Extraction and Characterization of Proteins

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Abstract

The activity of invertase and the quantification of albumin and casein were performed and analyzed after extraction of the said proteins from their respective sources. Isolation of proteins was initiated by the breakage of the cell wall / membranes in three different ways. Homogenization of invertase, albumin and casein were achieved via grinding process, addition of 1M acetic acid and acidification by 0.1M hydrochloric acid correspondingly. Extraction of invertase and casein involved precipitation through the utilization of 95% ethanol. The obtained crude extracts for invertase and casein were 0.86 g and 0.9092 g respectively. In contrast, albumin’s isolation depended on its solubility in saturated ammonium sulfate salt. Two trials were performed and the resulting weights of the precipitates were 19.32 g. for precipitate 1 and 2.80 g. for precipitate 2. The activity of invertase was ascertained through Benedict’s Test. Test tubes containing either a combination of glucose and fructose or sucrose exhibited an orange coloration—indicative of a weak positive result. Quantification processes for albumin were determined using Warburg-Christian and Bradford Assays. Both method involved spectrophotometry. The resulting protein concentrations in Warburg-Christian were 1.0042 mg/ml for precipitate 1 and 0.7427 mg/ml for precipitate 2. The Bradford Assay for precipitate 1 (test tubes 7, 8 and 9) yielded 7.47, 8.97 and 8.28 μg/ml and for precipitate 2 (test tubes 8 and 9) gave 6.44 and 4.73 μg/ml in that order. Casein’s concentration was deduced through the Warburg-Christian Method yielded 0.265 mg/ml.

Discussion of Data and Results

Invertase—also known as sucrase, saccharase or β-fructofuranosidase—is an efficient enzyme in the hydrolysis of sucrose. Despite abundance in molds, bacteria, plants and higher animals; the most common source of invertase has remained to be yeast, in which “invertase exists in more than one form—the lightweight (135 kDa) intracellular invertase enclosed within the rigid cell wall and the heavyweight (270 kDa) extracellular variety that coats the outside of the cell.” It is because of the existence of the rigid cell wall separating the two types of invertases that sand has to be mixed during the grinding process to ensure breakage of wall and to obtain a large amount of isolated invertase. The addition of hexane creates a non-polar environment for the enzyme as well as for other intracellular components. Subsequent pouring of water in portions not only introduces a polar medium but also minimizes the interference of proteases, species which cleave peptide bonds within polypeptide chains (endopeptidases) or sequentially digest amino acids from the C or N terminus (exopeptidases), on the isolation of invertase. Cheesecloth filtration, repetitive centrifugation and discarding of sediments are performed afterwards to remove unwanted components such as the added sand or the ruptured cell walls. The precipitation of invertase is made possible by immersion of the final supernatant in an ice bath and pouring of 95% ethanol. The method is based fundamentally on the principle regarding dielectric constants. Upon introduction of the alcohol, “the dielectric constant is lowered and the attractions between the ionizable groups of the enzyme are increased. Consequently, interactions among the proteins present are lessened and the solubility of the invertase in the medium is decreased.” Centrifugation is again employed to serve a similar purpose. However, instead of discarding the sediments as before, it is the supernatant that must be removed as most, if not all, of the invertase species in the solution are assumed to have undergone complete precipitation. Lastly, the precipitates are both weighed and analyzed of the physical properties (Experimental Results: weight = 0.85 g and physical properties = white powdery precipitate) before dissolving in 0.1M acetate buffer with pH 5 as the enzyme is...
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