8.a-7-2-6.
Molybdenum
Molybdenum dependent enzymic action is involved in the oxidation-reduction biology of nitrogen compounds.
Mo in heparin was (7/?*).
8.a-7-2-7.
Silver
Silver occurs in mammalian heparin. Ag (4/0.5*), and may be similarly involved in thiocyanate-related pathology.
8.a-7-2-8.
Lanthanum, Neodymium, Zirconium & Yttrium.
La 7/1*,W 5/0*, Nd 5/0*, Zr 5/0.3*,Y 3/0* were present in significant amounts in the studied heparin.
8.a-7-2-9.
Cerium and Antimony
Cation exchange replacement of cations by Tl+ appeared to scarcely remove Ce (7/3.5*) and Sb (2/1.7*); it must be assumed therefore that these elements occur in anionic or form unusully strongly bound adducts with heparin or are artifacts of the analytical procedure.
Inappropriate release of such stored metals may stimulate inappropriate, pro-disease, redox activity.
8.a-7-2-10.
Mercury and Cadmium
Mass spectral analysis shows that although mercury is below the detection limit in the above mammalian animal heparin, both mercury and cadmium have been reported (Muzzarelli 1983) to be present in chitosan evidently via marine pollution; intoxication from this source and dental fillings have been listed in Internet discussions of mercury toxicity to be a major causative factor in AD.
Further work is required to fully epidemiologically quantifiy such trace metal loading of heparin/heparan sulphate and other mammalian GAGs and to determine possible pathological links.
8.a-7-2-11.
Aluminium
The sample was not assayed for Al because of the use of an aluminium powder sampling procedure. Further work is required to assess Al contents of high ash and normal pharmaceutical heparin.
8a-7-3.
Nickel Effects.
Nickel etal provokes hypersensitivity in some persons. Nickel ions normally do not demonstrate well-defined aqueous solution redox behaviour, but heterocyclic N-containing ligands have been observed to stabilize otherwise difficult-to-produce Ni(III) (Lindol, 1975) which may redox cycle with Ni(II) leading to the formation of active oxygen and nitrogen species.
As Ni is employed in mammalian enzyme centres, relevant Ni-dependent heparan sulphate interaction might be anticipated. Further studies are required to test this possibility.
Ni in heparin was relatively high (170/?*).
8a-7-4.
Anions in Heparin.
Major, likely anionic 'contaminants' detected by mass spectrometry in the above heparin sample were also surprisingly largely removable by cation exchange resin treatment, including 2000ppm chloride, 130 ppm bromide, 10 ppm iodide and surprisingly 890 ppm fluoride (removable to 4ppm upon Tl+ exchange, therefore likely to be attached to heparin-bound cations such as Al3+).
19-F NMR studies might be of value in establishing the mode of binding of such fluorine to heparin.
Strong binding of heparin to iodide, chloride and sulphate has previously been described in a Patent claim (Fo-We, 1962). In a 1980 review Jaques stated that heparin exhibits "strong binding of counterions of sodium, potassium, ammonium, quaternary ammonium radicals, and coions such as sulfate, phosphate and acetate". Sulphate anions in heparin were found not to be removed by dialysis (Simon 1982) as would be expected if present as a consequence e.g. of de-N-sulphonation; unpublished observations of the author confirmed that a proportion of the 35S labelled sulphate in heparin may behave anomalously on gel filtration separation in keeping with the formation of a bound sulphate anion-heparin complex.
8.a-7-4-1.
Phosphorus
The above analysis also showed the presence of 440ppm phosphorus (which was reduced to 30ppm on cation exchange resin treatment), likely present as phosphate ((1348ppm as PO43- or other P(V) oxy-species derivatives) in heparin.
Additional interest of the high phosphorus content of heparin is related to a likely influence of such phosphate and related species in enhancing damaging metal redox behaviour.
Condensed phosphates if bound to heparin/heparan sulphate would be expected to provide strong metal ion binding sites.
Calcium in the studied heparin was 30000ppm which was reduced to 30 ppm upon passage through the cation exchange resin column in agreement with the chemical environment of heparin-bound phosphorus being possibly present as a calcium P(V)oxyspecies-heparin complex.
