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A Hypothesis (With Corrections)

A Hypothesis (With Corrections)

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First Manuscript  A Hypothesis: Heparin/Heparan Sulphates Modulate Protein Activities by Water Activity Regulation Including Nanohole Induced Non-Equilibrium Water and Hofmeister-Like Effects Generated by Interfacial Aqueous Phases with High Ionic Strength, Multi-Element Inorganic Salt Compositions
David Grant PhD MRSC
Aberdeenshire UK AB53 6SX (& University of Aberdeen)Heparan sulphate microstructure, evidently strictly regulated both temporarily and positionally duringdevelopment (being responsible amongst numerous other established functions for regulating severalgrowth factor activities [(1) cf Bernfield et al
 
1999; Hacker et al 2005] which are apparently dependenton the existence in syndecans, glypicans, agrin and other protoglycans of heparan sulphate sidechainshaving a great variety different linearly information-encoded sequences (dependent on the occurrencein this uronic acid (1->4) D glucosamine repeat disaccharides of sulphated iduronate, glucouronate and N-acetyl, N-sulphonate or unsubstituted glucosamine 2-deoxyglucosamine, residues) which are believed to enable several types of signalling including those involving binding to basic sites in proteins via specific information encoded sequences of sugars analogous to the pentasaccharideantithrombin-binding site heparin [(1) Bernfield et al 1999; Lyon & Gallagher 1997] (such aninformation code is more complex than that of DNA to which it is obviously is however similar in principle although the heparan sulphate system apparently lacks any ability to act as a template for itsself replication).It is now further suggested that binding of polysaccharides to proteins may require correct interstitialaqueous solutions (e.g. containing separate ‘soft-ice’ like phases) which are now believed to contributeto the inorganic co-solute multi-ionic environments** . With heparan sulphate it seems that a system other than molecular recognition by an antithrombin(AT)-like-fine tuned electrostatic quaternary N
+
protein link to individual codon sulphate mineral-likeanionic patterns (cf 
a
) must allow fine structure discrimination to be established. A required highdegree of selectivity by different microstructures present in different heparan sulphates seems to be the basis for their ability to select different proteins for their normal (e.g. growth factor orchestration)functions (1). The existence of some previously unsuspected unconventional molecular recognitionsystem is however apparently required to facilitate this. This possibility is suggested by the work of [(1a) Kreuger et al 2005] who found that most probable signalling oligosaccharides failed todiscriminate between their individual FGF growth factor isoform target binding sites when they were present as individual molecular signals (obtained from the high molecular weight by scission,separation and purification).This finding seems to be starkly contrary to the selectivity apparently achieved by the parent highmolecular weigh proteoglycans in their in vivo environments.This situation might have arisen (as suggested by Kreuger et al) from the ability of such larger molecules (but not the smaller segments) to form additional hydrogen-bonding, Van der Waals forcesand salt links (including those with inorganic ion bridges [cf 1a-1]) and perhaps involving the wholeheparan sulphate proteoglycan chain which may be involved.It is now proposed that the ultimate basis of heparan sulphate signalling is actually the induced water structure (nb this is commonly acknowledged to be promoted in some non-specific manner by theextracellular matrix) and that this is the ultimate cause of tissue integrity; tissue development may alsorequire input form ‘correct’ water structures which might be associated with the longer polysaccharidesegments molecules. This correct water structure for the proposed heparan sulphate signalling etc.functions could involve both hydrophilic and hydrophobic repulsive and attractive forces similar to theoften long range effects which have been identified to occur in aqueous solutions adjacent tohydrophobic surfaces and hydrophobic and hydophilic mica surfaces (cf. [(1b) Christenson & Claesson2001] for which puzzling long range effects have been identified which might conceivably mimic thehigh anionic density + hydrophobic N-Ac region systems of heparan sulphates). These types of water structures are also believed to critically depend on the presence of sub-microscopic sized nano holeswhich have been generated in water structures by the micro-bubbles (which exist adjacent to mica andsilicate surface studied by atomic force microscopy) pointing to the possible key role of such amechanism in the of formation of (presumable non-equilibrium, thermostatically unstable) water microstructures in the biochemical mechanism of tissue generation and its upkeep.
 
