Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Advances in pharmacology, biochemistry and biotechnology are increasingly dependant upon
affinity chromatography as a preferred separation technique for the purification and
characterisation of specific biomolecules.
In the past few years avidin-biotin technology has been widely and successfully used in the fields
of medicine, pharmacy, biology and biochemistry. The avidin-biotin complex (ABC) has been
used as a mediator for affinity chromatography, affinity cytochemistry, immunoassay,
histopathology, bioaffinity sensors, erosslinking and immobilisation studies.
The main reason for the popularity of the ABC and its growing usefulness in biotechnology is the
exceptionally high affinity (1015 M-l) and stability of the noncovalent interaction between avidin
and biotin. The use of the ABC is broadening as different biotin derivatives and
avidin-containing conjugates are becoming commercially available.
The aim of this work was to evaluate the usefulness of a plutonic" FI 08 and the ABC conjugate
to effect affinity separation. Towards this aim, the adsorption of plutonic" F108 onto
hydrophobic polysulphone membrane surfaces was studied. This information was used to
determine the theoretical maximum amount of pluronic" FI08 that will adsorb onto a unit
surface area of the membrane. It is known that the polypropylene oxide (PPO) centre block ofthe
pluronic" F I08 surfactant molecule governs the concentration of pluronic" F I 08 molecules that
will adsorb onto a given hydrophobic surface. If the maximum coating concentration of
plutonic" FI08 is known, one can assume that the maximum coating concentration of any
pluronic derivative, with the same PPO centre block size, will be the same. Adsorption studies
were carried out, the Langmuir adsorption isotherm was determined, and subsequently the
fractional coating was calculated.
The end-groups of plutonic" FI08 were modified as follows and the substituted pluronic was
adsorbed onto a membrane that was to act as the solid support matrix in the development of an
affinity system: Amino pluronic was synthesised by first tosylating pluronic" FI08, followed by azidation with NaN3 then reduction with LiAI~. The synthesised amino pluronic was then
biotinylated using N-hydroxysuccinimide biotin ester. The suitability of this synthetic route was
first assessed on a model compound, 2-methoxyethylamine, and validated by NMR (Nuclear
Magnetic Resonance) spectroscopy. The synthetic protocol was then used to derivatise the larger
pluronic molecule.
The affinity system was tested on two different hydrophobic surfaces: polystyrene and
polysulphone membranes (PSMs). Avidin-conjugated horseradish peroxidase was obtained and
used to interact with the immobilised biotin. The enzymatic reaction of the coupled peroxidase
converted the substrate, 2, 2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) to a
coloured product. The colour developed is proportional to the amount of biotin that was
immobilised on the hydrophobic surfaces studied.
Non-covalent immobilisation of the synthesised biotin-pluronic molecule was successfully
obtained onto the hydrophobic polystyrene as well as the polysulphone membrane surfaces. / AFRIKAANSE OPSOMMING: Vooruitgang in die farmakologie, biochemie en biotegnologie word al meer afhanklik van
affiniteits chromatografie as die verkose tegniek vir die suiwering en karaterisering van
spesifieke biomolekules.
Oor die afgelope jare het die avidien-biotien tegnologie homself as baie bruikbaar bewys in die
mediese, farmakologiese, biologiese en biochemiese velde. Toepassings waar die avidien-biotien
kompleks betrokke was sluit in die toepassing as 'n mediator vir affiniteits chromatografie,
affiniteits sitologie, immuno bepalings, histopatologie, bioaffiniteits sensors sowel as
kruisbinding en immobiliserings studies en vele meer.
Die hoofrede vir die gewildheid van die avidien-biotien kompleks en die groeiende bruikbaarheid
in die biotegnologie is die buitengewone hoë affiniteit (l015 M-I
) en stabiliteit van die
nie-kovalente interaksie tussen avidien en biotien. Die toepassingsveld van die avidien-biotien
kompleks word wyer met die verskeidenheid biotien derivate en avidien-bevattende konjugate
wat kommersiëel beskikbaar is.
Die doel van die werk wat hier gedokumenteer word is om die bruikbaarheid van Plutonic" FI08
en die avidien-biotien kompleks, vir gebruik in 'n affiniteits chromatografie sisteem, te evalueer.
Om hierdie doel te bereik is die adsorpsie van Pluronic" FI08 aan hidrofobiese polisulfoon
membraan oppervlaktes bestudeer. Die eksperimentele data wat gegenireer is, is gebruik om die
teoretiese maksimum hoeveelheid Pluronic wat per eenheids oppervlakte membraan adsorbeer te
bepaal. Dit is reeds bekend dat die polipropileen (PPO) middel blok van die Pluronic emulgant
die konsentrasie van die geadsorbeerde Pluronic molekules op 'n gegewe hidrofobiese
oppervlakte bepaal. Indien die maksimum bedekkingskonsentrasie VIr maksimum
oppervlakbedekking van Plutonic" FI08 bekend is, kan teoreties aanvaar word dat die
bedekkingskonsentrasie vir enige Pluronic derivaat met dieselfde grootte PPO blok dieselfde sal
wees. Adsorpsiestudies was uitgevoer om die Langmuir adsorpsie isoterm te bepaal.
Daaropvolgend was die fraksionele bedekking bereken. Amino-pluronic was gesintetiseer deur die eindpunte van Pluronic te derivatiseer. Hierdie
Pluronic derivaat was gevolglik geadsorbeer aan 'n membraan wat gedien het as die soliede
oppervlakte vir die ontwikkeling van 'n affiniteits chromatografie sisteem.
Amino-pluronic was gesintetiseer deur Pluronic eers te tosileer en daarna te asideer met NaN3 en
laastens te reduseer met LiAI~. Die produk was gebiotinileer deur gebruik te maak van
N-hidroksisuksinimied-biotien-ester. Die bruikbaarheid van hierdie sintetiese roete is eers bepaal
deur van 'n model verbinding, 2-metoksiëtielamien, gebruik te maak en dit met behulp van KMR
(Kern Magnetiese Resonans) spektroskopie te karakteriseer.
Die affiniteits sisteem is getoets op twee verskillende hidrofobiese oppervlaktes naamlik
polistireen en polisulfoon membraan oppervlaktes. Avidien gekonjugeerd met 'n peroksiedase
ensiem is gebruik om met die geïmmobiliseerde biotien te assosieer. Die ensiematiese reaksie
van die gekoppelde peroksiedase het die substraat
2, 2' -azino-di-(3-etiel-benzthiazolien-6-sulfoonsuur) (ABTS) omgesit na 'n gekleurde produk,
waar dit teenwoordig is. 'n Reeks wasstappe is gebruik om die gemodifiseerde peroksidase
ensiem wat nie aan die hidrofobiese oppervlakte gekoppel nie, weg te spoel. Hierdeur is die mate
van binding aan die hirofobiese oppervlakte gekwantifiseer deur die kleur te kwantifiseer wat
ontwikkelomdat die kleurontwikkeling direk proporsioneel is aan die hoeveelheid peroksidase
wat nog aan die membraan gekoppel is.
Nie-kovalente immobilisasie van die gesintetiseerde biotien-pluronic molekule is suksesvolop
beide die hidrofobiese polistireen oppervlakte sowel as die polisulfoon membraan verkry.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53522 |
| Date | 03 1900 |
| Creators | Liebenberg, Liesl Eileen |
| Contributors | Swart, P., Jacobs, E. P., Bredenkamp, M. W., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 95 pages : illustrations |
| Rights | Stellenbosch University |
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