [Heparan sulphate may serve as a vascular flow sensor via conformation changes elicited mechanically and electrostatically by Na + and Ca2+ cation binding] (29-14) M.J. Robinson, P. Tessier, R. Poulson and N. Hogg The S100 family heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfate glycosaminoglycans on endothelial cells J. 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 assembly J. Biol. Chem., 276 (38) 36035-36042 [Ca2+ dependence of extracellular microfibrils fibrillin-1 binding to heparan sulphate proteoglycan] (29-16) M.J. Stanley, B.F. Liebersbach, W. Liu, D.J. Anhalt and R.D. Sanderson Heparan sulfate-mediated cell aggregation J. Biol. Chem., 1995, 270 (10) 5077-5083 [Aggregation of syndecan-1-transfected cells mediation by divalent cations] (29-17) A.L. Jones, M.D. Hulett and C.R. Parish Histidine rich glycoprotein HRG- a novel adapter protein in plasma that modulates the immune vascular and coagulation systems Immunol. Cell Biol., 2005, 83 (2) 106-118 (29-18) L. Kerp Importance of zinc for histamine storage in mast cells Intern. Arch. Allergy Apppl. Immunol., 1963, 22, 112-123 Cf. L. Kerp and G. Steinhauser On a ternary heparin-metalhistamine-complex Klin. Wochschr., 1961, 39, 762-764 (29-19) B. Lages and S.S. Stivala Copper ion binding and heparin interactions of human fibrinogen Biopolymers, 1973, 12, 961-974

(29-20) R. Gonzáles-Iglesias, M.A. Pajares, C. Ocal, J.C. Espoinosa, B. Oesch and M. Gasset Prion protein interaction with glycosaminoglycan occurs with the formation of oligomeric complexes stabilized by Cu(II) bridges J. Mol. Biol., 2002, 319, 527-540 (29-21) Y. Yamane, S. Saito and T. Koizumi Effects of calcium and magnesium on the anticoagulant action of heparin Chem. Pharm. Bull (Tokyo) 1983, 31, (9) 3214-3221; Chem. Abs., 99, 205875e (29-22) M.A. Liebel and A.A. White Inhibition 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, 136893b Alzheimer’s 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. Aguiar, M.A. Dalboni, A.S. Garófalo and N. Schor Characterization of glycosaminoglycans in tubular epithelial cells: Calcium oxalate and oxalate ions effects 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 of oxalate which can induce the formation of (harmful) calcium oxalate crystals. A servo feedback system could 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. Schuschke, N.K. Karamanos and D.J. Klimis-Zacas Dietary manganese affects the concentration , composition and sulfation pattern of heparan sulfate glycosaminoglycans in Sprague-Dawley rat aorta Biometals, 2006, 19 (5) 535-546 (29-26) T.A. Fritz, M.M. Gabb, G. Wei and J.D. Esko Two N-acetylgluocosaminyltransferases catalyse the biosynthesis of heparan sulfate J. Biol. Chem., 1994, 269 (46) 28809-28814 [α-GlcNAc transferase I can use both Mn2+ and Ca2+ while α-GlcNAc transferase II can only use Mn2+. Mn2+ status may therefore affect heparan sulphate proteoglycan biosynthesis by this mechanism] (29-27) P. Jaya and P.A. Kurup Effect of magnesium deficiency on the metabolism of glycosaminoglycans in rats J. Biosci., 1986, 10, 487-493 (29-28) Y. Fujiwara and T. Kaji Suppression of proteoglycan synthesis by calcium ionophore A23187 in cultured vascular endothelial cells; implication of intracellular calcium accumulation in lead inhibition of

