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    Berit Smestad

    A thesis presented to the Faculty

    of Science of the University of London

    in candidature for the degree of Doctor

    of Philosophy,

    December, 1971* Chemistry Department, Royal Holloway College, University of London, Englefield Green, Surrey.

  • ProQuest Number: 10096786

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    ProQuest 10096786

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    I wish to thank Dr.E.E.Percival for her

    supervision, interest and help in carrying out this work,

    I would also like to thank Professor E.J,

    Bourne for his interest and for providing laboratory


    I am grateful to Professor R.G.S.Bidw'ell,

    Queens University, Kingston, Ontario, Canada, for providing

    facilities to carry out a part of this work in his laboratory.

    Finally, I am indebted to the following

    institutions for financial support:

    Institute of Pharmacy, Oslo, I969 and 1970.A.C. Houeiis legat, Oslo, I969,Norsk Parmaceutiskr Selskap, Oslo, I969.Professor S.A,Sexes legat, University of Oslo,1970,Norsk Medisinaldepot, Oslo, 1970.International Federation of University Women, Summer 1970 for work in Canada.

    The British Council, Oct.1970 - Dec.1971.


  • PART I

    Investigation of the Carbohydrates synthesised by the marine

    greed alga Acetabularia crenulata

    By means of sequential extractions with different

    solvents the following carbohydrates were isolated and


    1, Prom an alcoholic extract ^glucose, B-fructose, allulose,

    myo-inositol and an alcohol tentatively identified as

    allo~-cfuercitol. This is only the second time allulose has

    been found in Nature and the first time in any alga. An

    homologous series of fructose-containing oligosaccharides

    were also separated and characterised as 2,1linked units

    terminated by a molecule of sucrose at the potential reducing


    2. Aqueous extraction gave a mixture of a fructan (major) and

    a sulphated heteropolysaocharide.

    a) These two polysaccharides were separated on a column of

    DEAE~oellulose, Using the classical techniques the fructan

    was characterised as an inulin type polysaccharide,

    b) (i) The sulphated polysaccharide contained D-glucuronic acid,

    ^galactose, ^rhamnose and small proportions of xylose and

    4-0-methylgalactose, Each of the sugars were separated and

    characterised. The presence of the last sugar has not been a constituent of any green algal polysaccharide before*

    a.H.C.' TP TP.l

  • 11

    (ii) By extraction of stalks and caps' separately with cold

    and hot water and elution from the BEAE-cellulose with 0.5

    and 1.0 M potassium chloride similar sulphated polysaccharidebpAO-

    with variable proportions of the different sugars and portions

    of sulphate were separated,

    (iii) Structural studies by partial desulphation, mthylation,

    periodate oxidation and partial hydrolysis established the'

    essential similarity of these fractions and that the main

    structural features are: highly branched molecules containing

    1 ,3-lihked D-galaotose, ^-sulphate (rnajor) and 6-sulphate,

    1 ,2-linked L-rhamnose^and glucuronic acid, galactose and

    rhamnose all present as end groups. Glucuronic acid is linked

    to both rhamnose and galactose and galactose units are

    mutually linked in the macromolecule.

    3. A j3-(l -^4)linked maiman was extracted with alkali. Mthylation,

    periodate oxidation and gel filtration studies proved this

    structure and indicated some degree of branching and a higher

    molecular weight than those of previously reported for mannans

    from green algae,


    Photosynthetic studies on 1, Acetabularia mediterranea, 2,Fucus

    vesicuXosus and 3* Ulva lactuca^14-1. Pulse labelling experiments with CO^ on A .mediterranea

  • Ill

    followed by ethanolic extraction led to the separation of

    labelled sucrose, glucose, fructose and the first three

    oligosaccharides characterised in Part I (l), A possible

    biosynthetic interconver&ion of these carbohydrates is

    described from the results of these experiments*

    142. a) Pulse labelling experiments with COg on Fucus vesiouloaus

    a marine bcrown alga, show that of the low molecular weight

    carbohydrates formed by photosynthesis, mannitol is formed first

    The possible conversion of mannitol' into laminaran via mono-and

    di- glucosylmannitol is discussed*

    b) The polysaccharides, laminaran, xylogalactofucogluouronan (A),

    xylogluQur-onogalactofacan (b ), fucoidan (C) and alginio acid

    were extracted and separated by various fractionation techniques.

