WRL3217 Contiued Modif

20/4/05 Modified 18/5/08; 20/5/09
The Relevance to Metallomics of the Binding of Metal Ions to

Heparin/Heparan Sulphate.
Heparin may provide a high capacity multilelement binding matrix of especial relevance to biological metallomic research.
The heparanome and metallome are suggested to interact and modulate the activity of a range of
fundamental biological processes in animals.
David Grant*
(A hypothesis compiled from research carried out at Marischal College, University of Aberdeen*
re-copied from a note written 4/2/05 at Turriff AB53)
Summary
The metallome, a new scientific field designated by H. Haraguchi which concerns, inter alia the
relationships between the multielement profiles of biological and geological matrices, is now suggested
to be highly relevant to the heparanome, the system of heparin/heparan sulphate polysaccharides which
occur in multicellular animal and which may provide, in addition to the modulation of many protein
activities, a wideranging system of homeostasis for metal ions and H+ . The heparanome and the
metallome may cross react in vivo
Introduction
Recent discussion of the relevance of the large number (ca. 80) of elements routinely detected by mass
spectroscopic analysis in biological matrices has led H. Haraguchi (1) to elaborate some of the earlier
ideas of R.J.P. Williams, and to initiate a new scientific field dealing with the occurrence and relevance
of
multielement matrices for which the term “metallomics” has been suggested. Implicit in the concept of
metallomics is the possible significance of connections between geology, inorganic chemistry and
biology The metallome was suggested by Haraguchi to apply to biology principally for the provision of
metal ions required to generate active sites in proteins (1). It is now suggested that a similar fundamental
requirement of metal ions as essential cofactors for polysaccharides is also of relevance. Metal ions are
known to occur naturally in anionic polysaccharides (1a,b,c) including heparin (1d) which sequesters
Cu2+ Ca2+ and Zn2+ (1g) in a physiologically relevant manner. This phenomenon could be critically
relevant to the activity of the “heparanome”** (2) (the heparin/heparan sulphate and (putatively) metal
ion dependent signalling system in animal biology (2-9)). Heparin may contain a surprisingly large
amounts of the least abundant of the full range of the ultratrace non-physiological elements (1d) which
are also now known to consistently occur in biological fluids including blood serum (1). While it is now
fairly well established that Cu2+, Ca2+ and Zn2+ are essentially required cofactors for various heparanome-
related protein control mechanisms (10), (10b) (some examples of which are listed in Table I), the
various ultratrace inorganic ions which occur in biological fluids (1) but which currently have no known
physiological function, might conceivably also be involved in heparin/heparan sulphate signalling or its
anthropogenic perturbation.
Medical Use of Metal Ion Binding to Heparin/Heparan Sulphate

Binding of Eu3+ (11) can be used for heparin assay in blood samples.
Pathological lesions can be imaged (12) by an altered binding of metal ion radiolabels such as 67Ga.
Similar use of 111In and 99Tc labelled heparin (13), however, seemed to be less effective.
67Ga is believed to bind especially strongly to the heparan sulphates which occur at all adherent cell
surfaces but are subject to structural alteration as a consequence of pathological situations e.g. cellular
oncogenic transformation (14).
In vitro studies (12a) confirmed that radioimaging by metal ions is facilitated by an alteration in the
anionic density of heparan sulphates associated with pathological lesions (rather than, e.g., transferrin),
this being confirmed by an in vitro study which confirmed that a wide range of physiologically relevant
metal ions bind to heparan sulphate (11e). These findings also tend support the notion that heparan
sulphate probably exists in vivo as a form of specific metallomic matrix.
Participation of Metal Ions in Heparin/Heparan Sulphate Signalling.

Heparin/heparan sulphate, which contains a linear encoded information system, is produced by an as yet
not fully understood biosynthesis in the Golgi apparatus, which includes epimerisation, deacetylation and
sulphation stages (2-9) but also is subjected to a postsynthetic modification by both enzymic (15) and
non-enzymic (16) (16a) (16b) pathways which are apparently used for the creation and transmission of
encoded information in the form of anionic polysaccharides. These anionic polysaccharides are now
suggested always to occur in vivo in the form of (multiple) metal ion complexes. This information
processing system apparently also includes possibly complex metal ion-dependent inputs from enzymic
and non-enzymic scission by redox metal ion generated radicals as well as by specific redox metal ion
dependent deaminative cleavage reactions (17).
The postsynthetic coding alteration of 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 which 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 (18-20).
Metal Ions Can Link Heparin/Heparan Sulphate Domains to Proteins

