How ionic effects which influence water chemistry may be involved in the aetiology of protein

misfolding diseases
A suggestion as to how pentosan polysulphate inhibits TSEs
Note by D Grant 28/2/08

Summary
Insight into the early stages of generation of life and the evolution of multicellular organisms may be
gleaned from studies of the key role of polyanions which act as buffers to affect complex inorganic ion
profiles in seawater and blood serum; this process is suggested to be influenced by a polyanion
adsorbed water-structure modification mechanism.
(Both of these aqueous solutions/dispersions contain similar amounts of dissolved CaCO3 scale
inhibitors of chemical of structures similar to heparin and pentosan polysulphate).

Introduction
The ability of modern instrumentation to rapidly generate experimental data and the increasing number
of working scientists creates a log-jam of information. This is often of critical value to ongoing current
researchers who are often are unaware of this information because of the way information is currently
published stored and retrieved has major imperfections. The internet offers what seems to be a
comprehensive method of sourcing such information but in practice much of the information is also not
available in this way including older files which cannot now be accessed by usual search engines.
The lack of free general public access to much of the current scientific literature in recent years
compounds this problem.
Other problems arise from commercial secrecy. Information generated in the vast commercial research
establishment may not be generally circulated outside selected discussion groups. A problem often
arises of accessing medical information for non-medically qualified scientists. Another more general
problem is what constitutes a genuine scientist. It is surely wrong to restrict this definition to
individuals who are members of select groups requiring perhaps over zealous entry qualifications.
Essential information essential to allow progress in important scientific puzzles (water structure is a
prominent example) is withheld from pure science research initiatives. Another problem here is that
peer review system can eliminate useful information for publication of articles in a final form.
The new pre-publication initiative such as Nature Precedings is to be welcomed as it appears to be set
up to create permanent web-based archives of what is potentially useful information for the scientific
community and these notes are written in this context.

An example of the lack public dissemenation of information useful to biomedical research is a

discourse on water structure by G Wilse Robinson from 2005 but which cannot now be accessed from
the internet. This document touches briefly on the relevance of seawater to biology and is a useful
introduction to how the seawater multi-elements which become enriched at highly sulphated polyanion
such as heparin may be a key factor for the fuller understanding of the aetiology of prion diseases
The G Wilse Robinson concepts were extended by PM Wiggins, but the major problem still remains of
firmly establishing a sound scientific basis of the chemical nature of liquid water. For this scientists
need to access as much of the international and industrially held documentation as possible.
The two state liquid model theories include the (Wiggins) LDW/HDW theory and the Chaplin
icosahedral theory.
In any case it is evident that the nature of liquid water has a complex physical chemical uniqueness
which tends to add weight to the hypothesis that a wide range of flexible supramolecular structural
assembly and disassembly properties associated with biological solutions containing this liquid as a
major molecular component is the ultimate basis of biology at all levels. Pathologies (e.g. those
involving the deposition of plaques including transmissible spongiform encephalopathies TSEs) could
even evidently derive from the perturbation by pathological agents of such water supramolecular
chemistry.
It is now argued that the water structures present at very high ionic strength of multi-inorganic ion
associated with seawater/blood serum is the correct one on which to focus to elucidate why heparin and
pentosan polysufate a highly sulphated heparin analogue can act as n effective therapeutic agent for
Creutzfeld Jacob disease

Prompted by the old web document:

