Biochemistry - BIO119 - Module 1 PDF

1 BIO119 Course Lead: Ms Jean Course Lead: Christal Jean  2 Course Lead: Christal Jean  Biochemistry: 3 Biochemistry is the application of chemistry to the study of biol...

1 BIO119 Course Lead: Ms Jean Course Lead: Christal Jean  2 Course Lead: Christal Jean  Biochemistry: 3 Biochemistry is the application of chemistry to the study of biological processes at the cellular and molecular level. Course Lead: Christal Jean  Introduction to Cell Structure Module 1 BIO119 Course Lead: Ms. Jean Course Lead: Christal Jean  5 MODULE NUMBER: 1 INTRODUCTION TO CELL STRUCTURE Module Description The module focuses on the cell as the most basic unit of life. It gives a brief overview of the cell and its structures as an introduction to the course. It outlines the variations which exist between animal and plant cells. The module will review various organelles within the cells. Course Lead: Christal Jean  6 MODULE NUMBER: 1 INTRODUCTION TO CELL STRUCTURE Learning Outcomes: 1. Define terms associated with cells 2. Describe the organelles found in all kinds of cells 3. Outline the function of each organelle 4. Outline the method used for fractionation and separation of cell components 5. Differentiate between the features of eukaryotic and prokaryotic cells Course Lead: Christal Jean  Definitions 7 Genome: The genome is the entire genetic complement of an organism, that is, all the organic bases contained within the DNA. A genome is a set of complete DNA which include all genes in it. It contains sufficient information that is needed to develop and maintain an organism. The full complement of DNA is organized into 23 pairs or 46 chromosomes. Course Lead: Christal Jean  8 Definitions Co-enzyme: Organic Cofactors A coenzyme is an organic non-protein compound that binds with an enzyme to catalyze a reaction. Coenzymes are often broadly called cofactors, but they are chemically different. A coenzyme cannot function alone, but can be reused several times when paired with an enzyme. Course Lead: Christal Jean  Definitions 9 Pili: Protein structures that extend from the bacterial cell envelope. They function to attach the cells to surfaces. Sex Pili. Flagella: A slender threadlike structure, that enables protozoa, bacteria, spermatozoa etc to swim. Plasmids: A small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. Usually small circular, double-stranded DNA molecules in bacteria. Course Lead: Christal Jean Plasmids are sometimes present in archaea and eukaryotic organisms.  1 Definitions 0 Cytosol: The liquid found inside of cells. It is the water-based solution in which organelles, proteins, and other cell structures float. A complex solution, whose properties allow the functions of life to take place. Cytosol contains proteins, amino acids, mRNA, ribosomes, sugars, ions, messenger molecules, and more! Course Lead: Christal Jean Function of Cytosol 1 1 Cytosol serves as the medium for intracellular processes. Activities that take place in, or involve the cytosol include: 1. Enzyme activities. Enzymes often require certain salt concentrations, pH levels, and other environmental conditions to work properly. Course Lead: Christal Jean  1 Function of Cytosol 2 2. Signal Transduction. Messenger molecules may diffuse through the cytosol to change the functioning of enzymes, organelles, or even DNA transcription. These may be messengers from outside the cell, or messengers from one part of the cell to another. Course Lead: Christal Jean  1 Function of Cytosol 3 3. Structural support of the cell and organelles. Most cells rely on the volume of cytosol to create its shape and create space for chemicals to move within the cell. 4. In prokaryotes which lack membrane-bound organelles, virtually all functions of life including DNA transcription and replication, glycolysis, etc., happen in the cytosol. Course Lead: Christal Jean  1 Ribosomes: 4 Minute particles consisting of RNA and associated proteins found in large numbers in the cytoplasm of living cells. They bind messenger RNA and transfer RNA to synthesize polypeptides and proteins. Course Lead: Christal Jean Prokaryotic Ribosomes  15 Cell Structure and Function Course Lead: Christal Jean  1 Cell 6 Structure and Function Course Lead: Christal Jean  Lysosomes - “suicide bags” Course Lead: Christal Jean Course Lead: Christal Jean  1 Cell Structure and Function 9 The nucleoid, in bacteria, is not separated from the cytoplasm by a membrane. The nucleus, in higher organisms, consists of nuclear material enclosed within a double membrane, the nuclear envelope. Cells with nuclear envelopes are called eukaryotes (Greek eu, “true,” and karyon, “nucleus”) Cells without nuclear envelopes—bacterial cells—are prokaryotes (Greek pro, “before”) Course Lead: Christal Jean Three Domain System Recognizes three types of cells - Classification Schemes Three domain system, based on a comparison of ribosomal RNA genes, divides microorganisms into: – Bacteria (true