Ir spectra of a heparin solution peptized hydroxyapatite (obtained in studies of the inhibition of crystallization of this substance by heparin) confirmed the presence oxy-phosphate attached to the heparin chains in such a way as to significantly alter the heparin ir asymmetric S-O vibration absorbance bands (Grant et al 1992, unpublished).
8.a-7-4-2.
Silicon
The mass spectral results of heparin discussed above also showed 5900ppm Si, 100ppm being retained bound to the heparin after passage through a cation exchange resin. Clearly a large part of the silicon is likely to be present as silicate and be bridged to heparin by cations such as Ca2+ and Al3+ .
Schwarz (1973) found that polyuronides, including GAGs, contained strongly linked (molybdate-inactive) silicate, (e.g. as a crosslinking silicic acid derivative, 29-Si NMR would be usefully employed to determine the environment of Si in such complexes); this author reported that heparan sulphate had 427ppm Si which could not be removed with 8M urea.
Grant et al (1992f) presented experimental evidence for the importance of multi-phase phenomena in various cation (including calcium)-heparin interaction as an altenative explanation to the Manning hypothesis for the existence of critial binding behaviour. Such a phase change mechanism may explain why inorganic anions are often irreversibly bound to the highly anionic polysaccharides (of additional likely relevance to understanding the mechanism of DNA and RNA cation binding where a similar situation pertains) and may further be of relevance to the possible gating of ion, including proton, conduction by such polymers as well, perhaps being relevant to understanding of the apparently unrelated phenomena such as the physical chemistry of the formation of pathological amyloid (heparan sulphate containing) deposits in which metal ion loading may have some causative participation (cf, Ca2+ but not other M2+ except non-physiological Cd2+ mediated the association of human serum amyloid P component with heparan and dermatan sulphate (Hamazaki 1987)).
A mechanism (Long & Williamson 1979) of control of cellular proliferation by heparin/heparan sulphate interactions with Ca2+ might be extended to include the co-binding of phosphate and silicate to Ca2+ (perturbable by such cations as Al3+?) to heparin/heparan sulphate and be of further possible relevance to the involvement of GAGs in calcification.
8.a.-7-4-3.
Boron.
The mass spectrometric results showed that the starting heparin contained <25ppm B, likely as borates or related anions, reducible to <1ppm after cation exchange.
8.a-7-5.
The Binding of Aluminium & Fluoride to Heparin.
A previous indication by Kojima et al (1983) that Al3+ binds strongly to heparin/heparan sulphate was confirmed by Grant et al (unpublished results obtained using potentiometric and osmotic methods). Both heparin/heparan sulphate biochemistry and (more controversially) the effect of aluminium intoxication, have been implicated in neurodegenerative diseases such as AD (e.g. Lehmann 1992).
Fluoride intoxication at high levels is reported to alter the GAG content of bones (Susheela & Jahr; Priince & Navia) which might include the biological effects of Al3+-polysaccharide binding (perhaps causing perturbation of Ca2+-heparan sulphate binding?).
Biologically-relevant effects of Al3+ plus F- include adenylate cyclase activation by F- (involving trace Al3+ or Be2+ co-catalysis cf, e.g, by Martin, 1988).
8.a-7-6.
Importance of Nucleation Factors & Lipids in Induction of Polysaccharide Supramolecular Structures.
Further ideas of relevance of binding of anions to heparin/heparan sulphate might be an involvement of these polysaccharides in various currently poorly-understood physiological nucleation phenomena and disease-related alteration in such activity due to erroneous supramolecular structural distorting effects.
Since Ca/Mg pyrophosphate granules are believed to nucleate calcification (e.g. Taylor et al 1990) such pyrophosphate granules attached to glycosaminoglycans may have similar properties.
Nucleation initiating catalysts may control specific glycosaminoglycan phase change; iron impurities are believed to be initiators of carrageenan supramolecular structure formation (Williamson FB, Aberdeen, personal communication).
8.a-7-6-1.
