Extraction and Characterization of Proteins

Topics: Protein, Amino acid, Solubility Pages: 12 (3647 words) Published: February 5, 2011


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 based on Bradford Assay. In general, the assay indicates that better extraction and purification techniques should be employed. Appropriate quantification techniques should also be used to obtain more accurate results.

Discussion of Data and Results

Proteins are a class of nitrogen-containing organic compounds considered as one of the basic functional units essential to life. Among its many roles include the transport of neutral molecules, ions and electrons such as hemoglobin that transports oxygen; the formation of biological materials such as muscle, hair and bone; or the regulation of activity by binding to other proteins. In this experiment, one of the objectives is to isolate various proteins from known rich sources. The experiment involved the extraction of invertase from baker’s yeast, albumin from fresh egg white, and casein from milk. Spectrophotometric methods were then applied in the characterization and quantification of casein and albumin.

Invertase splits sucrose into glucose and fructose (invert syrup) and can be applied to improve the shelf life of confections. Albumin (ovalbumin) is a globular glycoprotein which is the major component of egg white. It has an isoelectric point of 4.6. Casein is the major protein in bovine milk, constituting about 80% (w/w) of the total protein. The principal forms of casein (α- and β-casein) are phosphoproteins with the phosphate being present mainly in the form of O-phosphoserine residues. Its isoelectric point is 4.6. (Boyer, 2000)

The isolation of proteins from natural sources often requires disruption of the cell membranes in a suitable medium to release the cell contents. To separate proteins from other cellular components, several techniques can be used such as centrifugation, salting out, isoelectric precipitation, or precipitation using organic solvents or acidic protein precipitants. To purify proteins further, various techniques can be employed like column chromatography, density gradient centrifugation, electrophoresis and enzymatic digestion. To isolate and purify a certain protein, one or several of the techniques mentioned may be used, depending on the type of the impurities and the protein to be purified. The protein in its purified form is obtained and stored in crystalline form. (Boyer, 2000)

The solubility behavior of proteins is largely affected by the ionic strength. In general, this behavior is described by a curve like the one below. As the ionic strength increases, protein solubility increases at first. This is refered to as “salting in”. Although, beyond a certain point, the solubility begins to decrease and this is known as “salting out”. (Boyer, 2000)

Figure 1. Solubility of proteins

If the ionic strength is low, the activity coefficients of the ionizable groups become surrounded by counter-ions which prevent the interactions between the ionizable groups. Hence, the interactions between protein molecules are decreased and the solubility is increased (in general, a solute is soluble whenever solute-solvent interactions exceed solute-solute interactions). If the ionic strength is high, the activity coefficients of the ionizable groups of the protein are increased such that their...
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