endothelial proteoglycan synthesis J. Health Sci., 2002, 48, 460-466 Cf., Y. Fujiwara, and J. Kaji Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan by proliferating vascular endothelial cells in culture Toxicology, 1999, 133 (2,3) 159-169; Chem. Abs., 131, 154569 Cf. T. Kaji, C. Yamamoto and M. Sakamoto Effect of lead on the glycosaminoglycan metabolism of bovine aortic endothelial cells in culture. Ibid., 1991, 68, 249-257 (29-29) A. Cárdenas, A. Bernard and R. Lauwerys Incorporation of [35-S] sulfate into glomerular membranes of rats chronically exposed to cadmium and its relation with urinary glycosaminoglycans and proteinuria Toxicology, 1992, 76, 219-231 (29-30) D.M. Templeton Metal-proteoglycan interactions in the regulation of renal mesangial cells: Implication for metal induced nephropathy Proc. Trace Element Health Disease IUPAC Int. Symp., 1990, p. 209-219; Ed., Aito A. (29-31) M.F. McCarty Reported anti atherosclerotic activity of silicon may reflect increased endothelial synthesis of heparan sulfate proteoglycans Med. Hypotheses, 1997, 49, 175-176 Cf., R.M. Iler, The Chemistry of Silica , Wiley, 1979, cf. p. 762 (29-32) K. Pawlowska-Góral, M. Wardas, W. Wardas and U. Majnusz The role of fluoride ions in glycosaminoglycan sulphation in cultured fibroblasts Fluoride, 1998, 31, 193-202 (29-33) C.P. Dietrich, H.B. Nader, V. Buonassisi and P. Colburn Inhibition of synthesis of heparan sulfate by selenate: possible dependence on sulfation for chain polymerization FASEB J., 1988, 2, 56-59

(32) D. Grant, W.F. Long and F.B. Williamson Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization Biochem. J., 1989, 259, 41-45. Cf., Degenerative and inflammatory diseases may result from defects in antimineralization mechanism afforded by glycosaminglycans. (33) D. Grant, W.F.Long and F.B. Williamson Med. Hypotheses, 1992, 38, 49-55. [Multi-element containing anionic polysaccharides may be present as inorganic-crystal-like anionic structures at solution interfaces]. [In a range of related studies (reported as yet only as conference posters and in M.Sc. theses form

Aberdeen University) it was reported that inorganic crystal surfaces can discriminate (or “read off”) polysaccharide microstructures]. {It is of interest in this context that unliganded free iron ions can catalyse the de-N-sulphonation of heparin suggesting that iron overload could diminishes heparan sulphate tissue protection}. (34-1) H. Morita, A. Yoshimura, K. Inui, T. Ideura, H. Watanabe, L. Wang, R. Soininen and K. Tryggvason Heparan sulfate of perlecan is involved in glomerullar filtration. J. Am.Soc. Nephrol., 2005, 16, 1703-1710.

APPENDIX Supplementary References
Listed Alphabetically According to First Named Author

Ayotte L., and A.S. Perlin Nmr spectrosocpy observations related to the function of sulfate groups in heparin. Calcium binding vs. biological activity. Carbohydr. Res., 1986, 145 (2) 267-277. Bodini M.E., and D.T. Sawyer Electrochemical and spectroscopic studies of manganese(II), (III) and (IV) gluconate complexes 2. Reactivity and equilbriums 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) also similar reactions from V(IV), Cr(II), Ti(III) and Fe(II) discussed]. Burger, K., F. Gaizer, M. Pékli, G.Takácsi Nagy and J. Siemroth The effect of cations on the calcium ion coordination of heparin. Inorganica Chimica Acta, 1984 92, 173-176. [Ca and Zn selective electrodes show complex effects of Li, Na, K and Mg on Ca binding; Zn more strongly bound than Ca and Zn binding strongly reduces K binding]. Casu B., P. Oreste, A. Naggi, G. Torri, G. Zoppetti, G. Sporteoletti and F. de Santis European Patent Application No. 0245813 (1987). Clamp M., B. Fry, M. Kamal, X. Xie, J. Cuff, M.F. Lin, M. Kellis, K. Lindblad-Toh and E.S. Lander Distinguishing protein-coding and noncoding genes in the human genome. PNAS, USA, 2007, 104, 19428-19433 doi:10.1073/pnas.0709013104. Cochran D.L. Glycosaminoglycan stimulation of calcium release from mouse calvariae. Specificity for hyaluronic acid and dermatan sulfate. Calcif. Tissue Int., 1994, 41, 79-85. [Hyaluronic acid was more stimulatory of Ca2+ release from bone cultures than heparin which although inactive alone caused the release of Ca2+ in the presence of parathyroid hormone; diminished heparin/heparan sulphate affected by ageing, is associated with increased proportion of hyaluronate and dermatan sulphate

