11. As part of a research project in biochemistry you are charged with purifying
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11. As part of a research project in biochemistry you are charged with purifying a dehydrogenase enzyme from a species of bacteria. Your advisor informs you that four isoforms of enzyme exist in the cell: three soluble isoforms and a membrane-associated isoform. Two of the soluble isoforms occur naturally as monomers while the other functions as a homo-tetramer. Analysis of the genes for the monomeric forms indicates significant differences in their respective amino acid compositions, most notably in the percentages of the following amino acids (Glu, Asp, Thr). Generate an outline of the purification scheme you would utilize to isolate the more soluble of the monomeric enzyme isoforms. For each methodology employed provide a chemically based rationale for its inclusion, i.e., briefly explain how each step gets you closer to a pure solution of the desired enzyme. (10) a.Explanation / Answer
Cell biomass collection (temperature at 4°C), cell extract preparation The initial step in purification is a separation of soluble proteins from insoluble cellular material by differential centrifugation After centrifugation, the soluble proteins remain in the supernatant. The supernatant fraction still contains a mixture of enzymes then subject it to further purification methods. Liquid chromatography: The HPLC system, have a Q-Sepharose fast-flow column with UV/VIS detector Collection of fractions and check enzyme activity Perform dialysis to concentrate the desired product Native Gel Electrophoresis (Native-PAGE): Dissolved molecules in an electric field move, or migrate, at a speed determined by their charge: mass ratio. Example, if two molecules have the same mass and shape, the one with the greater net charge will move faster toward an electrode. (In the present case there is variation in amino acid composition). (Native-PAGE - mobility depends on both the protein's charge and its hydrodynamic size) Affinity chromatography: Here ligands can be substrates of an enzyme or other small molecules that can bind to the specific proteins. Then collection of isoforms Then, go for two-dimensional Gel-electrophoresis: In this case, purification of isoenzyme is being performed isoforms have similar function but different amino acid composition. Therefore, they will differ in the acidic and basic group and they will differ in charge. First, they will be separated by charge then followed by on the basis of mass
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