Extraction and Characterization of Proteins
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)
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)
References: http://www.glue.umd.edu/~NSW/ench485/lab6a.htm http://matcmadison.edu/biotech/resources/proteins/labManual/chapter_4/section4_2.htm http://www.science.smith.edu/departments/Biochem/Biochem_353/Bradford.html http://www-class.unl.edu/biochem/protein_assay/bradford_assay.htm