G Wilse Robinson had noted in 1998 that the establishment of a molecular level understanding of
liquid water was the most important remaining unsolved problem in science*. He mentioned that
the Nobel laureate A Szent–Gyorgi has already noted that “ water is the matrix of life” but also
recognized that this water was not simply pure water but the rather different chemical composition
of seawater known to be a highly complex solution of many inorganic ions and dispersed colloids
including polyanionic humic matter which can affect the attainment of thermodynamics equilibria
between the hydrogen bonded liquid water aggregates and inorganic ions which occur in such
solutions. Such natural polyanions in seawater (which can also include anionic polysaccharide from
marine algae and nowadays also perhaps the phosphonate-based herbicides and laundry detergents
which resist biodegradation) avidly sequesters the inorganic ions present as well as acting as a potent
crystallisation inhibitor by adsorption at surfaces of the nuclei which might otherwise catalyse the
attainment of thermodynamic equilibrium solubility such sparingly soluble crystalline phases as
CaCO3(calcite) and BaSO4 (this is a credible mechanism by which the polyanions act as part of a
natural a buffer system by which the multi-inorganic elemental composition of seawater is maintained
throughout geological time). A similar mechanism where anionic polysaccharides substitute for humic
acid may also contribute to inorganic ion homeostasis in multicellular organisms including humans for
which for the inorganic elements in extracellular fluids, it should be noted also demonstrate a perhaps
surprising approximate log-log correlation between about fifty (perhaps more) inorganic elements
present in both seawater and blood serum. It can even be convincingly argued that many degenerative
disease processes which are both solutions are also supersaturated with respect to CaCO3(calcite)
precipitation.associated with pathological “calcification” events are triggered by deficiencies in the
amounts of fine structure of such inhibitors (and can be alleviated by the direct or indirect
administration of such substances or their mimetics).
[It has been found that the anionic polysaccharides such as heparin and alginates and fulvic acid salts
(humic acid fractions) show mass spectroscopically determined 40+ inorganic contents exponentially
related to the multi-elemental composition of seawater (the relationship seems to involve a preferential
sequestration of those ions which occur in least abundance)].
Heparan sulphates are key players in the necessary extension of genetic information holding and
processing required for the evolution of multicellular animal organisms, plants seem to use analogous
systems. Anionic polysaccharides seem to have become highly evolved to enable and enhance genetic
information processing in eukaryotic organisms.

The groundbreaking study by Hofmeister in 1898 of the phenomenon of salt promoted protein
denaturation, enabled salts to be ranked in a Hofmeister or lyotropic series.
The presence in seawater of such dissolved inorganic salts which can become highly concentrated at
natural polyanionic (e.g. polysaccharide) surfaces might achieve locally-sufficiently high salt
concentrations to cause protein denaturation by the Hofmeister mechanism.
{Evidence that heparin, perhaps the most anionic biopolymer, creates at it s surface a high ionic
strength is that akaganeite forms in aqueous heparin. This form of β FeO.OH is known to require high
ionic strength the (there are heparin-like segments in heparan sulphate the major information sorting
biopolymer of extracellular matrix cell surface and links to the cytosol and genome}
.Organisms which have intracellular compartments in direct contact with aquatic habitats appear to
include in their protective mechanisms an evolved alteration in the sulphated polysaccharide contents
of such tissues; such heparan and chondroitin sulphates could be extracted in a direct mathematical
logarithmic correlation with the salinity of the habitat (as reported by Nader et al. for fifteen lower
multicellular animal organism species whose tissues are permeated by environmental water). This
increase in the putatively molecular chaperone polysaccharides, both could inhibit the but might also
promote the establishment a precise fine-tuned water structure environment which more generally
allows extracellular proteins to assume their correct folding patterns. Similar considerations will, of
course, apply to the cytosol.
Further evidence that precision of water structure and efficient action of the mechanism which allow it
to be achieved which, of course, for higher animals is centred on kidney function, but which still
retains a critical role for complex polysaccharide structures including those containing heparan
sulphate in glomerular basement membranes.
Such organisms also cannot substitute substantial amounts of D2O for H2O. {This seems less true for
bacteria however}. The origin of the toxic action of D2O tends to confirm the importance of correct
water structure for healthy biological functions.
.Spectroscopcially identified hydrogen bonded water absorption band shifts can be directly indicative
of the supramolecular structure changes in aqueous saline solutions.
While it is still commonly stated in many biochemical research papers that the origin of the Hofmeister
series is currently unknown, this statement would be hotly disputed by the applied science laboratory
pioneers of fundamental water chemistry research (WAP Luck and coworkers and their predecessors)
who had concluded that the lyotropic series arises because protein folding is driven by water chemistry
and the balance of water structures which show up as highly reproducible vibrational spectroscopic
absorption energies attributable to different kinds of hydrogen-bonded water molecules. This system
is disturbed according to the Hofmeister series for solutions containing only salt and water.
In agreement with the two-state water theories there are two prominent bands which Luck has
ascribed to water inside clusters and water on the edges of clusters, the former being more strongly
hydrogen bonded; these can now be re-assigned to LDW and the less hydrogen bonded to
HDW. Luck noted that under conditions of temperature where the HDW became abundant the
sapphire cell windows were attacked in confirmation of the suggestions by Wiggins that HDW is
highly reactive.
(The variation of the spectra of water is discussed at length in a 1976 review; this included the effect
of salts which seemed to be similar to a change of temperature on the evident equilibria which exited
between the different water structures which gave rise to different vibrational spectral absorptions and
similarly for the apparent effect of such water structures can be followed by the generation of turbidity
from the formation of polyether micelles).

