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The history of Alginate chemistry - Structure 4

Structure - Poly M

Further fine structure information has been obtained from the action of certain alginate lyases upon the alginate chain. An extracellular poly α-L-guluronate lyase was used to degrade the α-L-guluronic acid and random segments and subsequently measure the chain length of the poly β-D-mannuronic acid blocks. Average chain lengths of about 24 residues were obtained initially (Turvey & Boyd, 1978) but later work indicated shorter block lengths (Turvey, 1983). Products from the action of L-guluronic acid lyase and from the the action of D-mannuronic acid lyase were separated by HPLC and examined by high resolution proton NMR. This work suggested an average block length of around seven residues. Seven residues is not long enough for a guluronic acid block to cationically bind to another, fifteen being the calculated minimum (Kohn, 1975). Hence the proposed binding mechanism of chains in a calcium alginate gel needed to be modified to include two separate poly α-L-guluronic acid blocks separated by one β-D-mannuronic acid residue. The study also showed that the mixed block segments were not strictly alternating (Boyd & Turvey, 1978; Kashiwabara et al 1969; Davidson et al 1977; Madgewick et al, 1973; Nakada & Sweeney, 1967; Nisizawa et al, 1968). An alginate lyase has been extracted from a marine bacterium and shown to cleave endolytically (1-4)- β-D-mannuronic acid polymers to oligomers containing a 4,5 unsaturated terminal residue. The overall effect of the enzyme is to reduce poly mannuronic acid to its trimer, the depolymerisation stops at this point (romeo & Preston; 1986).

The glycosidic linkage is 4C1 di-equatorial in mannuronic acid

Poly α-L-guluronic acid blocks and poly β-D-mannuronic acid blocks give very different circular dichroism spectra. A recorded spectra can be fitted to a computer matched combination of the three individual block's spectra. The correlation of results is very good compared to other techniques but the resolution of the spectra for samples with large portions of heteropolymeric blocks was found to be poor (Morris et al, 1973, 1975, 1980a; Craigie, 1984).

The long chain structure of alginate was confirmed by X-ray diffraction analysis on alginate fibres (Astbury, 1945). It was also proposed that the unit cell of the alginate structure consisted of four mannuronic acid residues and perhaps four water molecules. On drying the x-ray diffraction pattern deteriorates but the change is complately reversible on re-exposure to the atmosphere. The availability of the almost pure polyguluronic acid and polymannuronic acid segments has allowed the structure of the two segments to be analysed by X-ray fibre diffraction and infra red spectroscopy of oriented films. The repeat distance along the alginate chain can be measured and a model conformation built to fit these experimental distances, this has shed light on the structure of both the polyguluronic acid and the polymannuronic acid segments. The structure of the poly mannuronic acid segments are very similar to that of cellulose and other β-1,4 linked polysaccharides. The mannuronic acid residues are in the 4C1 conformation and consequently its diequatorially linked. The linkages give the polymer segments containing polymannuronic acid a flattened, ribbon like structure. It has been proposed that this structure is further stabilised by the formation of hydrogen bonds between the proton on the hydroxyl at carbon 3 in one ring with the ring oxygen of an adjacent residue. Another from of hydrogen, between the carboxyl groups hydroxyl and the oxygen atom attached to C3 of a parallel chain causes the poly mannuronic acid chains to bond into sheets of anti parallel residues.