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Mike has determined that enzyme he is attempting to purify has an isoelectric point of 4.5 (pI = 4.5). He has decided to examine anion exchange chromatography as a potential purification step. He tested out using 2 different buffer and linear NaCl gradient on HPLC (like what you did). His results are shown below. Which buffer should Mike uses for his purification (both buffer has pH higher than enzyme’s pI)? Why?

Mike has determined that enzyme he is attempting to purify has an isoelectric point of 4.5 (pI = 4.5). He has decided to examine anion exchange chromatography as a potential purification step. He tested out using 2 different buffer and linear NaCl gradient on HPLC (like what you did). His results are shown below. Which buffer should Mike uses for his purification (both buffer has pH higher than enzyme’s pI)? Why?

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
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Mike has determined that enzyme he is attempting to purify has an isoelectric point of 4.5 (pI = 4.5). He has decided to examine anion exchange chromatography as a potential purification step. He tested out using 2 different buffer and linear NaCl gradient on HPLC (like what you did). His results are shown below.

Which buffer should Mike uses for his purification (both buffer has pH higher than enzyme’s pI)? Why?

The image displays a line graph titled "pH 5 data," illustrating experimental results for absorbance and enzyme activity across different fractions. ### Graph Details: - **X-Axis (Fraction Number):** - Marked from 1 to 30, representing different fractions collected during the experiment. - **Y-Axis:** - Range from 0 to 2.5. - Represents two different metrics: - Absorbance at 280 nm (A280) - Enzyme activity. ### Data Representations: - **A280 (Absorbance):** - Indicated by a line with diamond-shaped markers. - Peaks around fraction numbers 3 and 19. - **Enzyme Activity:** - Represented by a line with square markers. - Shows a significant increase between fractions 2 to 6, followed by a peak around fraction number 19. - Activity diminishes toward the later fraction numbers. ### Interpretation: This graph provides insight into how the protein concentration (as indicated by A280) and enzyme activity vary across the collected fractions at pH 5. Noticeable peaks indicate fractions where enzyme activity and protein concentration are at their highest.
Transcribed Image Text:The image displays a line graph titled "pH 5 data," illustrating experimental results for absorbance and enzyme activity across different fractions. ### Graph Details: - **X-Axis (Fraction Number):** - Marked from 1 to 30, representing different fractions collected during the experiment. - **Y-Axis:** - Range from 0 to 2.5. - Represents two different metrics: - Absorbance at 280 nm (A280) - Enzyme activity. ### Data Representations: - **A280 (Absorbance):** - Indicated by a line with diamond-shaped markers. - Peaks around fraction numbers 3 and 19. - **Enzyme Activity:** - Represented by a line with square markers. - Shows a significant increase between fractions 2 to 6, followed by a peak around fraction number 19. - Activity diminishes toward the later fraction numbers. ### Interpretation: This graph provides insight into how the protein concentration (as indicated by A280) and enzyme activity vary across the collected fractions at pH 5. Noticeable peaks indicate fractions where enzyme activity and protein concentration are at their highest.
**pH 8 Data** This graph displays the relationship between fraction numbers and two variables: A280 absorbance and enzyme activity at pH 8. - **X-axis (Fraction number):** Numbers ranging from 1 to 30 representing different fractions collected during an experimental phase. - **Y-axis:** Measures the levels of two variables: - **A280 (diamond markers):** Represents absorbance at 280 nm, commonly used to measure protein concentration. - **Enzyme Activity (square markers):** Indicates the level of enzymatic activity in each fraction. - **Data Points:** - **A280:** Peaks appear at fraction numbers 1, 3, 6, 11, 16, 21, and 26, indicating higher protein concentration at these points. - **Enzyme Activity:** A significant peak is observed at fraction number 27, suggesting peak enzymatic activity in this fraction, with lower activities around it. This graphical representation helps visualize where maximum protein concentration and enzyme activity occur, highlighting fraction 27 as having the highest enzymatic activity under the given conditions (pH 8).
Transcribed Image Text:**pH 8 Data** This graph displays the relationship between fraction numbers and two variables: A280 absorbance and enzyme activity at pH 8. - **X-axis (Fraction number):** Numbers ranging from 1 to 30 representing different fractions collected during an experimental phase. - **Y-axis:** Measures the levels of two variables: - **A280 (diamond markers):** Represents absorbance at 280 nm, commonly used to measure protein concentration. - **Enzyme Activity (square markers):** Indicates the level of enzymatic activity in each fraction. - **Data Points:** - **A280:** Peaks appear at fraction numbers 1, 3, 6, 11, 16, 21, and 26, indicating higher protein concentration at these points. - **Enzyme Activity:** A significant peak is observed at fraction number 27, suggesting peak enzymatic activity in this fraction, with lower activities around it. This graphical representation helps visualize where maximum protein concentration and enzyme activity occur, highlighting fraction 27 as having the highest enzymatic activity under the given conditions (pH 8).
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