Hydrophobic Polyanion Binding Conditions.
Physiological phase changes in the pericellular environment clearly involve the physical chemistry of lipids. Whereas under normal physiological saline conditions, metal ions are often only relatively weakly bound by mono and polysaccharides, in non-aqueous media including pericellular lipid-rich environments, large conformational alterations are found upon cation sugar interaction, suggesting that this is unusually strong in character (cf Tajmir-Riahi 1989),
The nmr and infrared spectra of various heparin metal ion complexes provide some insight into details of polyanion-cation-water interaction.
13-C nmr spectra are influenced by a time averaging of fast cation exchange processes exemplified by a dominant effect of Na in mixed Na/Ca heparin and by a surprisingly masked effect of paramagnetic ions althought an apparent specific binding of paramagnetic Cu(II) shows up more clearly in 1-H nmr spectra (Rej et al 1990).
205-Tl nmr shows a relatively uninformative distorted single broad absorption indicating a poorly resolved distribution of cation environments.
Solid samples studied by ir provide evidence of cation-specific interaction with clear dependence of the importance of cation-dependent water binding under these conditions.
Binding of heparin to basic polypeptides in hydrophobic media show the importance of glue-like activities of water molecules associated with the cation/polyanion complex (Grant et al 1992).
8b HEPARAN SULPHATE, BRAIN DEVELOPMENT, FUNCTION & NEURODEGENERATIVE
DISEASES INCLUDING ALZHEIMER'S DISEASE (AD).
8b.-1 Introduction.
Heparan sulphate proteoglycans, nitric oxide (Salvemini et al 1998), and perhaps redox systems which will include Zn2+ effects (perturbed by Al3+ ?) ions are implicated in the pathogenesis of AD and related diseases as well as in normal memory function.
A chronic inflammatory state of the brain may occur in AD and might explain why patients on anti-inflammatory drug therapy for rheumatoid arthritis appear less susceptible to AD (McGeer et al 1990) agreeing with a likely involvement of immunologically produced nitric oxide/nitrous acid /redox metal heparan sulphate damage in AD.
Inappropriate modulation of heparan sulphate by nitric oxide metabolites influenced by trace metal ions (perhaps including inappropriate initiation of polyanion phase change as discussed in the previous section) might instigate neurological malfunction and contribute e.g. to the formation of amyloid deposits in various amyloidoses which have the common non-covalently linked basement membrane heparan sulphate PG component (Narindrasorasak et al 1991). Neutralization of inappropriately formed nitric oxide generated nitrous acid by ascorbate reduction or by sacrificial deaminative cleavage of glucosamines may provide essential protection against such effects.
Iron (iron ions are especially potent potential redox and phase change damaging agents) is reported to be selectively accumulated with aluminium in neurofibrillar tangles (NFI) of AD (Perl et al 1992).
Membrane spanning heparan sulphate proteoglycans are thought likely to be important regulators of activity-dependent modulation of neuronal connectivity (Laun et al 1999) perhaps linking cytoskeletal elements with the pericellular environment.
Nitrous acid also can potentially disrupt neurotransmitter monoamine oxidase activity directly deaminately cleaving dopamine and its metabolites and cause DNA cross-linking (Kirchner & Hopkins 1991).
Liver amyloid was reported to show a very high level of de-N-sulphonated heparan sulphate chains absent from other heparan sulphates (Linker & Hovingh 1977) allowing uncontrolled nitrous acid cleavage.
8b-2.
Heparin/Heparan Sulphate in AD.
Fukuchi et al 1998 recently reviewed the involvement of heparin/heparan sulphate (in beta-amyloid protein, neurite plaque, NFT tau protein fibril formation and ApoE4 lipid processisng) in AD but did not deal with the effects of nitric oxide and its metabolites in this disease which is now suggested (as a main theme of these notes) to damage heparan sulphate molecules under conditions pertaining to such diseases.
8.b-2-1.
Learning and Heparin/Heparan Sulphates.