which would tend to augment Ca2+ mobilisation]. Dais P., Q.J. Peng and A.S. Perlin A relationship between 13-C-chemical-shift displacements. and counterion-condensation theory, in the binding of calcium ion by heparin. Carbohydr. Res., 1987, 168, 163-179. Davis T.A., B. Volesky and A. Mucci A review of the biochemistry of heavy metal biosorption by brown algae. Water Res., 2003, 37 (18) 4311-4330. T.A. Davis, F. Llanes, G. Volesky, G. Diaz-Pulido, L. McCook and A. Mucci 1H-NMR study of Na alginates extracted from Sargassum spp. in relation to heavy metal biosorption. Appl. Biochem. Biotechnol., 2003, 110, 75-90. [Cell wall constituents such as alginate and fucoidan are chiefly responsible for the passive removal of toxic heavy metals such as Cd, Cu, Zn, Pb, Cr and Hg]. T.A. Davis, F. Llanes, B.Volesky and A. Mucci Metal selectivity of Sargassum spp. and their alginates in relation to their alpha-1 guluronic acid content and conformation. Environ. Sci. Technol., 2003, 37, 261-267. Cf. O. Raize, Y. Argaman and S. Yannai Mechanism of biosorption of different heavy metals by brown marine macroalgae. Biotechnology and Bioengineering, 2004, 87, 451-458; B. Larsen, Proc. Int. Seaweed Symp Xth, Gothenburg (1980) de Gruyter, Berlin, 1981, pp 7-34; Cf., F. Mo, T.J. Broback and I.R. Siddiqui Carbohydr. Res., 1985, 145, 13. [Alginate from brown seaweed occurred as a mixed Na-Mg-Ca-Sr salt]
In agarophytes, the polysaccharide agarose present in the extracellular mucilage is believed to provide such a function as well as to create a firm hydrated gel].

Dunstone J.R. Ion-exchange reactions between acid mucopolysaccharides and various cations. Biochem. J., 1962, 85, 336-351. Cf., J.E. Scott In The Chemical Physiology of Mucopolysaccharides. Binding in solutions containing acid mucopolysaccharides; Ch.12, p. 171-187; Ed., Guiliano Quintarelli; Churchill, London (1968). Fransson L.-Å., I. Carlstedt, L. Coster and A. Malmstrom Binding of transferrin to the core protein of fibroblast protoheparan sulfate. Proc. 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 Heparin was subjected to numerous in vitro metal ions binding studies; these confirm the existence of complex polysaccharide binding mechanisms for metal ions. It was found that while the Manning electrostatic polyelectrolyte counterion binding theory correctly predicts the tendency for heparin to attract counterions more strongly accordingly to the value of the counterion charge, to show a characteristic discontinuity in the binding isotherms for individual counterions and to predict that a wide range of counterions can simultaneously bind to this polyanion, it was found that not all equally charged counterions bound at similar strength, that the discontinuity for individual counterion binding occurred at ratios of cation/anion which disagreed (12) with Manning theory predictions. Electrostatic binding theories also fail to account for a well-established paradox that ultra-anionic heparin to binds strongly to SO42- (13) and other anions. In depth studies of the mechanism

of binding of counterions to heparin (12) however suggested an alternative model of counterion binding to heparin involving a key role for a phase change engulfment processes triggered by an altered hydration and hydrogen bonding associated with the non-electrostatic association of inorganic ions with the polyol group of polysaccharides; this process may be the origin of the ability of polysaccharides to bind a wide range of inorganic ions. This binding process is however subject to additional modulation of binding by the presence of the additional electrostatic fields present in heparin (and heparin-like segments of heparan sulphate). It should be noted that heparin and heparan sulphate are uniquely complex and, amongst the anionic polysaccharides, show greatest structural diversity (to which the bound inorganic ions, it is now suggested, must also contribute). This flexible diversity apparently facilitates the performance of complex signalling and control functions throughout a wide range of activities in animal biochemistry. It is therefore predicted that the precise physical chemical and ultrastructural details of the counterion profile (including an ability to engage in variable rapid site exchange and the possible perturbation of this by anthropogenic input) could be highly relevant to the in vivo molecular (/supramolecular) mechanisms which control the heparanome. Grant D., W.F. Long and F.B. Williamson Analysis by infrared spectroscopy of the association of water and metal ions with heparin. Biochem. Soc. Trans., 1983, 11, 96. Grant D., C.F. Moffat, W.F. Long and F..B. Williamson Altered water structure in mixtures of heparin and metal ions. Biochem. Soc. Trans., 1984, 12, 302. Grant D., W.F., Long and F.B. Williamson A role for glycosaminoglycans in cellular adhesion of relevance to the cancer state. Biochem. Soc. Trans., 1985, 13, 389. Grant D., W.F., Long and F.B. Williamson Pericellular heparans may contribute to the protection of cells from free radicals. Med. Hypotheses, 1987, 23, 67-72. Grant D., W.F. Long and F.B. Williamson A model of two conformational forms of heparins/heparans suggested by infrared spectroscopy. Med. Hypotheses, 1987, 24, 131-137. Grant D., W.F. Long and F.B. Williamson Effect of heparin on dismutation of superoxide anion. Biochem. Soc. Trans., 1988, 16, 1030-1031. Grant D., W.F. Long and F.B. Williamson A model of Ca2+ -heparin interaction. Biochem Soc. Trans. 1988, 16, 1028-1029. The presence of more than one counterion (studied with Na+ + Ca2+) caused a blurring of The chemical distinctions between the counterions which tended to undergo random fast exchange distribution with a similar binding strength. Grant D., W.F. Long, C.F. Moffat and F.B. Williamson Infrared spectroscopy of chemically modified heparins. Biochem. J., 1989, 261, 1035-1038. Grant D., W.F. Long and F..B. Williamson Biochem. J., 1989, 259, 41-45. Heparin-polypeptide interaction. Near-i.r spectroscopy in an anhydrous dispersant allows the involvement of polymer-associated water to be assessed.