A effect of the binding to heparin/heparan of (non-redox) metal ions may be to assist heparan sulphate-
protein binding which at least in some cases, has an absolute requirement for specific divalent metal ions,
which are apparently needed to create a correct linkage between the two types of polymers to facilitate
normal heparin/heparan sulphate biochemical control processes.
Cf. the anticoagulant heparin/heparan sulphate antithrombin (III) binding sequence which is the major mammalian blood
anticoagulant mechanism which operates in conjunction with the action of divalent metal ions which similarly can affect growth
factor receptor activation by a process which could further be relevant to the toxic actions of e.g., barium ions in the aetiology of
degenerative diseases in which abnormal growth factor activities are apparent.
Table I collects some of the reported evidence for an absolute requirement for the presence of specific
divalent metal ions (e.g. Ca2+ or Zn2+) putatively required to generate the required polysaccharide
conformations in order to achieve the correct interactions with their target proteins.
These actions, it may be supposed, may be perturbable by such toxic ions as Pb2+, Cd2+ and Ba2+.
It must be emphasised that although metal ions are known (Table I) to have critical roles in 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 (10b). 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.
A direct observation of Ca2+ ions linking annexin-V and heparin is shown by the X-ray structure of a
heparin oligosaccharide annexin-V complex (10c). Heparin/heparan sulphate and Ca2+ are required for in
vivo structure building of functionally active annexin-V aggregates at the plasma membrane.
The structure of fibroblast growth factor receptor dimer (10e) shows a similar divalent cation dependent
interlinking of heparan sulphate to protein.
The Role of Heparin/Heparan in Controlled Assembly/Disassembly

of Solid Phases
The assembly, disassembly and inhibition of crystalline and semi-amorphous substance formation is
strongly affected by the presence of anionic polysaccharides, which includes the important function of
heparin/heparan sulphate for the inhibition of pathological calcification (21) and also for an apparent
antioxidant mechanism involving the removal of Cu2+ (22), Fe2+ (23) and Fe3+ (24) ions from the solution
phase including by the binding of these ions to heparin/heparan sulphate, and their assembly into redox-
inert aggregates (25) similarly to how chitosan and hyaluronan have recently been reported (26) to
achieve a similar antioxidant protection. Heparin may bind paramagnetic ions in aggregated insoluble
forms (26a).
Discussion
Polysaccharides, occurring abundantly throughout biota, characteristically bind to and may, in vivo,
naturally contain a wide range of both nutrient and toxic elements present in amounts which shows an
approximate correlation with the amounts of such elements in blood serum and seawater (1).
This circumstance seems relevant, inter alia, to a full definition of the scope of metallomics (1) a new
branch of biometal science, which aims to compare and evaluate the relevance of various multielement
profiles throughout biology and geology.
Electrostatic theory (e.g. that of Manning) predicts that polyelectrolytes will bind oppositely charged
counterions equally for equally charged counterions. In depth studies of the mechanism of binding of
counterions to heparin (26a) dis not, however, support the Manning electrostatic binding theory, despite
the similarity of the strength of binding found to be achieved of many similarly charged cations to
heparin which had been predicted by this theory. The Manning electrostatic attraction theory cannot
account for the well-established the ability of the ultraanionic heparin anions to strongly to associated
with SO42- anions (27). The non-electrostatic binding activity of polyol groups within all
polysaccharides may, however, be responsible for this. Such a general mode of binding by all
polysaccharides evidently become modulated by additional electrostatic fields present in anionic
polysaccharides exemplified by heparin/heparan sulphate. These formally ultra-anionic polysacchairdes
also show great structural diversity (to which inorganic ions it is now suggested also contribute). This
flexible diversity apparently enables these molecules to perform a wide range of signalling and control
functions in animal biochemistry. It is therefore predicted that the precise physical chemical and
ultrastructural details of the counterion profile (and its ability to engage in variable rapid site exchange
(cf. 28)) and the possible perturbation of this by anthropogenic input, could be highly relevant to the in
vivo molecular (/supramolecular) structure forming and physiological activities of these polysaccharides.
Specific metal ions (e.g. Ca2+) have previously been reported to be absolutely required for several
hepain/heparan sulphate protein binding and regulation functions, some examples of these effects being
listed in Table I. Inorganic cations and anions have also been reported to modulate heparan sulphate
primarly biosynthesis as exemplified in Table II and specific cations may be are required for
postsynthetic structural modification (cf. Table I).
Table I
Examples of facilitation by divalent cations of the binding of heparin/heparan

to proteins.
Cation Process Involving Heparin/Heparan Sulphate | References
_____________________________________________________________________________________
M2+ ions Assembly of Annexin-V Capila et al. 2001, ref. (10c)

at phospholipid membranes
-------------------------------------------------------------------------------------------------------------------------------------
Mn2+ Serum lipid aggregate formation

