Praktikum Biokimia Farmasi : Penentuan Aktivitas Spesifik Enzim

Adam Hermawan

6 Mar 202217:48

Summary

TLDRThis 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.

Outlines

00:00

🔬 Enzyme Activity and Protein Concentration Determination

This paragraph introduces the objectives of a laboratory practice focused on determining the specific activity of the enzyme Glutathione S-Transferase (GST) and the protein concentration using the biuret method. It explains the importance of knowing the protein concentration in the reaction mixture for accurate enzyme activity measurements. The paragraph also delves into a brief overview of proteins, their structure, and the different types of amino acids that compose them. It outlines various quantitative protein analysis methods, including titration, gasometry, and spectrophotometry, highlighting the biuret method's advantages and limitations.

05:02

🧪 Preparation and Analysis of Glutathione S-Transferase

The second paragraph details the process of preparing a cytosolic fraction containing glutathione transferase from mice, which is then verified using LED. It describes the steps for determining enzyme activity in the liver of mice using the CDN method and creating a standard curve with BSA using the biuret method. The paragraph also explains the process of isolating cytosol, including the use of centrifugation and specific buffers, and storing the reaction mixture for further laboratory practice. It emphasizes the importance of measuring protein concentration and enzyme activity in the context of studying GST, a phase 2 metabolism enzyme involved in the detoxification process.

10:03

📊 Measuring Enzyme Activity and Creating a Standard Curve

This paragraph discusses the methodology for measuring the activity of GST using 1-chloro-2,4-dinitrobenzene as a substrate and recording the absorbance at 346 nm to determine the enzyme's activity in micromoles per minute per millimeter. It also explains how to create a standard curve using BSA and the biuret reagent, which involves mixing reagents, observing the reaction, and measuring absorbance at 550 nm. The paragraph provides a formula for calculating the protein concentration in the cytosolic fraction and emphasizes the importance of accounting for dilution factors in the calculations.

15:04

🏥 Significance of Specific Enzyme Activity in Pharmacology

The final paragraph explores the significance of measuring specific enzyme activity, particularly for GST, in the field of pharmacology. It discusses the role of GST as a phase 2 metabolism enzyme that catalyzes the conjugation of glutathione with various substrates, including xenobiotics. The paragraph highlights the potential of GST as a therapeutic target in anti-tumor agents, given its overexpression in tumors and its ability to detoxify chemotherapy drugs. Targeting GST could enhance the effectiveness of chemotherapy by reducing its detoxification, leading to a more aggressive treatment against cancerous cells.

Mindmap

Keywords

💡Specific Activity of Enzyme

The specific activity of an enzyme refers to the amount of enzyme catalyst that catalyzes one micromole of substrate per minute under standard conditions. It is a measure of enzyme purity and efficiency, with a higher specific activity indicating a more pure enzyme preparation. In the script, the specific activity of the enzyme glutathione S-transferase (GST) is determined as part of the laboratory practice, which is essential for understanding the enzyme's role in metabolic processes.

💡Glutathione S-Transferase (GST)

Glutathione S-transferase, or GST, is a group of enzymes involved in the body's detoxification processes. It catalyzes the conjugation of reduced glutathione to various electrophilic compounds, thereby facilitating their excretion. In the video, GST is highlighted as a phase 2 metabolizing enzyme, which is crucial for the body's ability to process and eliminate xenobiotics, such as drugs and carcinogens.

💡Biuret Method

The Biuret method is a protein assay technique used to determine protein concentration in a solution. It involves the reaction of peptide bonds with copper ions to form a purple-colored complex, the intensity of which is measured at 560 nm. In the script, the Biuret method is employed to measure the protein concentration in the cytosolic fraction, which is necessary for calculating the specific activity of the enzyme GST.

💡Cytosolic Fraction

The cytosolic fraction refers to the part of the cell's cytoplasm that is soluble in water and does not include organelles or large molecules. It is an important component in biochemical assays, as it contains many enzymes and proteins of interest. In the script, the preparation of the cytosolic fraction from rat liver is described as a step in the process of measuring enzyme activity.

💡Enzyme Activity Assay

An enzyme activity assay is a biochemical method used to measure the rate at which an enzyme catalyzes a chemical reaction. It is fundamental in understanding enzyme kinetics and function. The script describes the process of determining the activity of the enzyme GST using a method involving the measurement of a product formed during the reaction.

💡Protein Concentration

Protein concentration is a measure of the amount of protein present in a given volume of solution. It is a critical parameter in biochemical research, as it allows for the normalization of enzyme activity measurements. The script emphasizes the importance of determining protein concentration using the Biuret method before calculating the specific activity of the enzyme.

💡Spectrophotometry

Spectrophotometry is the technique of measuring the absorption or transmission of light by a substance to quantitatively determine its concentration. In the script, spectrophotometry is used to measure the absorbance of the product in the enzyme activity assay and the color intensity in the Biuret protein assay.

💡Protein Polymerization

Protein polymerization, or polypeptide formation, is the process by which amino acids are linked together by peptide bonds to form proteins. This concept is fundamental to understanding the structure and function of proteins. The script mentions this process in the context of explaining the composition and structure of proteins.

💡Amino Acids

Amino acids are the building blocks of proteins, consisting of an amino group, a carboxyl group, and a side chain that varies between different amino acids. There are 20 standard amino acids that make up the proteins in organisms. The script discusses the composition of proteins from amino acids and their role in forming polypeptide chains.

💡Electrophilic Compounds

Electrophilic compounds are molecules that have an affinity for electron-rich areas and can react with nucleophiles. In the context of the script, electrophilic compounds are substrates for the GST enzyme, which catalyzes their conjugation with glutathione, making them less reactive and more easily excretable.

💡Conjugation Reaction

A conjugation reaction in biochemistry refers to the process of combining two molecules to form a single product. Specifically, in the script, it describes the conjugation of glutathione with electrophilic compounds, which is a detoxification mechanism facilitated by the GST enzyme.

Highlights

The purpose of the lab is to determine the specific activity of the enzyme Glutathione S-Transferase (GST) and the protein concentration using the biuret method.

Proteins are polymers made up of amino acids as monomers, with 20 different types involved in their structure.

Amino acids consist of an amino group and a carboxyl group, with side chains that can be nonpolar, polar uncharged, or polar charged.

Quantitative protein analysis methods include volumetry, gasometry, and spectrophotometry, each with its principles and applications.

The biuret method involves the reaction of peptide chains with the biuret reagent, resulting in a purple complex measurable at a wavelength of 560 nanometers.

The biuret method's advantages include its speed and simplicity, but it is less sensitive due to not all protein groups reacting positively.

GST is a phase 2 metabolism enzyme that catalyzes the conjugation of glutathione with various substrates, including xenobiotics.

GST plays a role in the detoxification process, converting electrophilic compounds into more reactive forms that can be conjugated with glutathione.

The lab procedure includes the preparation of cytosolic fractions containing GST from mice, which will be confirmed by LED.

The determination of GST enzyme activity is performed using the CDNB method, measuring the formation of the GS-DNB conjugate at 346 nm.

A standard curve of BSA with the biuret method is created to determine protein concentration in the cytosolic fraction.

The calculation of specific enzyme activity involves dividing the reaction rate by the protein concentration in the cytosolic solution.

The importance of measuring protein concentration and enzyme activity is highlighted, especially in the context of pharmacology and detoxification.

GST's role in protecting cells from chemotherapy-induced cell death and its potential as a therapeutic target in cancer treatment is discussed.

The lab concludes with the significance of GST in pharmacology, its relevance in anti-tumor agents targeting, and its potential impact on cancer therapy.