Nitric oxide neurotransmitter activity may be involved in learning (Hawkins et al 1998, cf also Breen 1992, Fukuchi et al 1998). N-CAM glycoprotein function in synaptic connectivity was found to be heparin-dependent (Bullock & Rose 1992) (Cole et al 1986) with a possible memory function release of heparan sulphate (Sugara & Der 1994); injection of heparin into the adult rat hippocampus induced seizures (Mudher et al 1998); the heparan sulphate proteoglycan syndecan-3 was implicated in neuron extracellular matrix interaction in the cellular mechanism responsible for synaptic plasticity, associated with cortactin src type and fyn tyrosine kinases, playing a crucial role in long term potentiation (LTP) (enzymic cleavage of heparan sulphate as well as the addition of heparin prevented development of LTP in rat hippocampal cells studied by Laun et al, 1999); heparan sulphate may also provide resevoirs for growth factors, zinc and neurotransmitters.
8.b-2-2. Zinc in AD.
Zinc is a potential modulator of heparin/heparan sulphate activity.
Zinc is an intracellular messenger causing protein kinase C (PK) to translocate to polymerized actin components in the cytoskeleton (Zalewski et al 1990).
A neurological function in sustained cellular responses involves PKC is long-term potentiation of synaptic transmission in the hippocampus (Ackers et al 1986); this activity is also heparan sulphate dependent (Laun et al 1999).
Phosphorylation of cytoskeletal elements modulatable by heparin/heparan suphate may be implicated in short-term memory.
Involvement of zinc APP-heparan sulphate interactions (Multhaup 1994) in AD therapy (Masler et al 1993) suggest possible modulation of APP activity by heparin/zinc. Zinc supplementation could apparently impair congnitive function in AD patients, restorable on cessation of the zinc suppplementation (cf Smith et al 1994).
The reported anti-heparinase activity of Zn2+ (cf Patent Spec RO96508) needs to be investigated further, but indicates a possible role of Zn2+ in the modulation of heparin/heparan sulphate signalling via alteration of its degradation rate.
Are there linkages between Zn2+ - heparin/heparan, Zn2+- ascorbate, Zn2+- finger linked biochemistry Zn in SOD (Cu,Zn SOD), and effects of Zn2+ on NO biochemistry?
Zinc disturbance in AD may include a function of specialized Zn2+-GAG binding (Grant et al 1992f).
Zn2+ may counter a neurological protection afforded by heparin (considerd to be against proteolytic cleavage of APP, the integral transmembrane protein that is released from cells in culture following proteolytic cleavage) but nitric oxide related mechanisms might also be relevant.
Selective Zn2+ binding to heparin (e.g. Woodhead et al 1986), an unusual proton binding effect produced uniquely by Zn2+ with heparin (Grant et al 1992f) (and perhaps a role in proton conduction) may have relevance for zinc-dependent heparan sulphate processing.
Zn2+ catalysis of de-N-sulphonation of heparin by co-binding of metals and the role of Zn fingers in proteins in modulation of gene expression may be part of overall Zn2+ controlled mechanisms which include, at some stage, heparan sulphate-linked redox ascorbate and nitric oxide biochemistry and nitric oxide modulation of metal-protein binding (cf Draper et al 1996).
Zn2+ at 50 nM (the effect saturated at 70 micro M) promoted heparin binding to amyloid precursor protein APP and abolished a protective effect of heparin against its proteolytic cleavage. Zinc dietary supplementation was reported to impair cognitive function in AD patients (Masler et al (1993).
Heparan sulphate was separated from other GAGs by its greater readiness to form Zn2+ cross- linked phospholipid precipitates (Wu & Cohen 1984).
Parrish & Fair (1981) reported that Zn2+ was bound to heparin but less readily by chondroitin 4 and 6 sulphates, dermatan sulphate or hydaluronic acid.
NMR studies by Whitfield & Sarkar (1991) suggested that Zn2+-carboxyl interactions controlled the conformation of the iduronate rings of heparin monsaccharides and Li+ ions appear similarly to affect iduronate conformations in heparin preparations (Grant et al 1991; perhaps these observations are relevant to heparan sulphate mechanisms in neurological physiology.