low density lipoprotein (LDL) Lindahl & Hook 1978, ref. (4)
phospholipid linking M2+ ions
-----------------------------------------------------------------------------------------------------------------------------------
M2+ ion Modulation of FGF-2 activity Kan et al. 1996, ref. (10e)
Ca2+, divalent cation required for dimerisation of
Mg2+ 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)
-----------------------------------------------------------------------------------------------------------------------------------
Ca 2+
Inhibition of heparin binding to thrombin Spreight & Griffith 1983, ref. (29-3)
------------------------------------------------------------------------------------------------------------------------------------
Ca2+ Fibronectin Hayashi & Yamada 1982, ref. (29-4)
-------------------------------------------------------------------------------------------------------------------------------------
Ca2+ β2 and β3 integrins, Discussed by Kan et al. 1996, ref. (10e)
-------------------------------------------------------------------------------------------------------------------------------------
Ca 2+
Platelet/endothelial
cell adhesion molecule-1 (PECAN-1) Discussed by Kan et al. 1996, ref. (10e)
-------------------------------------------------------------------------------------------------------------------------------------
Ca2+ Cadhedrin, Discussed by Kan et al. 1996 ref. (10e)
-------------------------------------------------------------------------------------------------------------------------------------
Ca2+ L-selectin, Koenig et al. 1998, ref. (29-5)
Norgard-Sumnich et al. 1993, ref (29-6)
--------------------------------------------------------------------------------------------------------------------------------
Ca2+ Serum amyloid P (SAP) Nielson et al. 1994, ref. (29-7)
-----------------------------------------------------------------------------------------------------------------------------------
Ca2+ Porcine brain synaptosome Shinjo et al. 2004, ref (29-8)
Zhao & Zhang 2003, ref. (29-9)
Ca-ATPase inhibition
-----------------------------------------------------------------------------------------------------------------------------------
Ca2+ Recycling of heparan sulphate proteoglycans by parathyroid cell lines
is dependent on extracellular Ca2+concentration Takeuchi et al. 1990, ref. (29-10)
-----------------------------------------------------------------------------------------------------------------------------------
Ca2+ - NEUROLOGICAL ACTIVITY -Heparan Sulphate
Ca2+ Signaling in neurons induced by S100A4 Kiryushko et al. 2006, ref. (29-11)
Glycosaminogycans (especially heparan sulphate)
may act as co-receptors of S100 proteins in neurons
-------------------------------------------------------------------------------------------------------------------------------------
Ca2+/Na+ Smooth muscle Na+/Ca2+ exchanger
heparin fragments bearing C4-5 unsaturation Schinjo et al. 2004, ref. (29-8)
at the non-reducing end, produced by
bacterial lyase activity
Table I, cont.
Heparin binds with high affinity to Knaus et al. 1990, ref. (29-12)
voltage-dependent L-type Ca2+ channels
cf. putative Ca2+/Na+ vascular flow sensor

via induced heparan conformation alteration Siegel et al. 1998, ref (29-13)
Zn2+ (and Ca2+ ) Modulation of heparan sulphate binding
Zn2+ Inhibition of FGF-2 activity Ricard Blum S et al. 2004 ref. (10b)
---------------------------------------------------------------------------------------------------------------------------------
Zn 2+
Modulation of MRP-8/14 S100 activity Robinson et al. 2002, ref. (29-14)
-----------------------------------------------------------------------------------------------------------------------------------
Zn2+ Fibrillin-1, Tidemann et al. 2001, ref. (29-15)
-----------------------------------------------------------------------------------------------------------------------------------
Zn 2+
H-kininogen. Stanley et al. 1995, ref. (29-16)
-------------------------------------------------------------------------------------------------------------------------------------
Zn 2+
Histidine rich glycoprotein Jones et al. 2006, ref. (29-17)
-------------------------------------------------------------------------------------------------------------------------------------
Zn2+ Histamine Kerp 1963, ref (29-18)
-------------------------------------------------------------------------------------------------------------------------------------
Cu 2+
Fibrinogen Lages & Stivala 1973, ref. (29-19)
-------------------------------------------------------------------------------------------------------------------------------------
Cu 2+
Prions 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)
enables heparan sulphate oligosaccharide generation e.g. , Ding et al.., 2002, ref. (16a)
A possibly related process is the

Mn2+ dependent soluble guanylate cyclase inhibition by sulphated polysaccharides 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 recox metals such as Cu and Fe,
known to occur in phosphate buffers, might promote this reaction.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------
Amyloid Formation
Zn2+ 50nM Zn2+ promotion of binding of heparin to
Amyloid Precursor Protein (APP)
Inhibition of APP proteolysis by heparin abolished by Zn2+ Masters et al. 1993, ref. (29-23)
---------------------------------------------------------------------------------------------------------------------------------------------
Inorganic Crystal Formation

Calcium Oxalate Crystals or precursor high oxalate concentration Borges et al., 2005, ref. (29-24)
Table II
Examples of Reports of The Effects of Inorganic Cations and Anions on Heparan Sulphate Biosynthesis
Mn2+ Promotion of heparan sulphate A.Z. Kalea et al., Biometals 2006, 19,
biosynthesis 535-546 (ref. 29-25)
----------------------------------------- ------------------------------------
α-GlcNAc transferase I can use both Mn2+ T.A. Fritz et al.,J. Biol. Chem., 1994, 269,
and Ca2+ but 28809-28814 (ref. 29-26)
α-GlcAc transferase II can use only Mn2+
Mg2+ Effect of Mg2+ deficiency on the P. Jaya and P.A. Kurup,J. Biosci., 1986,
metabolism of glycosaminoglycans, 10, 487-493 (ref. 29-27)
including heparan sulphate
Ca2+ Suppression of proteoglycan including

heparan sulphate synthesis by calcium
ionophore A23187 in cultured
vascular endothelial cells
------------------------------------------ --------------------------------------- Y. Fujiwara and T. Kaji , J. Health Sci.,
Pb2+ Implication of intracellular calcium 2002, 48, 460-466
accumulation in Pb2+ inhibition of (ref. 29-28)
endothelial proteoglycan including
heparan sulphate biosynthesis
Cd2+ Inhibition of the incorporation of [35-S] A. Cardenas et al.,Toxicology