An important additional finding of WAP Luck et al studies was that the effect of small inorganic ion
solutes on water structure is additive. The complex multi-inorganic-ion composition of seawater and
blood serum will then not be trivial phenomenon but part of strategy to replicate ancient multi salt
environments conducive to correct protein folding. This will of course also apply to prions.

The effect of solutes on the misfolding of the cellular prion protein PrPC to give the monomer unit of
the scrapie factor PrPSc (which is believed to be the basis of transmissible spongiform
encephalopathies (TSEs ) and related diseases, has been reported to obey the Hofmeister series (which
includes an important contribution of anions, which had been left out of earlier attempts to investigate
this effect).
Prions are also subject to control of folding by known chaperone modulators of water structure.
Pentosan polysufate may also behave similarly but from knowledge gained during studies
of the ability of pentosan polyslfate to inhibit viruses it can be suggested that this drug could act
by several simultaneous mechanisms.

Apart from the Hofmeister effect of ions on water structure, anionic polymers and other biopolymers
become strongly hydrated in aqueous solutions (this shows up as non-freeing water in e.g. 1H-NMR),
The somewhat unusual mode of hydration of the most anionic of such polymers is also strongly
implicated in the detailed mechanism by which general Hofmeister effects can modulate prion folding
and hence control the aetiology of TSEs.
Such highly anionic polyelectrolytes which include DNA, heparin and humic acids are known to bind
counterions in a water-structure-related-fashion.
This in some ways seemed to resemble a pure physics rather than (due to Manning) was found to be
inadequate.
The importance of this water-centred activity to biochemistry is that it may control the activity of the
molecular chaperone systems especially those which are based on highly anionic polysaccharides
(using a wide definition of the term chaperone). The principle example of this polyanionic system is
that provided by the heparan sulphate proteoglycans which can be regarded as an extension of the
genome.
[The heparan sulphate system of polysaccharides held by core proteins (differences in which are used
as a basis of classification) comprises a carefully conserved and apparently chemically stabilised
system of anionic patterns provided by alternating substituted uronic acid [iduruonic and glucouronic]
containing sugar-OSO3-, sugar-N-SO3- and sugar COO- anionic groups [together with NAc groups]
arrangements which are, together with in a pre-determined amount of non sulphonated GlcNH2 group,
strictly regulated [developmentally regulated]in regard to expression). The may also exist a further
carefully orchestrated system of postsynthetic generation of altered anionic density (e.g. by 6-O-
sulphatase) glycosidic bond scission by heparanase and nitrosative deaminative scission at
unsubstituted GlcNH2 groups (dependent on nitric oxide, redox status, itself dependent on the presence
of ascorbate and also a redox cycling of copper ions)].
Other more traditional classes of chaperone molecules are heat shock proetins and ‘chemical
chaperones’ such as α, α -trehalose, sucrose, DMSO and betaine). That these can greatly influence the
folding of prion proteins (and were suggested of potential therapeutic agents for TSE diseases from this
ability) was first reported by the Caughey group). This can be taken as a hint also that water structure
is the prime unseen molecular system which is mediated by these chaperones or osmolytes. (E.g. the
effect of such osmolytes as trehalose seems to show up by a profound effect on the vibrational bands of
water; although this is at first sight of a different character from the straightforward application of a
rapid HDW/LDW equilibrium it is apparent that alteration of water activity is the ultimate modus
operandi of these chemical chaperones [Their effect might tentatively be assigned as a kinetic and not a
thermodynamic equilibration shift one e.g. local viscosity enhancement at protein surface water though
perturbation of the LDWHDW rate of attainment of this equilibrium and may create a barrier to
misfolding by providing a immobile polywater layer at the protein surface]).