It might further be hypothesized that similar effects might include nulceation of phase change related to cycloskeletal elements involved neurological function and in memory function, further modulatable by nitric oxide.
8.b-2-3
Cognitive Function in AD.
Therapeutic Use of Heparin/Heparan Sulphate
A heparan sulphate containing GAG preparation ('Ateroid') has been reported by Ferrero et al (1989) to improve cognitive function in AD patients.
A mechanism which might be relevant to such effects by blocking by exogenous heparin of heparan sulphate beta amyloid peptide interaction was suggested by Leveugle et al (1994).
Vascular malfunction involving heparin/heparan sulphate and nitric oxide is also possibly involved the the aetiology of neurodegenerative diseases.
Heparin induced endothelial cell cytoskeletal reorganization was suggested to be a possible mechanism for vascular relaxation (Mandil et al 1995) and nexin-2 an isoform of the AD amyloid beta protein precursor secreted in large quantities by platelets upon vascular injury may be potentiated by heparin/heparan sulphate. Glia-derived nexin-1 binds to heparin (Rovelle et al 1992).
The nmr spectrum of heparin was perturbed by Gd3+ binding in a less selective manner than by Cu2+ (Rej et al 1990). Cu in heparin may contribute both to its SOD and angiogenic activity (Raju et al 1982).
8.b-2-4.
Neurite Outgrowth Stimulation by Heparin/Heparan Sulphate.
Neurite outgrowth is stimulated by heparin (e.g. Sedden et al 1994) and heparan sulphate (Dow et al 1992). A heparin-binding domain in APP is also thought to be involved in regulation of neurite outgrowth (Smith et al 1994) in which there is a likely autocrine role of heparan sulphate proteoglycans (Dow & Riolelle 1992) perhaps via a thrombospondin route (O'Rouke et al 1992).
8b-2-4-1..
Possible Heparin/Heparan Sulphate-Dependent Growth Factor Effects in AD.
Since the brain has long been recognized as a plentiful source of mitogenic acitivity and is a rich source of FGFs (Gimenea-Gallego et al 1985) subject to modulation by heparan sulphate (or by exogenous heparin), heparan sulphate proteoglycans are likely also to control bFGF activity in developing brain (Hondermark et al 1992) and be of relevance to neurodegenerative disease as is implied by the usefulness of a bFGF binding assay (Peiry et al 1992) to study the incidence of heparan sulphate in plaques in AD and other neurodegenerative diseases.
Regulation of nerve growth factor (NGF) (is likely to be heparan sulphate-dependent (Damon et al 1992). Cellular therapy to achieve NGF replacement has been proposed for AD therapy (Hawkins, press report 2000). NGF was reported to change the invasive properteis of neuro-ectoderm melanoma cells, possibly dependent on heparanase activity (Maschetti et al 1999); such activity, as discussed above, may also be Zn2+-dependent.
8.b-2-5.
Extracted Brain Heparan Sulphate - Lack of Correlation with AD.
Although brain heparan sulphates differed from those of other organs (Lindahl et al 1995), little difference was detected between the sugar composition and partial sequences of heparan sulphates from AD and control brains, indicating that the amount and primary microstructure of heparan sulphates may not contribute directly to AD; however the complexity of heparan sulphate biochemistry, involving the possibility of multiple cation and anion binding as well as multi-phase behaviour, does not rule out some future establishment of a direct heparan sulphate (supramolecular) structural link with neurodegnerative disease pocesses.
It is suggested that, in addition to the polysaccharide microstructure, the inorganic content of the normal and AD heparan sulphates needs to be be examined by e.g. spark source mass spectroscopy. Of particular interest is aluminium and silicon contents since there are putative links of these with the disease.
(cf silicic acid has been suggested to exert a protective role in aluminium neurotoxicity cf Birchall & Chappell 1989).
8.b-2-6.
Possible Transferrin Receptor Function, Aluminium & Heparin/Heparan Sulphate in AD.
Transferrin receptors which contain heparan sulphate side chains (Fransson et al, cf Gallagher et al 1986 and Hu & Reogoeczi 1992) are 67-Ga3+ markers for brain aluminium transport and coincide with the areas of the brain vulnerable to AD (although unaltered in abundance in AD) (Edwardson et al 1990).
