Question

In the course of purifying an enzyme, a researcher performs a purification step that results in an increase in the total activity to a value greater than that present in the original crude extract. Explain how the amount of total activity might increase.

Short answer

Total activity might increase due to removal of inhibitors or increased enzyme concentration.

Step-by-step solution

Understanding Total Activity

Total activity of an enzyme is calculated by multiplying the enzyme activity per unit volume by the total volume. This gives an overall measure of enzyme activity present in the entire sample.

Activity Calculation in Crude versus Purified Samples

In the crude extract, enzyme activity can be diluted by the presence of non-enzymatic proteins and other substances. After purification, these contaminants are reduced, potentially increasing specific activity because the enzyme becomes more concentrated.

Impacts of Purification Techniques

Certain purification techniques can change the enzyme conformation slightly, exposing more active sites, increasing its activity. Also, removal of inhibitors present in the crude sample can help restore or even boost the enzyme's activity.

Accounting for Activity Increase

During purification, inhibitors that were present in the crude extract might have been removed. This removal can result in the enzyme's true activity being realized, causing an apparent increase in total activity post-purification.

Key concepts

Total Activity

Total activity is a concept used to quantify the overall enzymatic activity within a sample. It's calculated by multiplying the enzyme activity per unit volume by the total volume of the enzyme solution. This calculation gives a comprehensive measure of the enzyme's potential to catalyze reactions in the entire sample, regardless of its concentration or purity level.
The total activity provides insights into how much enzyme is present and active in a given sample. During enzyme purification, the total activity can sometimes increase. This could be due to the removal of substances that were inhibiting the enzyme's activity, revealing the enzyme's full catalytic potential that wasn't initially detected in the crude extract.

Specific Activity

Specific activity is a crucial measure in the enzyme purification process. It is defined as the enzyme activity per amount of protein. In mathematical terms, specific activity can be expressed as:\[\text{Specific Activity} = \frac{\text{Enzyme Activity}}{\text{Total Protein Content}}\]This value provides a good indication of the enzyme's purity. Higher specific activity means that there is more enzyme activity per unit of protein, indicating less contamination from non-enzymatic proteins.
During purification, specific activity often increases because impurities and other proteins are removed. Consequently, the proportion of active enzyme molecules in the sample increases. This is reflected in the specific activity, even if the total protein content in the sample decreases. It's important to track specific activity to ensure that the purification steps are effective.

Purification Techniques

Enzyme purification involves separating the desired enzyme from other proteins and impurities. Effective purification techniques are crucial to isolate the enzyme while preserving its activity. Some commonly used purification methods include:

• Precipitation: Involves adding salts or solvents to selectively precipitate the enzyme out of solution.
• Chromatography: Techniques such as ion exchange, affinity, and gel filtration are used to separate enzymes based on size, charge, or binding affinity.

These techniques not only concentrate the enzyme but can also enhance activity by removing inhibitors. They might even induce slight conformational changes in the enzyme structure, potentially exposing more active sites. This can lead to greater enzymatic activity in the purified sample compared to the crude extract.

Enzyme Inhibitors

Enzyme inhibitors are substances that decrease the activity of enzymes. Presence of inhibitors in a crude extract can significantly reduce observed enzyme activity. These inhibitors might be proteins, ions, or small organic molecules that interfere with the enzyme's active site or overall structure.
During purification, inhibitors might be removed, leading to an increase in total activity of the enzyme. This can happen because the enzyme can now perform its function without hindrance. It's essential to identify and remove inhibitors to ensure the true functionality of the enzyme is measured and maintained over the course of purification. Understanding these impacts can help in designing effective enzyme assays and purification protocols.