The most efficient anti-TSE agent identified to date is pentosan polysufate, a heparin analogue.
This molecule seems to bind directly to prions and this is held to be responsible for the inhibition of
their misfolding. The binding process is likely to be similar to that of heparin to involve critical water
molecules at rate determining sites perhaps with influence on both the rate of binding and on
disturbance of the LDLHDW balance which putative determine the folding and misfolding
processes.

PPS might also act in other ways to inhibit TSEs. PPS also strongly inhibits a number of viruses.
PPS could inhibit those viruses or nucleic acid fragments which can act as catalysts to generate the
poly-PrP conformation held to be responsible for TSE infectivity. Multiple modes of action seem to
contribute to how PPS inhibits virus infectivity and proliferation (e.g. in the case of HIV-1 where cell
entropy via gp120 , viral replication via reverse transcriptase and cell aggregation were amongst the
systems which have been reported to be inhibited by this polyanion).

That PPS is so effective can be attributed to its similarity of heparan sulphate which generates PPS-like
fragments during tissue protective functions.
The heparin/heparan sulphate system, as well as providing for a management system for regulating
growth factor activity required for the formation of aggregates of cells during the assembly of organs
during development, growth and wound healing, also provides for a non-specific immune as well as
cellular immune anti-pathogen protection mechanism (against viruses, prions, bacteria and protozoa
infections and direct or assistance in enzymic-based protection against reactive oxygen and nitrogen
species, neutralisation of snake venom and other toxins and perhaps including toxic metal ions).
Included in this wide-specificity protection mechanism is the aforementioned ability of heparin/heparan
sulphate to strongly inhibit the formation of pathology-associated solid particles including insoluble
calcium (e.g.CaCO3 and Ca phosphate and urate crystals).

Insight into the mechanism of such processes can be achieved by in vitro studies of studies, the simples
strategy is to study the details of the binding of Ca2+ ions by those polyanions which have found
commercial use as calcification inhibitors.
A recent report of the use of calorimetric titration to elucidate the mechanism of attachment of
multivalent inorganic cations to a range of polyanionic scale inhibitors showed that rather than a
formerly held hypothesis that Coulombic forces were involved as a driving force for the binding of
Ca2+, the advantageous free energy release from entropic energy was a more likely origin. This
entropy driven process was controlled by the alteration of the water layer which is also bound to such
polyanions.
Earlier studies had found that various sugars including uronic acids bound Ca2+ not apparently by a
process centred on interaction between sugar-C(O)O- groups and Ca2+ but by binding which seemed to
implicate C-OH groups.
These results were also in agreement with a number of Ca2+ ion binding studies conducted with on
heparin, modified heparin and a range of other polyanions studied by equilibrium dialysis,
electrometric titration, polarimetery, infrared and NMR methods. The process was confirmed more
likely to be controlled by water structure changes and not by Coulombic interactions effects as had
been previously believed. [E.g. the Manning Coulombic hypothesis which suggests that heparin (a
polymer system of formal high polyanionic nature) should collect an inner sphere of counterions by
straightforward Coulombic electrostatic attraction which was also sensitive to the along-chain
polyanion charge density in such a manner that a discontinuity was predicted to occur in the binding
isotherms (between the inner sphere and outer sphere electrostatic attraction effects)]. A detailed
investigation of what appeared to be an experimental confirmation of this discontinuity showed that a
heparin surface “water structure” phase change mechanism provided a more credible explanation.
That this hydration is present in a real separated almost solid phase (not an imaginary phase boundary
which can still be described as a liquid water variant as discussed by PM Wiggins for pore surface
water hydration in cellulose acetate etc.) is suggested by the lack of paramagnetic broadening of
polymer NMR resonances under moderate paramagnetic ion loading of the polysaccharide and also the
lack of paramagnetic broadening of methanol present as an internal standard when paramagnetic ion
saturation of the polysaccharide produces highly distinct paramagnetic ion broadening of the
polysaccharide resonances.
Furthermore the prediction of the Manning hypothesis that all equally charged counterions of whatever
chemical nature should bind equally was found definitely not to be the case.
Another fairly good confirmation that electrostatic binding was not the reason for attraction of
inorganic ions to heparin was that the sulphate anion SO42- had been well established to bind strongly to
heparin (a range of other negatively charge ions also bind strongly). The negatively charged inorganic
ions should be strongly repelled by a strongly anionic binding site if electrostatic interaction is the
origin of inorganic ion binding to heparin and related molecules.
Another use to which the seemingly irrational behaviour of heparin in achieving the simultaneous
binding of oppositely charge ions is that a heparin-modified chromatographic SiO2-based
chromatographic column packing can achieve an efficient separation of both cations and anions on a
single column.