sulphate into glomerular membranes of 1992, 76, 219-231 (ref.29-29)
rats chronically exposed to Cd2+ and its
relation with urinary glycosaminoglycans
and proteinuria
Hg2+, Ni2+ Inhibition of glomerular heparan sulphate D.M. Templeton, Proc. Trace Element
biosynthesis. Health Disease, IUPAC Int. Symp.,1990,
Metal-proteoglycan interactions in the p. 209-219 (ref. 29-30)
regulation of renal mesangial cells:
implication for metal induced
nephropathy.
SiO2 Putative promotion of heparan sulphate M.F. McCarty
biosynthesis Med Hypoth.,1997, 49, 177-179
Cf., R.M. Iler The Chemistry of Silica ,
Si may always occur in heparin/heparan Wiley, 1979, cf. p. 762 (ref. 29-31)
sulphate
F- F- ions inhibit heparan sulphate sulphation M. Pawalowska Goral et al., Fluoride,

1998, 31, 193-201 (ref. 29-32)
SeO4 2-
Inhibits heparan sulphate biosynthesis C.P. Dietrich et al., FASEB J., 1988, 2, 56-
59 (ref. 29-33)
The reports listed in Table I and Table II, taken together, suggest that heparanome associated metal ions
could normally be essential components of the modus operadi of this complex information system in
which inorganic ions may perhaps act as part of a servo feedback messenger system acting in response to
achieve alterations in extracellular metal ion concentrations (similar to the known dependence on
extracellular Ca2+ concentration of heparan sulphate biosynthsis in rat parathyroid cells (29-10)).
The X-ray crystal structure of a heparin oligosaccharide annexin-V adduct indicates how interlinking Ca2+
ions might influence the aggregation of annexin V at plasma membranes and how this activity might be
perturbed by Ba2+ which is reported to disturb Ca2+ binding to annexin-V (2) 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 (30).
The ability to bind a wide range of elements by heparin/heparan sulphates is found to be shared with
other anionic polysaccharides such as those (e.g. alginates and carrageenans) occurring extracellularly in
marine algae. This is the basis of the use of the algal biomass of kelp for plant and animal nutrition (3-4)
and for its use for the removal toxic heavy metal ions from polluted waters (5,6) .
Heparin, however is believed to be the most highly anionic polysaccharide, is also readily available since
it is manufactured as a largely protein-free pharmaceutical agent for blood anticoagulation, mainly
attributable to its content of the antithrombin (III) binding sequence which is also present in
anticoagulant-type-HSPG present 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).
Heparin-like structures are known to also to occur in segments of similar structure present in the highly
sulphated domains of heparan sulphate (e.g. at endothelial surface and, in liver (31) and in glial cell
progenitors (32).
The physiological role of multi-element binding to heparin may suggest that heparin and related
polysaccharides also function as a nutrient sequesters able rapidly to release when required bound Fe2+,
Cu2+, Mn2+, Mg2+ and Ca2+ by ion exchange.
A possible role as a endogenous heavy metal detoxifier can also be suggested from the lower observed
efficiency of release of some toxic ions (e.g. Pb2+, Ce4+).
The phase change process in which redox iron ions are made unavailable by the promotion by heparin of
the oxidation of Fe(II) to Fe(III) and formation of stable colloidal particle formation is a process which
also has been reported to occur with with hyaluronate for the elimination of excess Fe(III) in living
organisms has been proposed (26), may be an original function of such polyanionic polysaccharides
since this will also protect against free radical damage to DNA (by e.g. Fenton reactions of Fe, Cu, V,
Mn, Cr and Ti ion promoted free radical induced damage).
Similar activity may also be relevant to the reported in vivo and in vitro inhibition of lipid peroxidation
by heparin (23a,b).
The gathering and safe handling of major and trace nutrients, it might further be suggested, could have
provided the driving force for the early evolution of heparin-like polysaccharides having highly evolved
metal ion dependent functions.
Heparin has been subjected to numerous in vitro metal ions binding studies (e.g. by Grant et al. (1f) and
further listed in the Appendix, which confirm the existence of complex binding mechanisms for metal
ions).
In cartilage, the association of metal ions and their aggregates with anionic glycosaminoglycan
proteoglycans is thought to be involved in the modulation of calcification and swelling (33). A study of
such effects by NMR relaxation seemed to indicate they arose simply from a physico-chemical
association between divalent counterions and chondroitin or dermatan sulphate polysaccharide chains but
the NMR detected effect of metal ions on heparin showed a much greater alteration consistent with a
much greater both monovalent and divalent cation metal ion association (34).
Such enhanced polyanionic attraction exerted by heparin seems to be augmented by non-electrostatic
forces, since although heparin is the most highly anionic of mammalian biopolymers, it can evidently
sequester elements present in both cationic and anionic forms (27).
The multi-elemental character of heparin is correlated to modern seawater suggests that the requirement
for sequestration of elements from seawater at the time of the early evolution of multicellular animals
prompted the evolution of heparan sulphate proteoglycans (including the evolution of the isomerase
enzyme to convert the glucuronic acid residues of precursor bacterial-like polysaccharides into the
iduronate moieties which are better able to bind metal ions further evolved into the complex
morphogenic mechanism
for the development of animals. The amounts of glycosaminoglycans most especially of heparan
sulphate present in marine invertebrates was found to increase with the salinity of aquatic habitats (24a)
The ionic filtration activity of glomerular basement membranes is also believed also to be dependent on
their heparan sulphate containing anionic sites (24b).
The heparan sulphate nutrient element sequestration function could have encouraged the evolution of
various additional mechanisms by which evolutionary pressure could be transmitted via heparan sulphate
microstructure e.g. by modulation of this by the Hofmeister effects of ionic environments, oxidative
(16a,b) and/or nitrosative (16,17) stress or cation-induced alteration in enzymic biosynthesis (this is
known to depend on extracellular Ca2+ (29-10), Mg2+ (29-27) and Mn2+ (29- 25) which can be altered by
the presence of inappropriate levels of heavy such metal ions as Pb2+ (29-28) and Cd2+ (29-29) which
inhibit, respectively the biosynthesis of the core protein and reduce the sulphation of the polysaccharide
chains).
Marine algal polysaccharides are known to bind a large variety of counter ions and although this seems to
occur rather non-specifically and at only moderate affinity, this type of binding seems to be that most
suited to create a potential reservoir and buffer system for major and trace metal ion nutrient binding and
pH control, a situation which also seems to pertain to heparin/heparan sulphate in animals.
The following polysaccharide ‘metallomic’ derived hypotheses can then be suggested:
1) anionic extracellular polysaccharides which contain highly conserved chemically

