Extraction and Characterization of Proteins

3660 Words Feb 18th, 2011 15 Pages
EXTRACTION AND CHARACTERIZATION OF PROTEINS

Abstract

Different techniques and principles for protein extraction and characterization were demonstrated in this experiment. Various proteins were extracted from different sources: 1.67 g yeast invertase, 1.03 g egg white albumin, and 5.15 g of milk casein. Activity assay for invertase was performed using Benedict’s test and the enzymes inverting action on sucrose was confirmed. Warburg-Christian Method and Bradford Assay were also employed to determine the protein concentration in the albumin and the casein samples. The concentrations for the albumin and casein samples were found to be 0.519 and 0.327 mg/mL, respectively based on Warburg-Christian Assay; and 6.5x10-3¬ and 1.9x10-2 mg/mL
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As a result, the interactions between protein molecules exceed protein-water interactions, and the solubility decreases. Moreover, the differences in the amino acid sequence makes proteins differ in their salting in and salting out behavior. This is the basis for the fractional precipitation of proteins by means of salt. Most commonly used salt is ammonium sulfate because it is available in highly purified form and because of its high solubility. (Boyer, 2000)

Proteins can also be fractionally precipitated by adjusting the pH, temperature and dielectric constant. Variations in pH also change the state of ionization of the functional groups, and hence, the net charge of the protein. Usually, the protein’s solubility is at its least at the isoelectric point pI and increases on either side of the pI. At the pI, the net charge of the protein is zero and the protein molecules do not repel each other. As a result, protein-protein interactions are increased and the solubility is at its lowest. (Boyer, 2000)

Meanwhile, if temperature is increased denatures the proteins. Proteins then unfold and the non-polar groups which were previously in the interior of the molecule become exposed. This leads to a decrease in the solubility of the protein in aqueous environment. Addition of ethanol, methanol, acetone and the like decreases the dielectric constant and thus decreases protein’s solubility. (Boyer, 2000)

The

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