The temperature dependence of the energetics of Ca2+ binding (and also Zn2+ binding) was most easily
explained by alteration of the entropy of water molecules at the polyanion surface.

This can also allow a “scale” inhibitor model of TSE to be suggested. It can be predicted that those
molecules which inhibit scale formation (including arterial calcified plaque) should be evaluated as
potential anti-PrPSc agents.

*A traditional view of the biological role of water was that this solvent as simply acted an inert carrier
of active biopolymers and the functioning of multicellular organisms needed only to consider the
activity of such polymers per se rather than the existence of complex interactions between these
polymeric systems and the now established multi-structural nature of water. The old view had been
challenged most notably by Bernal and Ling., but the concept of water driven biology was clouded by
the problems associated with the experimental difficulties and the large effect of impurities inherent in
the requirement for very rigid scientific protocols required to establish the basis of water biochemistry.

The common use of 1H NMR medical imaging of tissues and near infrared (NIR) spectroscopy to
evaluate commercial quality of biologically-derived materials depends largely on alteration of water
proton hydrogen-bond affected chemical environments, but these methods, while theoretically capable
of discriminating between different micoenvironments of water molecules, do so by a mechanisms
which can give partially misleading results; the highly successful use of these methods of probing
water structure relies essentially on empirical standardisations which, for biological tissue evaluation
by NMR is highly dependent on the interactions of water with paramagnetic ions so that while water
structure known from vibrational spectroscopic studies to differ between normal tissues and tumours;
the different manganese and iron contents of these tissues is what actually shows up in magnetic
resonance images. The use of normal transmission spectroscopy for studying water structure by NIR
suffers from a lack of a full understanding of the multiple factors (which include proton tunnelling and
Fermi effects and high absorbance of water layers at (curved) surfaces

References
Sinn CS Dimova R Antonietti M (2004)
Isothermal titration calorimetry of the polyelectrolyte/water interaction and binding of Ca2+.
Effect determining the quality of polymeric scale inhibitors
Macromolecules 37 3444-3450
[Inadequacy of Coulomb description i.e. ion as point charge and water as a homogeneous dielectric
medium is at least misleading, as it disregards the predominant thermodynamic effect of the chemical
speciation of the compounds in respect to the structure of water]

Helbert JR Marini MA (1964)

[Non-covalent binding of sulphate to heparin]
Biochim Biophys Acta 83 122

Lortat-Jacob H (2006)
Interferon and heparan sulphate
Biochem Soc Trans 34(3) 461-466

Kan M et al., (1996)

Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of fibroblast
growth factor receptor complex
J Biol Chem 271 (42) 26143-26148

Grant D et al. (1987)

Infrared spectroscopy of heparin-cation complexes
Biochem J 244 143-149
Cf also Grant D et al Biochem J 1991 275 193-197; ibid., 1992 287 849-853; ibid 1992 282 601-604;
ibid 1992 283 243-246; ibid 1992 285 477-480; Biochem Soc Trans 1991 18 1281-1281; ibid 18
1282-1283; ibid 1992 20 361S ibid 1991 19 390S ; ibid 1996 24 203S 1996 24 204S