Grant D., W.F. Long. C.F. Moffat and F.B. Williamson Infrared spectra of chemically modified heparin Biochem. J., 1989, 261, 1035-1038. The presence of more than one counterion (studied with Na+ + Ca2+) caused a blurring of the chemical distinctions between the counterions which tended to undergo random fast exchange distribution with a similar binding strength. Grant, D., W.F. Long., C.F. Moffat and F.B. Williamson Infrared spectroscopy as a method of investigating the conformation of iduronate saccharide residues in glycosaminoglycans Biochem. Soc. Trans., 1990, 18, 1277-1279 Grant D., W.F. Long and F. B. Williamson Infrared spectroscopy of heparin suggests that the region 750-950cm-1 is sensitive to changes in iduronate ring conformation; Biochem. J., 1991, 275, 193-197 Grant D., W. F. Long and F.B. Williamson Examination of cation-heparin interaction by potentiometric titration Abstracts 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. Williamson A possible ring conformational change of iduronate residues detected by ir spectroscopy of aqueous solutions of lithium heparin Biochem. Soc. Trans.,1991, 18 (6) 1281-1282; Grant D., W.F. Long and F.B. Williamson The dependence on counter-cation of the degree of hydration of heparin Biochem. Soc. Trans., 1991, 18 (6) 1283-1284 Grant D., C.F. Moffat., W.F. Long W.F. and F.B. Williamson Ca2+ -heparin interaction investigated polarimetrically Biochem. Soc. Trans., 1991, 19 (4) 391S Grant D., C.F. Moffat., W.F. Long and F.B. Willliamson A relationship between cation-induced changes in heparin optical rotation and heparin-cation associated constants Biochem. Soc. Trans., 1991, 19 (4) 392S Grant D., C.F. Moffat, W.F. Long and F.B. Williamson Optical rotation changes in chemically modified heparins as a guide to anionic groups involved in Ca2+ binding Biochem. Soc. Trans., 1991, 19, 393S Grant D., C.F. Moffat, W.F. Long and F.B. Williamson Carboxylate symmetric stretching frequencies and optical rotation shifts of heparin cation complexes Biochem. Soc. Trans., 1991, 19 (4) 394S Grant D., W.F. Long and F.B. Williamson I.r. spectrosocpic analysis of heparin-polypeptide interaction Biochem. Soc. Trans., 1991, 19 (4) 395S Grant D., W.F. Long, C.F. Moffat and F.B. Williamson Polarimetry of mixtures of Cu(II) ions and chemically modified heparins Biochem. Soc. Trans., 1991, 20, 2S