67-Ga3+ binding which was much more strongly bound to heparan sulphate than to other GAGs (Kojima et al 1983) likely mimicking effects of Al3+.
Al 3+is also implicated in dialysis dementia and Guam disease.
Al3+ may inhibit calcium-mediated proteolysis of cytoskeletal proteins and induces NF to form complexes throught to be via phosphate ester binding (Nixon et al, 1990).
Cis-aconitate is thought be be a likely pathological carrier of Al in AD and Al-citrate backs up transferrin and ferritin binding (Lehmann 1992).
8.b-2-7.
Fenton Reaction /Free-Radical Damage in Neurodegenerative Diseases.
Complex interactions involving heparin/heparan sulphate are seen to be involved directly and indirectly in control of both redox balance, antioxidant and other enzymic activity.
Redox balance of which glutathione, ascorbate, nitric oxide and multiple antioxidant activity is part, is suggested as a theme of these notes to be a dominant factor in heparin/heparan sulphate biochemistry, pro-disease, abnormal heparan sulphate arising from its perturbation which include abnormal iron, aluminium and silicon loading which could adversely affect the pro-antioxidant activity and proteolytic modulatory actions of heparin/heparan sulphate.
Al3+ directly deactivates SOD (Shainkin-Kestenbaum et al 1989) and brain proteases (Nixon et al 1990) and perhaps other relevant Ca2+, Cu(II) and Zn2+ dependent mechanisms.
In AD, Down's syndrome, amylotrophic lateral sclerosis, Parkinson's and Guam dementias (Leveugle et al 1994), affected neurons apparently oversynthesize lactoferrin (involved in iron and aluminium transport).
Antioxidant SOD genetic insufficiency has been identified in the familial form of amylotropic lateral sclerosis (Rosen et al 1993, cf Calder et al 1995), in agreement with an augmented superoxide initiated damage involving reactive nitrogen species including peroxynitrite (Cookson & Shaw 1999, cf Love 1999 who reviewed oxidative stress in brain ischemia).
Iron, phosphate (and aluminium) effects, however, may also be implicated in this.
As discussed in section 3.5 above, reactive oxygen free-radicals generated by iron-Fenton reactions are likely to damage GAGs. Further evidence of this is provided by Nagasawa et al 1992 who observed such damage to hyaluronic acid and heparin/heparan sulphate in less sulphated regions. Another example is the Cu(I) hydroxyl radical formation damage to hyaluronic acid in metallosis of the eye (Sterk et al 1985).
8c.
Supramoleular Structure of Heparin/Heparan Sulphate PGs and their Binding Potential.
Ca2+ (Liang et al 1982, Dais et al 1987) and Zn2+ binding the heparin may be critcially dependent on the cooperative effects of supramolecular structrure and arrayed N-sulphonate groups (Grant et al 1992c,f) to which crosslinking by small amounts of e.g. Al3+ could lead to disruption of the requirements for protein folding and binding.
Nitric oxide damage to heparan sulpahtes in neurodegenerative disease may enhance such inappropriate binding activities.
8c-1.
Silicates in GAGs.
Silicates have been found to be associated with aluminiumin AD plaques (Edwardson et al 1990). Although aluminium and silicate involvement in AD may be related to Zn2+ or Ca2+ homeostasis, silicon is an essential nutrient although the details of its biochemistry are still obscure and (inorganic) silicates are believed to occur in normal association with glycoaminoglycans (Iler,1979).
It is conceivable that silicate aquesition may be a normal function of GAGs. The effect, if ,any of Al3+ on this is worthy of study.
Differences in the amounts of bound silicates and phosphates by glycosaminoglycans from different species organs and diseases and work-up procedures might have contributed to difficulties in comparing metal ion binding and other activities of such GAG preparations.
9.
Involvement of Heparin/Heparan Sulphate in Modulation of Prion Protein Activity.