Lee KS Caughey B (2007)

A simplified recipe for prions
PNAS USA 104 (23) 9551-9552

Saa P Castilla J Soto C (2006)

Ultra-efficient replication of infectious prions by automatic prion misfolding cyclic amplification
J Biol Chem 281 (46) 35245-35252

DebBurman SK Raymond G.J Caughey B Lindquist S (1997)

Chaperone-supervised conversion of prion protein to its protease resistant forms
PNAS USA 94 (25) 13938-13943

Greenwood AD et al (2005)
Cell line dependent RNA expression profiles of prion-infected mouse neuronal cells
J Mol Biol 349 (3) 487-500

Gonzalez-Iglesias R et al (2002)
Prion protein interaction with glycosaminoglycan occurs with the formation of oligomeric complexes
stabilized buuCu(II) bridges
J Mol Biol 31 319 (2) 527-540

Fransson LA et al (2004)
Novel aspects of glypican glycation
Cell Mol Life Sci 61 1016-1024

Sulkowski B (1992)
Spontaneous conversion of PrPC to PrPSc
FEBS Lett 307 (2) 129-130

Haraguchi H (2004)
Metallomics as integrated biometal science
J Anal At Spectrom 19 5-14

Kuberan B et al. (2004)

Light induced 3-O sulfotransferase expression alters pineal heparan sulfate fine structure
J Biol Chem 279(7) 5053-5054
Bohrer D (2004)
Aluminium level in serum analysis and its importance in hemodialysis treatment
RBAC 36(2) 99-103

Templeton DM (1992)
Metal-proteoglycan interaction in the regulation of renal mesangial cells: implication for metal
induced nephrosis
Proceedings Trace Element Health Disease Ed Aito A Cambridge Royal Society of Chemistry
Cambridge UK p209-214 Chem Abs 111 294071z

Wiggins P (2008)
Life depends opn two kinds of water
PloS ONE 3(1):e1406

Further insight into the critical role played by water structure (an the elucidation of the role of
hydrophobic and hydrophilic hydration in protein folding is given by the two state liquid water model
recently reviewed by PM Wiggins in which the two forms are suggested to correspond to two forms of
ice distinguished by linear and bent hydrogen bonds present at low temperature).
The different forms of water structures which show up in vibrational spectra also allowed them to be
identified following their enrichment by the ability of pores formed in silica gels and cellulose acetate
and dextran membranes and membranes to separate out the low and high density forms from the
equilibrated two form matrix of liquid water. Such behaviour also allowed such pores to act as
selective binding sites for K+ relative to Na+ and also to selectively become enriched in L-
amino acids from racaemic mixtures providing a credible model for the possible roles such pores
(perhaps especially those present in naturally formed amorphous silicas e.g.. a characteristic feature of
thermal volcanic vents) in prebiotic evolution. A further indication of such basic biochemical
involvement of the two forms of water is that the hitherto puzzling aspects of the mechanism by which
enzymes function including for energy transduction during ATP ADT interconversion which
previously had been described by biochemists in terms of high energy ‘squiggle’ phosphate bonds.

[It should be emphasised that the LDW/HDW theory deserves a higher scientific status to the
discredited Deryagin “polywater”, but the general situation remains that artefacts can cloud the
interpretation of vibrational spectra of aqueous solutions. The “polywater” present in ‘SiO2’(H2O)n
complexes also is biologically relevant since Si also in the likely form of ‘SiO2’ occurs in polyuronides
incuding heparin /heparan sulphate and Si levels in serum seem to be strictly regulated as well as being
known to be an essential nutrient. Such Si maybe part of a complex seeding process by which the
phase changes which allow the heparan sulphate system to operate (by way of water structures
generated at the polysaccharide surfaces). Particulate ‘SiO2’ also is a useful additive in ionomer proton
conductors used commercially in fuel cells etc which have a similar counterion-dependent affinity for
water as heparin and for which the cation dependent water clusters evidently, by linking up between
adjacent anionic sites, are thought to be the molecular basis of the proton conduction and ionomer
functions.