Grant D., W.F. Long and F.B. Williamson IR spectroscopy of heparan sulphates isolated from the surfaces of normally and virally transformed fibroblasts NIR News,1992,19-21 Cf., Grant D., W.F. Long and F.B. Williamson NIR spectroscopy shows that animal cell adhesion to non-biological solid surfaces may require surface water structuring NIR News, 1992, 22-24 (Proc. Int. Conf., Aberdeen, Near Infrared Spectroscopy, 1991 pub. 1992 Ed. Murray I,, and Cowe I.A. VCA Weinheim Germany; Chem. Abs., 118 164498z) Grant D., W.F. Long and F.B. Williamson Multiple-specular-reflectance i.r. spectrocospy of glycosaminoglycan-cetylpyridinium complexes Biochem. Soc. Trans., 1992, 20(1) 4S Grant D., W.F. Long and F.B. Williamson A putative role for colloidal silicates in primitive evolution deduced in part form their relevance to modern pathological afflictions Med. Hypotheses., 1992, 38, 46-48 (Later discussions took this hypothesis further. The original background to this hypothesis was the observations partly reported in Brit Pats 1143014 and 1136016 of biological-like self assembly of silica sols. Different types of silica sols underwent self seeding of new particle growth and therefore a system of different silica sols (generated by slight difference in initial conditions) seemed to be potentially capable of competing with each other for the acquisition of low molecular weight (water soluble) silicic acid and thereby suggested a hypothesis for the generation of high levels of molecular complexity and cellular structures such as the precursors of modern organisms. The natural ability form structures of increasing 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 for obtaining nutrient for growth (initially silicic acid) might have been achieved by the evolution of motility this needing energy generation e.g. via mechanisms using sequestered inorganic energy-rich phosphate structures in the pores of the silica sols; primitive pro-biological polysilicates would have been capable from the start of multi-ion adsorption from seawater; evolution of polysaccharides proteins and nucleic acids would have followed from advantages produced by these systems for gaining nutrients. Cf., Grant D., W.F. Long and F.B. Williamson Degenerative and inflammatory diseases may result from defects in antimineralization mechanisms afforded by glycosaminoglycans Med Hypotheses, 1992, 38, 49-55 This article argued that polyanions similar to polyphosphates and glycosaminoglycans may 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. Williamson The binding of Pt(II) to heparin Biochem. Soc. Trans., 1996, 24, 204S Grant D., W.F. Long, G. Macintosh and F.B. Williamson Antioxidant activity of heparin Biochem. Soc. Trans., 1996, 24 (2) 194S Grant D (1997) Unpublished The ability of nitrite (which is believed to be the major nitric oxide metabolite which is active in the above heparan sulphate signalling pathways) to react with unsubstituted glucosamines now known to be formed during the primary biosynthesis as well as by post-synthetic events in heparan sulphate chains and thereby to create a range of signalling oligosaccharides is dependent on metal ion catalysis (iron ion can, e.g. catalyse a de-N-sulphonation process which is required to generate active sites for

nitrite cleavage). Inorganic cofactors which can affect nitrosative signalling by the heparome probably also include, apart from metal ions, a range of gas molecules (which may include hydrogen sulphide, hydrogen monoxide, hydrogen peroxide) as well as nitric oxide. Grant D., W.F. Long and F.B. Williamson Incompletely published (but displayed as conference posters) of studies conducted by procedures similar to these described in ref. (32) showed that the carrageenans and other anionic polysaccharides as well natural polyanions such as humic materials, by binding to nascent crystallization nuclei also inhibit CaCO3 crystallization; the highly efficient action of humic polymers is probably of major relevance 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 intensive agriculture]. Grant D. (2000) http://web.ukonline.com.uk/dgrant/dg5 Grant D. (2000) Ascorbate and nitric oxide in redox control of heparan sulphate http//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 at uniquely accessible extracellular sites in contrast with the DNA location. The suggested normal physiolgical heparan sulphate multielement sequestration behaviour seems intrinsically to contrast strongly with the perceived situation with DNA which it might be anticipated, must be shielded from potential disruption of its encoded information e.g. by Fenton reactions inducible by contact with damaging metal ions which is prevented in eukaryotes by holding DNA intracellularly, shielded by the cytoskeleton and histones and subject to the protective actions of antioxidants, high affinity metal ion binding proteins and specific damage correction repair mechanisms. Hence although both heparin/heparan sulphate and DNA contain conceptually similar linearly encoded information systems, quite different regulation of metal ion interactions may be required for proper function of these two linearly information encoded biopolymer systems].

Grant D. (2005) Metallome-Heparanome Crosstalk Hypothesis Although there are a large number of possible in vivo relevant multielement biological ligands additional to 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 the extracellular 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-anionic biological 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 ICPMS) 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, such polysaccharide 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 blood serum). The multielement character of heparin and heparan sulphate is of obvious relevance to the mechanism of heparanome protein interactions designed for subsequent easy release of a wide variety of metal ions required, e.g., for specific nucleic acid, protein or polysaccharide structure building and related functions. an absolute requirement for specific divalent metal ions can potentiate signalling by growth factors and could be critically relevant for fundamental studies of the biological roles of heparin/heparan sulphate--metal ion--protein and heparan 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 sea water are now thought to be of fundamental interest to the fuller understanding of the roles of metal ions in biology. Highly anionic extracellular polysaccharides could provide suitable metallomic ligands. Heparin, it is now suggested is perhaps the single most relevant such ligand since this is the most ultra anionic polysaccharide in biology. The uniquely high multielement binding capacity heparin can uniquely provide insight into mammalian metal ion presence. Further study of the mass spectrosocpic multielement evaluation of polysaccharides derived from human and animal tissues is warranted}.