Prion proteins have a heparin/heparan sulphate perspective (by differentially binding to heparin-like molecules, the synthesis and metabolic fate of prion proteins in scrapie infected cells is inhibited by reversing their phenotype back to normal (Gabizone et al 1993)).
Part of the normal cellular function of prion proteins may be conceivably of relevace to memory function as they are evidently implicated in neurodegenerative diseases, principally scrapie but also Kuru, Creutzfeld-Jacob and Gerstmann-Straussler-Scheinkler diseases (Prusnier 1995). The anti-scrapie effect of heparin and related molecules was established by workers (Diringer & Ehlers 1991) who do not, however, agree with the prion hypothesis (Diringer 1985).
A further factor which does not seem to have been considered is the effect of non-physiological metal intoxication of heparin/heparan sulphate from the perspective of prion protein biochemistry.
10.
WATER BINDING TO HEPARIN/HEPARAN SULPHATE.
The number of water molecules, strongly bound to sulphate half-ester groups of heparin was found to the highly dependent on the counterions present, averaging six for sodium, three for potassium and one for calcium. A quasi phase change mechanism of counter ion binding to heparin/heparan sulphate may have relevance to switchable ion and proton pumping. Commercial sulphonated polymers find use as proton conductors, such activity being dependent on cation-dependent water binding to the arrays of sulphonate groups (Colomban 1992) which is analogous to a simiilar effect of counterions on the stoicheometry of water molecule binding to sulphate half-ester groups in highly sulphated heparin-like molecules.
Hydration changes may also be relevant to the coil conformation changes thought to be responsible for signalling by sodium/calcium - heparan sulphate chains in the proposed mechanism of sensing of blood flow in vivo (Siegel et al 1998) and perhaps other servo-control mechanisms, evidently employing potentially involving heparan sulphate conformation -dependent effects, such as control of calcium/heparin binding sites in parathyroid cells (Takeuchi et al 1990).
10a
HOFMEISTER EFFECTS ON WATER STRUCTURE
The Hofmeister series ranks the relative protein denaturation abilities of aqueous salt solutions. Water aggregate structures present in various metal salt solutions, deducible from near infrared spectroscopy, also varied with the Hofmeister series (Luck 1969) indicating that the ionic content of aqueous solutions affects the aggregation state of hydrogen bonded water molecules thought to be composed, at physiological temperature, of several hundred H2O units but salt ions 'melt' these aggregates according to their Hofmeister activities. Flickering clusters of such labile hydrogen-bonded arrays may also behave in a determinisitic chaotic manner, e.g. being influenced by phase boundary conditions which although poorly understood, may be relevant to biology (which usually restricts such considerations to hydrophobicity indices) and even to the mechanism of memory storage and retrieval.
Heparinized surfaces may create bio-friendly non-denaturing surfaces by providing correct hydrophilicity/hydrophobicity in addition to providing anticoagulant protection as well as binding sites for antioxidants and growth factors. Heparin/heparan sulphate, as well as other biological polyanions, can be considered to provide Hofmeister-series activity modulation through Hofmeister-effect-empowered-hydration. This is thought to be related to the established morphological role of heparin/heparan sulphate and related polysaccharides in calcification and to a hypothetical role in e.g. cytoskeletal controls.
The entropic driven properties of quasi phase change behaviour (such consideration are also perhaps relevant to the reversible binding and diffusion of bFGF on HSPGs of basement membranes (Dowd et al 1999) and evently an unconventional discussion of water structure as an information carrier in organisms (Schwabl 1994) are possibly related the the unusual thermodynamic properties of water-rich gels perhaps underlying the evolution of sulphated polysaccharides for flexible ligand binding, the basis of developmental biology (Comper 1994).
Another example of such gel effect is the intestinal mucous coat with an unstirred water layer (Smithson et al 1981).
11.
MODULATION OF CRYSTALLIZATION BY HEPARIN/HEPARAN SULPHATE.
A biological function of glycosaminoglycans and related polysaccharides in calcification has been well established. A similar morphological role is possible with other biological mineral and protein structures. This includes cytoskeletal elements fundamental cell activity such as of the mitotic spindle (Roussel et al 1990) and perhaps also involves the nucleus (Bhavanandan & Davidson 1975).
