Problem 16 - Analytical Chemistry

Question

The activity of a glucose oxidase preparation is determined by measuring the volume of oxygen gas consumed as a function of time. A 10.0 -mg fraction of the preparation is added to a solution containing \(0.01 M\) glucose and saturated in oxygen. After 20.0 min, it is determined that 10.5 mL oxygen is consumed at standard temperature and pressure (STP). What is the activity of the enzyme preparation expressed in enzyme units per milligram? If the purified enzyme has an activity of 61.3 units/mg, what is the percent purity of this enzyme preparation?

Step-by-Step Solution

Answer

The enzyme activity is 0.0526 units/mg, and the purity is approximately 0.086%.

Step 1: Understand Enzyme Unit Definition

An enzyme unit is defined as the amount of enzyme that catalyzes the conversion of 1 micromole (\(\mu mol\)) of substrate per minute under specific conditions. At standard temperature and pressure (STP), 1 mole of gas occupies 22.4 liters, which means 1 \(\mu mol\) is 22.4 mL/1,000,000.

Step 2: Convert Oxygen Consumption to Micromoles

The oxygen consumption is 10.5 mL in 20 minutes. To find the micromoles, use the conversion: \(\frac{10.5 \, \text{mL}}{22.4 \, \text{L/mol}} \times 1000 = 10.521 \mu mol\).

Step 3: Calculate Enzyme Activity in Units

Activity is calculated as micromoles consumed per minute. Given 10.521 \(\mu mol\) is consumed in 20 minutes: \(\text{Activity} = \frac{10.521 \, \mu mol}{20 \, \text{min}} = 0.526 \mu mol/\text{min}\).

Step 4: Calculate Specific Activity

Specific activity is expressed as enzyme units per milligram. Given the use of 10 mg of enzyme, the specific activity is: \(\text{Specific Activity} = \frac{0.526 \mu mol/\text{min}}{10 \, \text{mg}} = 0.0526 \text{ units/mg}\).

Step 5: Calculate Percent Purity

Compare the observed specific activity with that of the purified enzyme (61.3 units/mg). The percent purity is: \(\text{Percent Purity} = \left(\frac{0.0526 \, \text{units/mg}}{61.3 \, \text{units/mg}}\right)\times 100\% \approx 0.086\%\).

Key Concepts

Glucose Oxidase

Glucose oxidase is an enzyme that plays a significant role in the oxidation of glucose to gluconic acid and hydrogen peroxide. It is commonly used in various biotechnological applications because of its ability to effectively catalyze this reaction. Often found in fungi, particularly in Aspergillus niger, glucose oxidase is extensively used in the food and pharmaceutical industries.
Its application extends to glucose monitoring in clinical diagnostics, where it helps in the determination of blood glucose levels. The enzyme's activity is crucial for such uses, as the amount of product formed per unit time will determine the efficacy of these applications. Additionally, it is used within the paper industry where its bleaching properties help in paper processing. Understanding this enzyme's kinetics is important when evaluating its application potential and effectiveness.

Enzyme Activity

Enzyme activity refers to the number of substrate molecules converted into product per enzyme molecule per unit time, measured in enzyme units. It indicates how effective an enzyme is in catalyzing a reaction under specific conditions.
For glucose oxidase, enzyme activity can be measured by observing the rate of oxygen consumption, as oxygen is consumed when glucose is converted into gluconic acid. This activity is expressed in terms of extit{micromoles of substrate converted per minute}.
Knowing enzyme activity allows scientists and technicians to compare different enzyme preparations and assess their effectiveness in catalyzing reactions. This information is crucial for processes like fermentation and clinical diagnostics where consistent enzyme performance is necessary.

Specific Activity

Specific activity is a crucial parameter when evaluating enzyme preparations. It refers to the enzyme activity per milligram of protein, typically expressed in units/mg. This measure helps to determine the purity of an enzyme preparation, where higher specific activity indicates a purer enzyme.
In our example, specific activity was calculated by dividing the enzyme activity by the weight of the enzyme used in the reaction. This calculation helps in determining how effective a particular amount of enzyme preparation is, indicating the presence of active enzyme versus inactive components.

• It aids in standardizing experiments.
• Allows comparison between batches or different enzyme preparations.
• Important in assessing enzyme purity and effectiveness.

Percent Purity

Percent purity is an indicator of how much of the enzyme preparation is active enzyme. It gives insight into the quality of the enzyme preparation being used, where comparing the specific activity of a sample to that of a purified enzyme determines purity.
Calculating percent purity shows what fraction of the enzyme preparation is the desired enzyme versus other proteins or impurities. In the provided solution, a specific activity much lower than that of the purified enzyme indicates low percent purity, suggesting the presence of impurities or inactive enzymes.

• Calculating percent purity involves comparing specific activities.
• Helps in quality assurance and control.
• Vital in applications that require high enzyme specificity.

Understanding and improving the percent purity could lead to more effective usage of enzymes in industrial and laboratory settings.