Myelin basic protein integrity may depend on such hydrophobic induced water structure (and dependon micro-bubbles for its existence) as it is strongly associated with lipid induced hydrophobic watersstructuring effects [(1c) Muelle et al 1999)] . It should be noted that the integrity of this protein hasalso been associated with heparan sulphate proteoglycans which enable the repair of damaged myelinsheaths, defects in which can be argued to give rise to neurological conditions such as multiplesclerosis. A scenario by which is this process becomes disrupted by a UV-vitamin D-thyroid factor dependent sulphate transporter which facilitates heparan sulphate sulphation could explain thegeographical incidence of multiple sclerosis in Australia and perhaps also the possible promotion of this disease by barium intoxication [(1d) Purdey, 2004].Bone Mineralization-Key Roles of Sulphated Polysacchrides. Effect of Fluoride as Possible ServoFeedback Regulator of Glycosaminoglycan (putative) Microstructure Generation.Recent research [(1e) Reid et al 2008] has confirmed the previously suspected key roles of sulphated polysaccharides which occur attached to the nascent hydroxyapatite crystallites including during boneformation. A more accurate description of bone is fluoride substituted hydroxyapatite.Both heparan sulphate and chondroitin sulphate might influence this. These polysaccharides areknown to alter their microstructure in response to the presence of fluoride [(1f) cf Pawalowska-Goral etal 1998]. Fluoride is also present in native heparin [(1g) Grant et al 1987]. As well as Ca (and putatively also PO
43-
) F
-
putatively engages in biofeedback signalling relating to control of calcification.Since the ultimate driving force in such crystallisation can be argued to be the regulation of water structure and this structure is also affected by the presence of glycosaminoglycans [(1h cf Grant et al1990)] This can be how it might rationally be proposed that these polysaccharides modulate this as thedriving force during such regulation.Water Structure May Directly Regulate Protein FoldingIt can also be logically suggested [e.g. (2a) Der 2008; cf Wiggins & Wilse Robinson etc] that proteinfolding (
 b
) must depend largely on the existence and the activities of interstitial water; TheHofmeister series can be explained by the effects of high concentrations of protein stabilisers or denaturant solutes on the surface tension of the interfacial water.This concept should also be extended it is now proposed to include the effect of nano-bubbles on water structure.Since heparin and heparin-like sites in heparan sulphate are ultra-anionic, high ionic strengthco-solute generating environments to those which promote the Hofmeister effect, it is also expectedthat the interstitial water and putatively also nano-bubbles which occur adjacent to heparin/heparansulphate polysaccharides and could likewise greatly affect how heparan sulphate attaches to an altersthe conformation of target proteins.Direct evidence that such interstitial water might participates in such actions was obtained from studiesof the binding of heparin to poly-L-lysine and poly-L-arginine [(2b) Grant et al. 1991] which indicatedthat binding was accompanied by large changes in the overtone stretching frequencies of the water molecules attached to heparin. Related studies by these authors of the binding of inorganic ions andinorganic solids to heparin** and heparan sulphate tended to confirm that such binding depended onhydration changes (rather than electrostatic attractive forces, e.g. as had previously been believed tocontrol this activity [cf. the Manning hypothesis]). Interstitial water and entropy changes thereof determined how counterions bound to heparin. Further direct evidence for the existence of a high ionicstrength environment adjacent to heparin is that crystalline akaganeite fibers (identified by X-raydiffraction) are produced at heparin surfaces (following binding of Fe
2+
to heparin, its oxidation to Fe
3+
could be Co
2+
dependent (such ions were also apparently commonly co-purified with heparin).[Formation of this fibrillar crystalline material {FeO.OH}is generally known to require the high ionicstrength conditions](Williamson FB Aberdeen, personal communication, unpublished work).Biological Fluids are Seawater-Like Multielement Matrices from which Heparin and AnionicPolysaccharides Selectively Sequester the Least Abundant Solute Ions.In a internet paper (
now no longer accessible?)
discussing how water structure influences proteinfolding G Wilse Robinson quotedSzengt Gorgi who stated that humans could be described as bags of skin filled with seawater.That seawater differs markedly from pure water points to the importance of such differences for  biochemistry and the need to centre biochemical studies on a more detailed model of impure water 
 