Grushka E. and A.S. Cohen The binding of Cu(II) and Zn(II) ions by heparin Anal. Lett., 1982, 15 (B16), 1277-1288 Hamazaki H. Ca2+-mediated association of human serum amyloid P component with heparan sulfate and dermatan sulfate J. Biol. Chem., 1987, 262, 1456-1460 (No binding was observed in the absence of added Ca2+ but other M2+ ions studied (M: Ba, Cu, Mg, Mn and Sr) did not promote binding) Herwats L., P. Laszlo and P. Genard How heparin binds sodium: a sodium-23 NMR study Noveau J de Chemie, 1977, 1 (2) 173-176 Hu W.-L., and R. Reogoeczi Hepatic heparan sulphate proteoglycan and the recycling of transferrin Biochem. Cell Biol., 1992, 70, 535-538 Iler R.K. The Chemistry of Silica Wiley, 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-sulfate and 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 drug Circulation, 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 this was regarded as ‘impurities’} N.b., heparin extracted from (mast cells) in mammalian tissue is a pharmaceutical agent and a convenient model for elucidating the behaviour of the structurally related heparan sulphate proteoglycan system management system which apparently most often depends for its activity on the information encoded heparin-like segments.

Karlinsky J.B. and R.H. Goldstein Regulation of sulfated glycosaminoglycan production by prostaglandin E2 in cultured lung fibroblasts J. 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 glycoprotein Thromb Haemostasis, 67 (1) 50 Chem. Abs., 114,187724t Kjellen L. and U. Lindahl Proteoglycans: structures and interactions Annu. 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 for membrane binding Eur. J. Biochem., FEBS., 1992, 210, 73-77 Liang J.N., B. Chakrabarti, L. Ayotte and A.S. Perlin An essential role for the 2-sulfamino group in the interaction of calcium ion with heparin Carbohydr. Res., 1982, 106, 101-109 Luck W. Ber Bunsenesgesel Phys, Chem., 1965, 69(1) 69 (Cf. also ibid., 826 and Kleeberg E. Ed., (Proc. Symp. 2-3 Aprl 1987, Marburg, FRG in Honor of the 65th Birthway of Werner A.P. Luck) “Interactions of Water in Ionic and Nonionic Hydrates”, Springer Verlag Berlin, 1987; Luck W.A.P. Is life mainly inorganic chemistry? Topics Curr. Chem., 1976, 5, 115-180 [Salt effects on the association of water: the explains the Hofmeister effect which may in turn explain the role of water clusters associated with sulphated polyanions as modulators of protein folding] [If life is mainly inorganic chemistry then the metallome is obviously a key player in he most fundamental processes determining the basic life processes] Lyon M.E. Specific heparin properties interfere with simultaneous measurement of ionized Mg and ionized Ca Clin. Biochem., 1995, 28 (1) 79 [Time dependent bias was observed in ionized Mg and Ca concentrations with Zn heparin but not with Li or electrolyte balanced heparin] Lyon, M., J.A. Deakin and J.T. Gallagher Liver heparan sulphate structure. A novel molecular design. J. Biol. Chem., 1994, 269 (15) 11208-112115. McKeehan W.L., X. Wu and M. Kan Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex J. Biol Chem., 1999, 274 (31) 21511-21514 Murata K., and Y. Yokoyama Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissue Atherosclerosis (Shannon, Irl.) 1989, 78 (1) 69-79; Chem. Abs., 111, 151343a [Age-dependent alteration in abundance of heparan sulphate at arterial walls] (In a related study by E. Feyzi , T. Saldeen, J. Larssen U. Lindahl and M Salmivirta Age-dependent modulation of heparan sulfate structure and function Biochem. J. 1998, 273, 13795-13798 it was proposed that lifespan in humans is determined by heparan sulphate microstructural alteration during ageing) Muzzarelli R.A.A Heparin-like substances and blood compatible polymers obtained from chitin and chitosan Polymer Science Technology, 1983, 23, 359-374 [This paper suggests that the multi-inorganic elements which are associated with these anionic polysaccharides derived from tap water]