complex interfaces (such as present in the antithrombin binding site of heparin)
generate specific conformations as a consequence of the binding of specific types
of metal ions.
Such stabilisation of anionic polysaccharide shapes by metal ions could have evolved to deal with metal
ion related processing both being a stimulus for the evolution of the polysaccharides themselves and for
the evolution of adducts between polysaccharides and other biological molecules;
2) heparan sulphate proteoglycans may be so structured as to facilitate the initial uptake, rapid transport
and release of nutrient elements and promote heavy metal detoxification processes.
References
(1)
H. Haraguchi
Metallomics as integrated biometal science
J. Anal. At. Spectrom., 2004, 19, 5-14
[The use of modern mass spectroscopic techniques suggests the common occurrence of 50+ 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) seems to have prompted Haraguchi to suggest that such studies should form the basis of a
new branch of science: “metallomics”.
The above review is largely concerned with the relevance of metallomics to proteomics.
Earlier reports had, however, also suggested that natural anionic polysaccharide-based matrices occur as
multi-element salts,
cf.
(1a)
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 and
(1b)
A. Wassermann
Cation adsorption by brown algae. The mode of occurrence of alginic acid
Ann. Bot. N.S., 1949; 13 (49): 81-88.
The studies reported in refs 1a and 1b clearly suggest that anionic polysaccharides in the cell walls of marine
alga
are in the form of multi-inorganic metal salts.
More recent studies 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,
Cf.,
(1c)
K. Truus, M. Vaher and I.Taure,
Proc. Estonian Acad. Sci. Chem., 2001, 50, 95-103,
and
Data for the multi-element analysis of Ascophylum nodosum which is cited in several internet sites (e.g. the
report of
the multi-elements in Ecklonia maxima given in http://www.gairesearch.co.za/kelp.html, and kelp elements
given in
http://www/alginure.co.uk/ascophylum-nodosum.html which were apparently provided by the Norwegian
Institute
of Seaweed Research.
Cf. also T.A. Davis et al. (Supplementary references)
(1d)
Multi-ion content data available to the author for heparin suggests that this material tends always to occur as a
extremely well-defined metallomic matrix, cf., the multi elemental composition data briefly reported by D.
Grant et
al., in Biochem. J., 1987, 244, p. 143 and more fully discussed by D. Grant in Chemistry Preprint Archive ,
2000,
October, 2000, (10), 94-103, showed that a sodium porcine mucosal heparin sample (prior to the final ion
exchange
reduction of multi-elements needed to achieve pharmaceutical grade heavy metal ion contents; this provided by
a
pharmaceutical industry source as being suitable for this type of academic research) was found by mass
spectrometric analysis to contain, (amounts in µg/g)
Ca (30000), Si (5900), Cl (5600), K (2000) Fe (1100), F (890) Cu (730), P (440) Ti (<390), Ni (<170),
Ba (140), Br (130), Zn (80), Sr (65), Co (<80), B (<25), Ga (20), As (15), Pb (16) , I (10), Cs (9), Mo (7), La
(7),
Ce (7), Ag (4), Sn (5), Nd (5) ,W (5), Zr (5), Rb (3) and V(3). Mass spectrometry of this heparin after passage
through a cation exchange column (in Tl form) also showed a similar multi-element presence (but with greatly
reduced amounts for the majority of the associated elements) which was similar in to the multi-elements
compositions of a range of pharmaceutical heparins deducible from data later reported by ALS (the major
international provider of such analytical data) posted at
http://www.analytica.se/hem20045/pdf/blood_collection_tubes.eng.pdf .
(These data were simply reported apparently to allow for a greater understanding of errors which may occur in
the attempted determinations of inorganic elements in heparinized blood sample fractions; the ICP-MS
elemental
data lists however enable, inter alia, an accurate measure of the multielemental contents of several lithium and
sodium heparins from the leachates obtained from a range of commercial heparinized blood collection tubes;
there
was no attempt, however, in this report, to discuss the wider relevance of these data which is now apparent).
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 the multielement source (the possibility of which requires
