Praktikum Biokimia Farmasi : Penentuan Aktivitas Spesifik Enzim

Summary

This script discusses the laboratory practice of determining specific enzyme activities, focusing on glutathione S-transferase (GST). It covers the preparation of liver cytosolic fractions from rats, the measurement of enzyme activity using CDNB as a substrate, and protein concentration using the Biuret method. The script also explains the importance of measuring enzyme activity and protein concentration in pharmacology, particularly in the context of detoxification and the potential targeting of GST in cancer therapy.

Takeaways

• 🔬 The lab session focuses on determining the specific activity of the enzyme Glutathione S-Transferase (GST) and measuring protein concentration using the Biuret method.
• 🧬 Proteins are polymers made up of amino acids as monomers, with 20 different types of amino acids that can be polar, nonpolar, charged, or uncharged.
• 📚 Quantitative analysis of proteins can be done through various methods including titration, spectrophotometry, and biuret reaction which is highlighted in the script.
• 🌡 The Biuret method involves the reaction of peptide chains with the biuret reagent, resulting in a purple complex that can be measured at a wavelength of 560 nanometers.
• 📉 The Biuret method has its advantages such as being quick and simple, but also has limitations like low sensitivity due to not all protein groups reacting positively.
• 🛠 GST is an enzyme involved in phase 2 metabolism, conjugating glutathione with various substrates, including xenobiotics, which can be detoxified in the body.
• 🐀 The lab procedure involves preparing a cytosolic fraction containing GST from rat liver, which is then used to determine enzyme activity using CDNB as a substrate.
• 📊 A standard curve using BSA (Bovine Serum Albumin) is created to measure protein concentration in the cytosolic fraction using the Biuret method.
• ⚗️ The specific activity of the enzyme is calculated by dividing the reaction rate (micromoles of product per minute) by the protein concentration in the cytosolic solution (milligrams per milliliter).
• 💊 GST's role in detoxification is significant in the field of pharmacology, especially as it can be a target for therapy in cancer treatment by enhancing the effectiveness of chemotherapy drugs.
• 📝 The importance of measuring enzyme specific activity is emphasized, particularly in the context of pharmaceutical research, where GST's activity can indicate the purity and effectiveness of the enzyme in detoxifying processes.

Q & A

What is the main objective of the lab practice described in the transcript?

The main objective of the lab practice is to determine the specific activity of the enzyme Glutathione S-Transferase (GST), establish the protein concentration using the biuret method, and understand the concept of enzyme activity in relation to the amount of protein used in the reaction.

What is the significance of determining the specific activity of the enzyme GST?

Determining the specific activity of GST is important because it provides a measure of the enzyme's efficiency and purity. It indicates how much substrate is converted to product per unit time and per unit amount of enzyme protein.

What is the biuret method used for in this context?

The biuret method is used to determine the protein concentration in the supernatant fraction of the cell lysate. It is a colorimetric assay that measures the absorbance of a complex formed between copper ions and peptide bonds in proteins.

What are the different types of amino acids that make up proteins?

Proteins are made up of 20 different types of amino acids, which can be categorized based on their side chains as nonpolar, polar uncharged, and polar charged.

How does the biuret reagent react with proteins to indicate a positive result?

The biuret reagent reacts with peptide bonds in proteins to form a complex that absorbs light in the visible spectrum, resulting in a color change to a blue-purple hue, indicating the presence of protein.

What is the role of GST in phase 2 metabolism?

GST plays a crucial role in phase 2 metabolism by catalyzing the conjugation of glutathione with various substrates, including xenobiotics, which helps in the detoxification process and the elimination of these substances from the body.

How is the enzyme activity measured in the lab practice?

The enzyme activity is measured by monitoring the formation of the product, in this case, the GS-DNB conjugate, spectrophotometrically at 346 nm over time, which allows for the calculation of the reaction rate and enzyme activity.

What is the significance of measuring the protein concentration in the supernatant fraction?

Measuring the protein concentration in the supernatant fraction is essential for calculating the specific activity of the enzyme. It ensures that the enzyme activity is normalized to the amount of protein present, providing a more accurate representation of the enzyme's efficiency.

What is the purpose of creating a standard curve using BSA in the biuret method?

The purpose of creating a standard curve using BSA is to establish a relationship between the absorbance values and known concentrations of protein. This curve is then used to determine the unknown protein concentration in the supernatant fraction.

Why is it important to know the specific activity of an enzyme in pharmaceutical research?

Knowing the specific activity of an enzyme like GST is important in pharmaceutical research because it can help in understanding the enzyme's role in drug metabolism and detoxification, which is crucial for developing drugs and therapies, especially in cancer treatment.