    The radioactivity in each was measured as was the radioactivity

    in the constituent sugars of the fucose-containing polysaccharides

    (all of which are sulphated). From the changes in the radioactivity

    of these polysaccharides in different samples it is postulated

    that (a ) is synthesised first and transformed into (C) via (E).

    Low molecular weight carbohydrates present in the acid extract

    are suggested as precursors for the acid polysaccharides. The

    residual material after acid and alkali extraction was hydrolysed

    and the radioactivity of the sugars in the hydrolysate was

  • IV

    measured. Glucose was the major radioactive sugar.

    3. Ulva lactuca, a marine green alga. Similar experiments on

    U.laotuca were carried out.

    a) Examincation of the 80^ ethanol extracts showed that sucrose

    is the first sugar to be synthesised and this is followed by

    glucose and fructose. Xylose, ribose(?) and myo-inositol also

    incorporated radioactivity.

    b) Starch and a sulphated glucuronoxylorhamnan were extracted

    and their radioactivities were measured. The former appears to be

    synthesised most rapidly and to be an active metabolite and the

    sulphated polysaccharide is laid down as a long terra storage

    product or as part of the skeletal structure of the alga.

    c) The residual material after ethanolic and aqueous extractions

    was examined in the same way. Glucose is again the major sugar

    and the results indicate that the carbohydrate is laid down in

    the cell wall after other constituents.


    PART I. Structural Studies of Carbohydrates in Algae

    Introduction Page 1Experimental 26General methods 26

    Carbohydrates of Acetabularia crenulata

    Introduction 34Experimental and extraction

    of the alga 39

    Low Molecular Weight Fraction

    Experimental 42Discussion 49

    The Fructan

    Introduction 51Experimental 53Discussion 5^

    Acidic Polysaccharide

    Experimental 61Discussion , 84

    The Mannan

    Introduction 102Experimental 103Discussion 113

  • CONTENTS . continued

    14PART II. Photbsynthetic studies with CO^ Qct carbohydrates in Algae

    1 Acetabularia mediterranea

    Introduction Page 113Experimental 119Discussion ' 121

    2. Fucus vesiculosus --- f

    Introduction 132Experimental 139Discussion 154

    3* Ulva lactuca

    Introduction 165Experimental 169Discussion 176


    Culture of A.mediterranea I8l

    References 183

  • P A R T I


  • Most of the plants growing in the sea belong to the

    class known as algae. They are one of the most primitive groups

    in the plant kingdom and evolved early in the earthfel history,

    and morphologically they.differ very little from those found as

    fossils. Although there are some freshwater species, they are

    mainly found in marine waters. They vary in form from unicellular

    plants, free-floating in the sea, to species which are several

    metres long and are fastened to rock with a root-system called a

    rhizoid. The algae are not differentiated into root, stem and

    leaves as are the land plants. Instead they have a thallus which

    varies in form from species to species, some looking almost like

    flowers with a stem and "leaves, and others looking like lettuce,

    for example, the sea lettuce, Ulva lactuca.

    The algae are classified mainly according to their colour,

    as the difference in pigments generally coincides with important

    morphological distinctions. They are divided into the following

    groups; Brown (Phaeophyceae), red (Rhodophyceae), green (Chloro-

    phyceae), and blue-green (Cyanophyceae), They all possess chloro

    phyll and photosynthesis as do the land plants. Generally the

    green and brown seaweeds grow nearest to the surface of the sea,

    while the red ones are found further down, but examples of all

    types can be found at all levels from the surface.

    Brown weeds were early collected for production of iodine,


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