further research), nevertheless the equilibration of heparin with the multielement composition of biogcial fluids
having multi-elemental compositions similar to blood serum seems to offer the most rational explanation of the
generation of similar (blood serum/seawater-like) inorganic element profiles in different commercial heparin
samples.
(1e)
Rodushkin F. Odman R. Olofsason and M.D. Axelsson
Determination of 60 elements in whole blood by sector field inductively coupled mass spectrometry
J. Anal. At. Spectrom., 2000, 15, 937-944.
[Other reports from this group which support the hypothesis of the occurrence of metallomic multi-
inorganic elements in biological matrices include:
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]
(1f) The results of in vitro studies of ion binding by heparin by D. Grant et al. also indicated that a range
of
inorganic cations and anions will likely to bind simultaneously to heparin.
Cf., D. Grant, W.F. Long and F.B. Williamson:
Infrared spectroscopy of heparin-cation 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,
P56 Abstract No 157;
(1g) The normal occurrence of a wide range of metal ions in heparin may also be concluded from the sporadic reports in the
literature of the presence of a variety of individual metal ions, e.g.,
G.F. Harrsion and A Sutton, Nature, 1963 (4869) 809, noted the presence of calcium, strontium and barium 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., 1d) 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. 1d)).
N.W. Alcock had also previously reported (Elem. Metab. Man Anim. Proc. Int. Symp., 4th., 1981 (Pub. 1982) Eds. J.M.
Gawthorme, J.M.M.M.C. Howell, C.L. White p. 678-680, Springer, Berlin; Chem. Abs., 96, 213646) the presence 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).
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.
(2)
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]
(3)
H.B. Nader, T.M.P.C. Ferreira, L. Toma,
S.F. Chavanto, C.P. Dietrich, B. Casu and G. Torri
Maintenance of heparan sulphate through evolution
Carbohydr. Res., 1988, 184, 292-300
(4)
U. Lindahl and M. Hook
Glycosaminoglycans and their binding to biological macromolecules
Ann. Rev. Biochem., 1978, 47, 385-417
(5)
L. Kjellen and U. Lindahl
Proteoglycans: structures and interactions
Annu. Rev. Biochem., 1991, 60, 443-465
(6)
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
(7)
M. Bernfield, M. Gotte, P.W. Park, O. Reizes, M.I. Fitzgerald, J. Lincecum, and M Zako
Functions of cell surface heparan sulfate proteoglycans
Ann. Rev. Biochem., 1999, 68, 729-777
(8)
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
(9)
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
(10)
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, initiated
by
the above review, include those described in ref. (10a) , refs (1f) and (21- 23) and other publications
of
D. Grant et al., listed in the Supplementary References}].
(10a)
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
(10b)
S. Ricard-Blum, O. Feraud, H. Lortat-Jacob 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
(10c)
I. Capila, M.J. Hernaiz, 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
(10d)
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
(10e)
M. Kan, F. Wang, M. Kan, T. Bao, 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]
(10f)
M. Kan, X. Wu, F. Wang and W.L. McKeegan
Specificity of factors determined by heparan sulfate in a binary complex with receptor
kinase
J. Biol. Chem., 1999, 274, 15946-15952
[Anticoagulant heparan sulphate is required for FGF receptor divalent cation
dependent association between heparan sulphate and the exodomain]
(11)
M. Rizk , Y. El-Shabrawy’ , N.A. Zakhari and S.S. Toubar
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]
(12)
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
(13)
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
(14)
E.g. N.E. Woodhead, W.F. Long and F.B. Williamson
The heparan sulphates of a 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, 389
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
(14a)
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
(15)
Heparanase degradation
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
(16)
Nitrous acid degradation of heparin/heparan sulphate - metal ion effects
K. Ding, K. Mani, F. Cheng, M. Belting and L.-A. 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.-A. Fransson