Nagasawa K., H. Uchiyama, N. Sato and A. Hatano Chemical change involved in the oxidative-reductive depolymerization of heparin Carbohydr. Res., 1992, 236, 165-180 Ohkubo Y., Tsukada F., Kohno H., and Kubodera A. (1989) Relationship between binding activity of 67-Ga and low sulphated acid glycosaminoglycans Nuclear Med. Biol., 16 (4) 343-346; Chem. Abs., 111, 111668d Panov V.P., and A.M. Ovespan (1984) Study of heparin salts by spectroscopic methods (translated) Vysokomol. Soedin. Ser., A 26(9) 1963-1970 ; Chem. Abs., 102, 113831q Parish R.F., and W.R. Fair Selective binding of zinc ions to heparin rather than to other glycosaminoglycans Biochem,.J., 1981, 193, 407-410 Percival E., and R.H. McDowell Chemistry and Enzymology of Marine Algal Polysaccharides Academic Press, London and New York (1967) cf., p. 19 Rabenstein D.L., J.M. Robert and J. Peng Multinuclear magnetic resonance studies of the interaction of inorganic cations with heparin Carbohydr. Res., 1995, 278, 239-256 Renne T., J. Dedio, G. David and W. Muller Esterl J. Biol. Chem., 2000, 275 (43) 33688-33696 [Zn2+ promotes the association of HS and (biotinylated H-) kininogen] Senofonte Violante N., and S. Caroli Assessment of references values for elements in human hair of urban schoolboys J. Trace Elem. Med. Biol., 2000, 14(1) 6-13 Schlemmer U. Studies of the binding of copper, zinc and calcium to pectin, alginate, carrageenan and gum guar in HCO3 - - CO2 buffer Food Chem., 1989, 32 (3) 223-234 Stringer, S.E., M. Mayer-Proschel, A. Kalyani, M. Rao and J.T. Gallagher Heparin is a unique marker of progenitors of the glial cell lineage. J. Biol. Chem., 1999, 274 (36) 25455-25460 Tajmir-Riahi H.-A. D-Glucose adducts with zinc-group metal ions. Synthesis and spectroscopic and structural characterization of Zn(II), Cd(II) and Hg(II) complexes with D-glucose, and the effects of metal-ion binding on the sugar anomeric structures Carbohydr. Res., 1989, 190, 29-37 Templeton D.M. Acceleration of the mercury-induced aquation of bromopentammine Co(III) by naturally occurring glycosaminoglycans Can. J. Chem., 1987, 65, 2411-2420

Timpl R.

Structure and biological activity of basement membrane proteins Eur. J. Biochem., 1989, 180, 487-502 Toida T.E. with R.J. Linhardt, et al. Detection of GAGs Cu(II) complex in capillary electrophoresis Electrophoresis, 1996, 17, 341 346; J. Chromatog., 1997, 787 (1-2) 266-270 Vandewalle B., F. Revillon, L. Hornez and J. Lefebvre Calcium regulation of heparan sulphate proteoglycans in breast cancer cells J. Cancer Res. Clin. Oncol., 1994, 120 (7) 389-392 ; Chem. Abs., 121,105487j Wiggins P(M) Life depends on tow kinds of water PLoS ONE 2008 Jam 9 3(1)e1406 Cf also <http://www.Isbu.ac.uk/water/monograph200904pw.pdf> And Physica A2002, 414, 458 Williams R.J.P. The biochemistry of sodium, potassium, magnesium and calcium Quart. Rev., 1970, 24 (3) 331-365 Yamaguchi S., T. Yoshioka, M. Utsunomiya, T. Koide, M. Osafune, A. Okuyana and T. Sonada Heparan sulfate in the stone matrix and its inhibitory effect on calcium oxalate crystallization Urol. 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. Hurley Heparin-binding properties of lactoferrin and lysozyme Comp. Biochem. Physiol., 1992, 103B (4) 889-895 [Biotinylated heparin binding to lactoferrin was dependent on Na, Ca, Cu, Zn and Fe cations] _____________________________________________________________________________________ A heparan sulphate-environment feedback system which can act to determine alteration in animal speciation must of course eventually alter DNA. This could be closely related to an interaction of mutational activity and nitrosative signalling processes. The current list of reported interactions between the heparanome and the genome seem to suggest this possibility but for a more complete understanding of the phenomenon of evolution of species it must also, it can be suggested, be established why the presence of liquid water is so essential for life. It can be argued that sugars and polysaccharides including heparan sulphate must play a key role in the regulation of water activity. (Cf. the interspecies dependence heparan sulphate contents of tissues of aquatic invertebrates were found to be related in a mathematically exact manner to the salinity and therefore potential osmolyte stress of their habitats (this information was reported by H.B. Nader et al. in ref. 7-2); a related phenomenon is the influence of inorganic ion concentrations upon Hofmeister effects, which in turn are ascribable to an action of such ions on water structure e.g. as discussed by W. Luck and P. Wiggins (cf. references cited in the Suppl. Refs. It can be demonstrated that heparin/heparan sulphate metal ion complexation impacts upon this phenomenon (e.g. as reported by Grant et al, in the references listed in the Suppl Refs are fully discussed in www.scribd.com/doc/26994439/Publication-2-Web).