Seeded calcite crystallization studies demonstrated that lower molecular weight or de-N-sulphonated heparins (such as might be produced as a result of nitric oxide/nitrous acid reaction) were much less effective calcification inhibitors than intact highly sulphated polymers (Grant et al 1989b).
A pro-disease effect of pathological in vitro crystallization and its prevention by heparin-like molecules has been discussed by Grant et al (1992b).
The ability of structurally sound heparan sulphates, but not pathologically altered heparan sulphates, both to inhibit crystallization and to act as seeds for the induction of correct protein folding could be related phenomena.
Rate determining processses during crystallization may be dependent on relative hydration energies in transition states and related cation and hydration-dependent polysaccharides may provide enthalpic driven proximity and flexibility for ligand binding requirements of multicellular organisms, which is at the basis of developmental biology (Comper 1994) and presently hypothesized to the an important fundement of the pathology of degenerative diseases and autoimmune processes.
Foreign particles (such as asbestos fibres) or abnormal solids (e.g. amyloid fibres) may provoke immunological reaction including nitric oxide/nitrite formation unless adequetly deactivated by suitable biopolymers including heparan sulphate which may restore acceptably inert structure.
12.
ESTABLISHED LABORATORY USE OF NITROUS ACID FOR HEPARIN/HEPARAN SULPHATE ANALYSIS
Nitrous acid has long been known to be a useful organic chemical reagent, employed by synthetic organic chemists to allow accurately defined molecular transformations to be achieved, e.g. yielding alcohols from aliphatic primary amines, diazo compounds from aromatic primary amines and nitrosoamines from secondary amines. Prior to the current awareness of nitric oxide and its metabolites as being highly pertinent to biochemistry and medicine, the high specificity of the nitrous acid deaminative cleavage of heparan sulphates encouraged chemists to employ this reaction to confirm the presence or absence of heparin/heparan sulphates in biological samples and to further characterize the fragments obtained by gel filtration etc.
The reaction is normally employed to allow scission of the N-sulphonated glucosamines leaving the N-acetyl glucosamines intact. Hydrazine treatment, however, selectively de-N acetylates which when followed by nitrous acid treatment at pH 4, provides further heparan sulphate fragments for analysis.
13
BINDING OF PATHOGENS TO HEPARAN SULPHATE
Such binding is common for many pathogens allowing therapeutic intervention to be achieved by heparin and heparinoids by blocking the heparin binding sites and by other mechanisms including inhibition of reverse transcriptases.
Helicobacter pylori (among the many heparan sulphate binding organisms) has a marked pro-cancer effect, enhanced in cyclotoxin associated antigen CagA strains. Exposure of gastric epithelial cells to H pylori induced activation of the transcription factor protein 1 and activation of the proto-oncogenes c fos and c jun. This might be a crucial step for the induction of neoplasia (Meyer-Ter-Veh et al 2000).
Heparin/heparan sulphate biochemistry might also be implicated in H pylori induction of cancer e.g. through perturbation of growth factors e.g. by the displacement of FGFs from heparan sulphate PG sites by bacterial heparan sulphate binding peptides (Ascencio et al 1995).
Antiproliferative effects of heparin on c fos and c jun in arterial walls appeared to be indirect perhaps via alteration of other cell cycle events (CA 120 124557c).
14.
REFERENCES.
Separate file.
15.
ACKNOWLEDEMENTS
Grateful thanks are due to Professor KEL McColl and Ms J Grant (Glasgow), Drs Rodger Worthington, Frank Willamson and Bill Long (Aberdeen) as well as to the Carnegie Foundation for the Universities of Scotland for assistance.
Spark source mass spectrometric results were kindly provided by Dr Bacon (Macaulay Inst Aberdeen) originally at the instigation of Colin Moffat (Aberdeen).
* Mass spectroscopic results are given thus : (parts per million by weight (ppm) in untreated heparin/ppm in heparin after cation exchange on a Tl+ loaded Abberlite IR 120 column).
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