Proteoglycan involvement in polyamine uptake
Biochem. J., 1999, 338, 317-323
(16a)
B. Lahiri, P.S. Lau, M. Pousada, D. Stanton, and I. Danishefsky
Depolymerization of heparin by complexed ferrous ions
Arch. Biochem. Biophys., 1992, 293, 54-68
Cf.,
J.P. Lahiev
Bio Metals, 1996, 9 (1) 10; Chem. Abs., 124, 109969t
[Fe(II) selectively degrades heparin]
(16b)
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
(17)
Cf., R.E. Vilar, D. Ghael, M. Li, D.D. Bhagat, L.M. Arrigo, M.K. Cowman, H.S. Dweck
and L.Rosenfeld
Nitric oxide degradation of heparin and heparan sulphate
Biochem. J., 1997, 324, 473-497
Cf., D. Ghael et al., 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]
{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
(to be the subject another communication)}.
(18)
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]
(19)
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]
(20)
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]
(21)
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
Med. Hypotheses, 1992, 38, 49-55
(22)
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]
(23a)
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) ref. (23b(
(23b)
Pericellular heparans may contribute to the protection of cells from free radicals
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], cf. also http://electra.chemistry.upatras.gr/fects/final/w-shops.htm]
(24a)
H.B. Nader, M.G.L. Medeiros. J.F. Paiva, V.M.P. Paiva, S.M.B. Jeronimo, T.M.P.C. Ferreira
T.M.P.C.
and C.P. Dietrich
A correlation between the sulphated glycosaminoglycan concentration and degree of salinity of the
habitiat in fifteen species of the classes Cructacea, 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 osmoeregulatory 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]
(24b)
H. Morita, A. Yoshimura, K. Inui, T. Ideura, H. Watanake, L. Wang, R. Soininen and K.
Tryggvason
Heparan sulfate of perlecan is involved in glomerullar filtration
J. Am.Soc. Nephrol., 2005, 16, 1703-1710
(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)
(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]
(26a)
13C n.m.r. spectroscopy of Cu2+- heparin suggests phase separation of the complex from Cu2+ in
aqueous solution
Ibid. 1992; 20: 214S and
Cation interaction with heparin at antithrombin-binding sites may differ
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 Med. Sci., 1986, 14, 903-4;
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 and
(26b)
D. Grant (2000) Seminar presentation and discussion University of Glasgow
(Discussions Relating to Ascorbate/Heparan Sulphate /Upper Stomach Cancer etc.)
http://web.ukonline.co.uk/dgrant/dg2/ 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)].
(27)
Binding of anions to heparin
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 Rudschau, 1982. 11, 452-455
and
L.B. Jaques
Heparin: an old drug with a new paradigm
Science, 1978, 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]
(28)
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+]
Tables I &II (Additional 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. Spreight 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-Sumnich, 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. Sorensen, 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. Schinjo, L.L.S. Tersario, V. Olivera, C.R. Nakaie, M.E.M. Ochiro, A.T. Ferreora,
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., 2003, 19, 373-378
(29-10)
Y. Takeuchi, K. Sasaguchi, 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. Klingelhogfer, E. Ludkanidin, V. Berezin, and E. Bock
Molecular mechanism of Ca2+ signaling 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 (380) 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., 2006, 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. Gonzales-Iglesias, M.A. Pajares, C. Ocal, J.C. Espoinosa, B. Oescg 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.C. Aguiar, M.A. Dalboni, A.S. Garofalo 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. Schuster, N.K. Karamanis 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 proteglycan 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. Cardenas, 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. Pawalowska-Goral, 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
(30)
M. Purdey
Chronic barium intoxication disrupts sulphated proteoglycan synthesis:
A hypothesis for the origin of multiple sclerosis
Med. Hypotheses., 2004, 62, 746-754
(31)
M. Lyon, J.A. Deakin and J.T. Gallagher
Liver heparan sulphate structure. A novel molecular design
J. Biol. Chem., 1994, 269 (15) 11208-112115
(32)
S.E. Stringer, 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
(33)
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
(34)
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]
Supplementary References
Of relevance to the hypothesis that heparin/heparan sulphate function as metallomic matrices
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 equilbria with molecular oxygen and hydrogen peroxide
J. Amer. Chem. Soc., 1976, 98 (26) 8366-8371
[Formation of hydroxyl radicals by Fenton type reactions from Mn(II) also
similar reactions from V(IV), Cr(II), Ti(III) and Fe(II) discussed]
Burger, K., F. Gaizer, M. Pekli, G.Takacsi 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. Orest, A. Naggi, G. Torri, G. Zoppetti, G. Sporteolettti and F. de Santis
European Patent Application No. 0245813 (1987)
Cochran D.L.
Glycosaminoglycan stimulation of calcium release from mouse calvariae.
Specificity for hyaluronic acid and dermatan sulfate
Calcif. Tissue, 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) 4311-4333
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., 2002, 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 Mucopolysacharides. Binding in solutions containing acid
mucopolysaccharides Ch.12, p. 171-187
Ed., Guiliano Quintarelli, Churchill, London (1968)
Fransson L.-A., 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
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
Ibid.,, 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