Loss of Essential Metal Ion Cofactors During Purification Although metal ions are known (cf., Table I) to have critical roles in the modulation 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 - heparan sulphate binding was discovered (ref. 14-2). Whilst binding to Zn2+ will normally occur in vivo, this binding was found to be it is abolished in vitro unless Zn2+ ions, evidently removed during column polysaccharide gel 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 unexpected source of experimental errors. In vivo, sufficient metal ions for facilitation of the correct protein-heparan sulphate interactions will normally 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 sodium chloride. --------------------------------------------The metal ion dependent postsynthetic alteration of information encoded in coded heparin/heparan is not restricted, as is DNA, by a requirement to preserve genetic information, so that while the entire signal in heparan sulphate chains is believed to be of physiological significance (e.g. as a sort of biological postcode for defining particular cellular types and locations) the details of this code have not yet been established owing to the lack of sequencing methods as good as those available for nucleic acids) the heparan sulphate chains are normally utilised after being specifically modified to generate fragments containing information packets designed to be read at distant sites. It is now suggested that inorganic elements are required to create the encoded information present in heparin/heparan sulphate system (including the oligosaccharide messengers). **The heparanome

The heparanome is the name recently suggested for the system of animal polysaccharides which contain evolutionary conserved (7-1) domains of mineral-like anionic arrays of sequences of highly sulphated polyiduronate/glucuronate glucosamine N-sulphonate residues. Studies of HSPG biochemistry suggest an especially important role for such information encoded sequences occurring in side chain polysaccharide structures which act like biological postcodes to facilitate the interaction with conserved HSPG binding sites in proteins. They have well established roles in morphogenesis (providing a reservoir and control system for basic fibroblast growth factor and its receptors, regulation of embryo assembly) mediation of adhesion and morphogenesis, the provision of links between cytoskeleton and extracellular matrix, modulation of antioxidant activity including the anchoring of endothelial antioxidant enzymes, the assembly of matrix phosphatidyl-inositol linkages, regulation of blood coagulation and apoptosis, modulation of synaptic and neurological activity, and are implicated in the mechanisms of memory, cognition and ageing. Although such activities are more pronounced for HSPGs than for other glycosaminoglycans the 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 fibrillogenesis including the transparency of the cornea. Glycosaminoglycans generally provide a regulation of calcification, cell migration, aggregation and development, a filtration barrier and stabilization of basement membrane, synaptic structures, endothelial surfaces, mediate of tranferrin uptake and have roles in antigen presentation. Heparin, a cocktail of heparan sulphate-like molecules produced by mast cells present in many animals (but not apparently in rabbits) is believed to be the most highly anionic polysaccharide system. It is manufactured as a largely protein-free pharmaceutical agent widely used for blood anticoagulation, an action which is mainly attributable to its content of an antithrombin (III) pentasaccharide binding sequence which is also present in anticoagulant-type-HSPG which can be induced to occur at vascular walls from which it can be released by proteolytic or nitrosative cleavage (processes which are also subject to perturbation by inappropriate multielements constituents of blood serum).

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----*Home based research continued from former institutional affiliated research at the University of Aberdeen (Marischal College) and discussions with F.B. Williamson and other former Aberdeen Polysaccharide Group members and others including R.J.P. Williams (Oxford University) and K.E.L. McColl (Glasgow University). Cf.,D. Grant, W.F. Long and F.B. Williamson:
Infrared spectroscopy of heparin-cation complexes Biochem. J., 1987, 244, 143-149 Similarity and dissimilarity in aspects of the binding to heparin of Ca2+ and Zn2+ as revealed by potentiometric titration Biochem. 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 cation Abstracts 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 157.

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