Department of Biochemistry and Molecular Biology

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Department of Biochemistry

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Browsing Department of Biochemistry and Molecular Biology by Subject "chromatography"

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    Purification and Characterisation of Lectin from Bowringia Miladbraedii Harms Seeds
    (Obafemi Awolowo University, 1985) Alakija, Augusus Adegbolamiyo Olufemi; Animashaun, T.; Aboderin, A.A.
    Lectin was extracted from Bowringia mildbraedii Harms seeds and shown to agglutinate red blood cells nonspecifically. The chromatography using different grades of Sephadex did not give a very good separation of the lectin. Purification of the lectin by Sepharose 68 chromatography followed by metal chelate affinity chromatography was compared with purification by Sepharose 6B followed by ion-exchange chromatography using DEAE cellulose. The latter method was preferred and yielded a protein which behaved as a single protein on polyacrylamide gel electrophoresis. It was deduced that the lectin occured as a tetrameric protein with two subunits A and B having approximate molecular weight of 14,000 and 16,000 respectively from experiments with SOS-PAGE in the presence of 2, β-mercaptoethanol. The molecular weight of the lectin is approximately 60,000. The B. mildbraedii lectin precipitated Afzelia africana polysaccharide with remarkable specificity and failed completely to form precipitin bands in agargel double diffusion plates with other polysaccharides tested even at varying concentrations. The haemagglutinating and polysaccharide precipitating activity of the lectin is appreciably inhibited by µ-methylD-mannoside, D-mannose and D-glucose.
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    Purification and Characterization of Lectins from Abrus Species.
    (Obafemi Awolowo University, 1985) Caiquo, Aspect Wallace Kyemenu; Animashaun, T.
    Saline extracts prepared from Abrus precatorius and Abrus fructiculosus seeds agglutinated red blood cells. The agglutination was inhibited by D-galactose and lactose. The lectins bound to Sepharose 6B and were eluted with D-galactose. These lectins were separated into an agglutinin and a toxin by chromatography on Diethyl amino ethyl (DEAE) cellulose. The toxin from Abrus fructiculosus seed was named fructin' to distinguish it from Abrus precatorius seed toxin, abrin. In sodium dodecyl - polyacrylamide gel electrophoresis, (SDS-PAGE) fructin and abrin gave single bands with molecular weights (proposed) 60,320 and 62,500 respectively. After treatment with mercaptoethanol, fructin and abrin were split into 2 bands each with molecular weights 32,360 and 30,200 (fructin), 36,520 and 28,800 (abrin). The agglutinins gave 2 bands each in SDS-PAGE with corresponding molecular weights of 57,480 and 55,650 (A. fructiculosus agglutinin) and 59,600 and 56,380 (A. precatorius agglutinin). The proposed native molecular weights are 113,130 (. fructiculosus agglutinin) and 115,980 ( A. precatorius agglutinin), Alen treated with SDS and f - mercaptoethanol, the agglutinins were split into 3 bands each with corresponding agar molecular weights 38,460, 33,500 and 28,180 (,. fructiculosus agglutinin) and 40,790, 35,590 and 31,260 (A.precatorius agglutinin). The saline extracts from the two seeds did not interact with Afzelia africana polysaccharide in agar gel double diffusion studies. The toxicity of fructin and A. fructiculosus agglutinin was established on mice, with fructin being about 150 times more toxic than the agglutinin. Antisera formed against abrin and A. precatorius agglutinin did not interact with saline extracts of A. fructiculosus, fructin and A. fructiculosus agglutinin.
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    Synthesis and Characterization of Poly-8-Methylamino-Adenylic.
    (Obafemi Awolowo University, 1987) Elekwa, Iheanyichukwu; Falayan, J.O.
    A polynucleotide, poly-8-methylamino adenylic acid, was synthesized and hybridized with poly U. Adenosine-5-monophosphate purchased from BDN Chemical Corporation was brominated with bromine in Na-acetate buffer pH 3.9, stirred for 24 hours. The excess bromine was removed with some drops of concentrated sodium sulphite solution. It was then evaporated to dryness, on a rotatory evaporator at 37 oC and characterised. The product 8-Bromo-adenosine-5-Monphosphate, was methylaminated with methylamine in water, and refluxed for 48 hours at 65oC on an oil bath. Excess methylamine was removed with dilute sulphuric acid, while the product was evaporated to dryness and characterized. The synthesized methylamino adenosine-5-monophosphate was converted to its diphosphate analogue by the phophoro-morpholidate process. Orthophosphoric acid was used to introduce the phosphate group. The converted methylamino-adenosine-5-diphosphate was polymerized with the enzyme polynucleotide phophorylase PNPase, (EC 2.7.7.8) from Micrococcus luteus that was purchased from Boehringner Chemical Corporation. This enzyme PNPase is only specific for the diphosphate form. The polymerization was carried out in a reaction medium of MgCl2.6H2O EDTA (Ethylene diaminetetra-acetic acid) and tris buffer pH 9.0. It was incubated for 21 hours at 36oC, and characterized. The Poly-8-methyamino adenylic acid so synthesized was hybridized with poly U purchased from BDH- Chemical Corporation. The Job’s continuous variation method in KH2PO4 + K2HPO4 buffer pH 7.00 at 37oC was adopted. The polymer formed at 1:1 hybrid with Poly U. This satisfies some of the requirements for synthetic polynucleotide inducers. Some physico-chemical methods used in the characterization, included infra-red, ultra-violet, proton nuclear magnestic resonance and chromatographic methods. Complete UV spectra were recorded in the range of 200-400nm on Sp 8-400 (uv/vis) spectrophotometer. The infra-red spectra recorded using a sodium chloride cell in the range of 200-4000 cm-1 on Sp 3 -300 infra-red spectrophotometer. And, the Proton Nuclear Magnetic Resonance was recorded on Perkin Elmer Spectrophometer. The spots on the paper chromatography were detected and developed under UV light (at 254 nm wavelength).