How Two State Water Chemistry May Be Invovled in TSEs.doc1 Corrected

8/9/2019 How Two State Water Chemistry May Be Invovled in TSEs.doc1 Corrected

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the Nobel laureate A SzentGyorgi 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 matterwhich 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 detergentswhich 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 indirectadministration 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 FeO.OH is known to require highionic 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.


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.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 spectroscopicabsorption 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 studiesof 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]


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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 chemicalchaperones such as , -trehalose, sucrose, DMSO and betaine). That these can greatly influence thefolding 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 LDWHDW 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 LDLHDW 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 onheparin, modified heparin and a range of other polyanions studied by equilibrium dialysis,

electrometric titration, polarimetery, infrared and NMR methods. The process was confirmed more


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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 aheparin 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 inorganicions 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


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[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 244 143-149Cf 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


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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 metalinduced 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 beidentified 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 theinterpretation of vibrational spectra of aqueous solutions. The polywater present in SiO2(H2O)ncomplexes 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.

How Two State Water Chemistry May Be Invovled in TSEs.doc1 Corrected

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