Activity assays were performed to determine the best purification method for a cell-free extract of Protein X. The results of the assays are shown below. Based on the data, what was the highest purification yield of Protein X?

Question

Activity assays were performed to determine the best purification method for a cell-free extract of Protein X.  The results of the assays are shown below.  Based on the data, what was the highest purification yield of Protein X?

Quizplus · Accepted Answer

11ecba2d_0e32_87df_a3bf_5300199e2bf7_MD0008_00 Extracts from cells are complex mixtures that contain many different proteins, lipids, and carbohydrate molecules. To isolate a specific enzyme, purification methods such as native gel electrophoresis, precipitation, and column chromatography are needed. The effectiveness of each purification method or step is confirmed by performing activity assays that measure the rate of the reaction catalyzed by the enzyme of interest. The activity values in an assay are measured in arbitrary units (u) that reflect the mass of the target enzyme (mg). Protein purity is represented as specific activity (u/mg), which is a ratio of the activity of the target protein and the total protein obtained in a purification step. Consequently, specific activity increases during purification as unwanted protein is removed and becomes constant when only the pure enzyme remains in solution. The yield achieved by a purification step is determined by calculating the ratio of total activity of the purified protein to the unpurified extract. Total activity is calculated as: 11ecba2d_0e32_87e0_a3bf_c195990f7360_MD0008_00 Total activity (TA)=total proteinmg×specific activity (umg) 11ecba2d_0e32_aef1_a3bf_d3c88f55fbb5_MD0008_00 TA of unpurified extract=800 mg×0.5 umg=400 u Therefore, the purification step with the greatest total activity would also have the highest yield. The total activity of the target enzyme was highest with the alcohol precipitation method: 11ecba2d_0e32_aef2_a3bf_23a1dca65353_MD0008_00 TA of alcohol precipitation=200 mg×1.0umg=200 u The ratio of purified protein activity to unpurified protein activity is: 11ecba2d_0e32_aef3_a3bf_cd46da7d7d2b_MD0008_00 Yield %=TA of purified proteinTA of unpurified extract×100 11ecba2d_0e32_d604_a3bf_eff760636b50_MD0008_00 Yield %=200u400u×100=0.5 ×100=50% Therefore, a maximum of 50% of Protein X was purified from the cell-free extract. (Choice A) This value is the specific activity achieved with the unpurified extract. (Choices B and D) These values represent the ratios of total purified protein to unpurified protein obtained with alcohol precipitation and column chromatography, respectively. Yield is determined by calculating ratios of total activity instead of total protein. Educational objective: Specific activity (u/mg), which reflects protein purity, is the ratio of the activity of the target protein and the total protein. The total activity is the product of the total protein and the specific activity. Yield is determined by dividing the total activity of the purified protein by the total activity of the unpurified extract.

Activity Assays Were Performed to Determine the Best Purification Method