A model of two conformational forms of heparins/heparans suggested by infrared
spectroscopy
Med. Hypotheses, 1987, 24, 131-137

Effect of heparin on dismutation of superoxide anion
Biochem. Soc. Trans., 1988,, 16, 1030-1031
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;
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;

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;

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
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
Grant D., C.F. Moffat, W.F. Long and F.B. Williamson

Carboxylate symmetric stretching frequencies and optical rotation shifts of
heparin cation complexes

I.r. spectrosocpic analysis of heparin-polypeptide interaction
Grant D., W.F. Long, C.F. Moffat and F.B. Williamson

Polarimetry of mixtures of Cu(II) ions and chemically modified heparins

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)
Multiple-specular-reflectance i.r. spectrocospy of
glycosaminoglycan-cetylpyridinium complexes
Biochem. Soc. Trans., 1992, 20(1) 4S

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

The binding of Pt(II) to heparin
Grant D., W.F. Long, G. Macintosh and F.B. Williamson

Antioxidant activity of heparin

Incompletely published (but displayed as conference posters) of studies conducted by procedures similar
to these described in ref. (21) 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. (2000)
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 ICP-
MS) show that heparin acts as an efficient multielement matrix, this was formerly thought to be of trivial
significance arising from contamination during extraction and work-up procedures. However, 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 however 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 mammalan 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)
[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]
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]
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]
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 anioic polysaccharides derives 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
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
Whitfield D.M., 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
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]
_____________________________________________________________________________________
Postscript. The following papers which were published after the initial drafting of this document are
relevant to the hypothesis that the metallome and the heparanome cross react.
Rudd T.R. et al., Influence of substitution patterns and cation binding in conformation and activity of
heparin derivatives
[The traditional view that signalling by heparin/heparan sulphate depends on the polysaccharide anionc
sequence is suggested to be incorrect; the binding of individual cations including K+ and Cu2+
dramatically alters activities (e.g. for regulation of fibroblast growth factor regulation); this indicates that
the heparanome-metallome interaction concept provides a plausible hypothesis for how heparan sulphate
exerts critical control behaviour in animal biochemistry]
Zcharia E et al., Newly generated heparanase knock-out mice unravel co-regulation of heparanase and
matrix metalloproteinases
PLoSD ONE 2009, 4 (4): e5181Epub 2009 Apr.10
**The heparanome
The heparanome is the name recently suggested for the system of animal polysaccharides which
contain evolutionary conserved (3) 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 betwen 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 implciated 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.
--------------------------------------------------------------------------------------------------------------------------------
-----
*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).

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20/4/05 Modified 18/5/08; 20/5/09

The Relevance to Metallomics of the Binding of Metal Ions to

Medical Use of Metal Ion Binding to Heparin/Heparan Sulphate

Participation of Metal Ions in Heparin/Heparan Sulphate Signalling.

The postsynthetic coding alteration of heparin/heparan is not restricted, as is DNA, by a requirement to

Metal Ions Can Link Heparin/Heparan Sulphate Domains to Proteins

The Role of Heparin/Heparan in Controlled Assembly/Disassembly

Examples of facilitation by divalent cations of the binding of heparin/heparan

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

Mn2+ Serum lipid aggregate formation

cf. putative Ca2+/Na+ vascular flow sensor

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

Metal ion modulation promotes scission of syndecan-1 heparan sulphate

A possibly related process is the

Inorganic Crystal Formation

Ca2+ Suppression of proteoglycan including

Cd2+ Inhibition of the incorporation of [35-S] A. Cardenas et al.,Toxicology

F- F- ions inhibit heparan sulphate sulphation M. Pawalowska Goral et al., Fluoride,

The following polysaccharide ‘metallomic’ derived hypotheses can then be suggested:

1) anionic extracellular polysaccharides which contain highly conserved chemically

Cf., S. Kojima, Y. Hama, T. Sasaki and A. Kuberoda

D. Grant, W.F. Long, G. Mackintosh and F.B. Williamson

Cf., M. Belting, S. Persson and L.-A. Fransson

J.R. Helbert and M.A. Marini

Tables I &II (Additional References)

Ayotte L., and A.S. Perlin

Bodini M.E., and D.T. Sawyer

Burger, K., F. Gaizer, M. Pekli, G.Takacsi Nagy and J. Siemroth

Dais P., Q.J. Peng and A.S. Perlin

Davis T.A., B. Volesky and A. Mucci

Grant D. et al. , Additional References

Grant D., W.F. Long and F.B. Williamson

Grant D., C.F. Moffat, W.F. Long and F..B. Williamson

Grant D., W.F., Long and F.B. Williamson

Grant D., W.F., Long and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., W.F. Long, C.F. Moffat and F.B. Williamson

Grant D., W.F. Long and F..B. Williamson

Grant D., W.F. Long. C.F. Moffat and F.B. Williamson

Grant, D., W.F. Long., C.F. Moffat and F.B. Williamson

Grant D., W.F. Long and F. B. Williamson

Grant D., W. F. Long and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., C.F. Moffat., W.F. Long and F.B. Willliamson

Grant D., C.F. Moffat, W.F. Long and F.B. Williamson

Grant D., C.F. Moffat, W.F. Long and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., W.F. Long, C.F. Moffat and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grant D., W.F. Long, G. Macintosh and F.B. Williamson

Grant D., W.F. Long and F.B. Williamson

Grushka E. and A.S. Cohen

Herwats L., P. Laszlo and P. Genard

Hu W.-L., and R. Reogoeczi

Karlinsky J.B. and R.H. Goldstein

Kazama Y. and Koide T. (1992)

Kjellen L. and U. Lindahl

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

Liang J.N., B. Chakrabarti, L. Ayotte and A.S. Perlin