(especially the effect of colloids in seawater and associated dissolved gases which might impartnanobubble structures and confer long range water structuring effects).The multi-inorganic nature of geological and biological phenomena including the seawater range of inorganic elements.The overlap of geology, inorganic chemistry and biological chemistry suggested a new branch of science was required (“metallomics”).This might , it can be argued rationally centre around the effect of water structure.[(3) Haraguchi, 2004] included heparin (but apparently only in regard to its sulphur content) in histabulation of topics for which a suggested new science [dealing with the] should be potentiallyconsidered of fundamental importance to biochemistry since biological fluids were multi-inorganic ionsolutions which were approximately similar to seawater and other natural waters.]It might further be suggested that since heparan sulphate seems to have co-evolved with multicellualr animals in the sea some 10
9
years ago a primitive role of cell surface heparan sulphates was to act as anutrient gatherer and buffer for seawater-like multi inorganic element containing salt solutions.This notion seems to be confirmed by a report from the Dietrich group of the existence of an exactmathematical relationship [(4) Nader et al. 1983) between the amounts of tissue heparan sulphates (andother sulphated polysaccharides) and the salinities of the habitats of fifteen species of aquaticinvertebrates, where habitat water might be required to directly bathe the heparan -sulphate- proteoglycan-lined tissues.A more evolved system in vertebrates which use complex hormone and dedicated kidney water ionhomeostasis is that these organs seem to have the ability to respond to the presence of small ions whichmight adversely affect their tissues by up-regulating heparan sulphate anti-crystal activity (an activitywhich is probably dependent on water structure modulation) [kidney epithelial cells have been reportedto alter their incorporation of radiolabelled sulphate into heparan sulphate and chondroitin sulphate inresponse to oxalate as well aas Ca oxalate crystals which are inhibitable by these polysaccharides [(4a)Borges et al 2005]].Anionic polysaccharides (and proteins) in animals when bathed in the multi-inorganic ion salt solution biological fluids will generate multi-inorganic ion/hydration water complexes similar to the anionic polysaccharides abundantly present in the cell walls of marine algae had been established [(4b)Wassemann, 1949] most likely to exist in this form
in vivo
rather than being present, as was originallysupposed, as free alginic acid.Although less chemically definable than the pure polyanionc polysaccharides (alginates, carrageenans,and the polysaccharide side-chains of glycosaminoglycans etc.) but nevertheless of considerableimportance to the homoeostasis of inorganic ions including carbonate, bicarbonate and Ca
2+
ions innatural waters is the system of humic/fulvic polymers (a system of polymethylene, polycarbonyl,caboxylated) material which comprises the largest system of organic polymers on earth.
[Added later,cf e.g. DA Hansell et al “Dissolved Organic Matter in The Ocean”, Oceanography 2009 22(4) “at 662PgC marine dissolved organic matter contains as much carbon as the atmosphere and is one of the Earth’s major carbon reservoirs”
] These natural polyanions bind numerous metal ions present inseawater etc. via abundant -COO
-
groups, which apparently gave rise to the geological deposits of fulvate organic matter.Determination by spark source mass spectrometry (SSMS) of the multi-inorganic element contents of geological fulvates and marine alginate showed obvious qualitative similarities to the SSMS resultsfor the multi-element contents of the animal polysaccharide heparin [(5) Grant et al 1987].Less information is currently available from the literature of similar studies of heparan sulphates(which are more difficult to obtain in large amounts) but studies conducted in the context of scitigraphic imaging (e.g. of tumours) has indicated that this procedure may depend upon the bindingof the radio-nuclides to heparan sulphate proteoglycans e.g. at cell surfaces; side experimentsestablished that
45
Ca in heparan sulphate could be replaced by a range of multivalent metal counterionsin a manner consistent with heparan sulphate being normally present in vivo in the form of a multi-inorganic matrix (5a).The apparent differences in the observed biochemical/physical properties of different brands of heparinseems to at least in part have its origin in the different degrees of ‘purification’ achieved by differentmanufacturers (6). It might even be suggested that such attempts at purification actually achieve

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