bacteria) – Archaea – Eukarya (eukaryotes) Protista Fungi Plantae Animalia Prokaryotic organisms belong either to the domain Archaea or Bacteria Course Lead: Christal Jean Archaea are prokaryotes that do not have peptidoglycan cell walls. Classification: Source of Energy Course Lead: Christal Jean The Eukarya are Subdivided into Four Kingdoms: Plantae Kingdom: Plants are multicellular organisms composed of eukaryotic cells. The cells are organized into tissues and have cell walls. They obtain nutrients by photosynthesis and absorption. Examples include mosses, ferns, conifers, and flowering plants. Animalia Kingdom: Animals are multicellular organisms composed of eukaryotic cells. The cells are organized into tissues and lack cell walls. They do not carry out photosynthesis and obtain nutrients primarily by ingestion. Examples include sponges, worms, insects, and vertebrates. Course Lead: Christal Jean The Eukarya are Subdivided into Four Kingdoms: Protista Kingdom: Simple, predominately unicellular eukaryotic organisms. Examples includes slime molds, euglenoids, algae, and protozoans. Fungi Kingdom: Unicellular or multicellular organisms with eukaryotic cell types. The cells have cell walls but are not organized into tissues. They do not carry out photosynthesis and obtain nutrients through absorption. Examples include sac fungi, club fungi, yeasts, and molds. Course Lead: Christal Jean Archaea Organisms within this domain are referred to as the extremophile prokaryotes, Some live in extreme environments. Archael cells have no nucleus (and so are prokaryotic) Initially classified as bacteria until unique properties were discovered that separated them from known bacteria, including: Unique lipids found in the membranes of their cells No peptidoglycan in their cell walls Ribosomal structure (small subunit) similar to the eukaryotic ribosome. Archaea a similar size range and metabolism as bacteria DNA transcription is more similar to that of eukaryotes Example: Halobacterium Course Lead: Christal Jean salinarum are a species of the archaea domain found in environments with high salt concentrations like the Dead Sea. Halophile Cell Structure and Function Course Lead: Christal Jean Course Lead: Christal Jean  Prokaryotic Cell Eukaryotic cell Endoplasmic reticulum absent Endoplasmic reticulum present Mitochondria absent Mitochondria present Cytoskeleton absent Cytoskeleton present Course Lead: Christal Jean Ribosomes smaller Ribosomes larger Prokaryotic Cells vs Eukaryotic Cells Prokaryotes are organisms made up of cells that: Lack a cell nucleus or any membrane-encased organelles. Eukaryotes are organisms made up of cells that: Possess a membrane-bound nucleus that holds genetic material as well as membrane-bound organelles. Prokaryotic Cell Eukaryotic cell Endoplasmic reticulum absent Endoplasmic reticulum present Mitochondria absent Mitochondria present Cytoskeleton absent Cytoskeleton present Course Lead: Christal Jean Ribosomes smaller Ribosomes larger Course Lead: Christal Jean Course Lead: Christal Jean Course Lead: Christal Jean Course Lead: Christal Jean Subcellular Fractionation Isolation of a particular subcellular organelle to study: - The organelle intact - A specific substance from the organelle A schematic representation of a typical animal cell, showing its compartmentalization into many different organelles. Reprinted from Alberts et al.. Subcellular Fraction Involves the homogenization or destruction of cell boundaries by different mechanical or chemical procedures. Followed by the separation of the subcellular fractions according to mass, surface, or specific gravity. Extraction: Mechanical or Chemical Procedures. Maximum disruption of whole cell; Minimum damage to subcellular components or organelles to be studied. Optimal conditions: Aqueous solution Osmotic pressure Temperature: 0 – 4 ͦ pH: 7.4 Followed by the separation of the subcellular fractions according to: mass, surface, or specific gravity. Homogenization Once cells are disrupted, their constituents are liberated in the sucrose solution. Homogenate – Cell-free system containing many intact organelles When carefully applied, homogenization leaves most of the membrane-bounded organelles intact. Centrifugation Process of separating soluble cell fluid from the particulate matter: Rate-zonal centrifugation Principle: Components in the homogenate have different: Mass-to-volume ratio (density) and size. Heavy & large bodies sediment under low speeds and low gravitational forces. Light and small substances require high speeds and high gravitational forces. Course Lead: Christal Jean Centrifugal Fractions: Nuclear – Nucleus Mitochondrial – Mitochondria, chloroplasts, lysosomes, peroxisomes Microsomal – Plasma membrane, endoplasmic reticulum, polyribosomes Soluble – Course Lead: Christal Jean Cytosol Subcellular Fractional: Assessment of Purity Morphology – Electron microscopy Marker Molecules – Enzymes or chemicals Immunological Techniques – Uses antibodies specific for organelle member Course Lead: Christal Jean  4 Main Types of Density Gradient Centrifugation 0 Isopycnic Separation Particles migrate through the solvent gradient until they reach the point where their buoyant density is equal to that of the gradient. Rate-Zonal Separation (Velocity gradient centrifugation) Particles are separated based on their size and mass. Course Lead: Christal Jean Course Lead: Christal Jean  Subcellular Fractionation 4 2 Centrifugation The process of centrifugation allows scientists to separate substances based on their shape and size. Samples are placed into a centrifuge — a machine that is designed to spin liquid solutions at a high speed. The mixing or rotating causes the mixture to experience a centrifugal force that pushes larger particles from the center toward the bottom, and smaller to the top. Larger components react to the force more than smaller components. Course Lead: Christal Jean  Subcellular Fractionation 4 3 Density Gradient Reagents A mixture or substance used in chemical analysis or experimentation. This reagent is used to assist in isolation or separation of the cells. These products: Speed up the process Increase the purity and throughput. By keeping particles from clustering and creating a set divider, the reagents increase the efficiency of density gradient centrifugation. Course Lead: Christal Jean  4 Subcellular Fractionation 4 Principles of Density Gradient Centrifugation Each particle has a specific set of physical characteristics; the properties of its biological makeup that can be used for separation and isolation. Density gradient centrifugation focuses on two — size and density. The length of time required for this process is dependent upon the size of the particles. Course Lead: Christal Jean  4 Subcellular Fractionation 5 In density gradient centrifugation the process is similar. Samples are placed into a centrifuge, but the end goal is not to sort them by size. The spinning from the centrifuge causes more dense particles to move to the outside edge. These particles have more mass and are carried further by their inertia. Less dense particles then settle towards the center of the sample. Creates a sorted solution that is layered by particle density from Course Lead: Christal Jean least to most.  4 Subcellular Fractionation 6 Differential centrifugation is a centrifugation separation method that is based on a particle’s mass. Since different sized cells already behave differently, the process is done with no special reagent or medium. Differential centrifugation is sometimes considered a simpler form of centrifugation. It is used for separating cells and organelles while density gradient centrifugation is used for molecules and particles. The main difference between the two centrifugation methods is the type of physical properties in which the process is based on. Differential centrifugation might be easier, but density gradient centrifugation is able to sort particles of a much smaller size. Course Lead: Christal Jean Course Lead: Christal Jean Course Lead: Christal Jean  4 Preparative Density Gradient Ultracentrifugation9 (Preparative Isopycnic Ultracentrifugation) - Usually higher speeds than differential centrifugation - Sample placed in solution that has a density gradient - Has greater resolving power than differential centrifugation A tissue sample may be subjected to differential centrifugation first, and then one of the resulting pellets could be subjected to density gradient ultracentrifugation to separate the various particles that are in that pellet. Course Lead: Christal Jean  Course Lead: Christal Jean Isopycnic (sucrose-density) Centrifugation  5 Separation of Cells - Fractionation of Tissue 1 Fractionation: to separate various cellular components (separate them into different “fractions”). Centrifugation of all types is beneficial to scientists because it harvests substances for experimentation or medical uses. It helps to remove contamination and impurities in samples so specific cells or particles can be isolated and studied. Course Lead: Christal Jean  5 Separation of Cells - Fractionation of Tissue 2 Further fractionation: The next step is to use one or more fractionation methods to remove all remaining impurities from your protein. Chromatography is an excellent and very commonly used method for protein purification. Chromatography is derived from the greek word “ kroma” which means colour. Course Lead: Christal Jean  5 Fractionation by Column Chromatography 3 Mobile phase – A buffered aqueous solution Stationary phase– A porous solid material (inside the column) with appropriate chemical properties. Resembles fine sand. Course Lead: Christal Jean  5 Fractionation by Column Chromatography 4 Protein mixture placed at top of column. Buffer is pumped into the column. Proteins dissolve in the buffer and are carried down the column by the buffer as it flows through the stationary phase. Different proteins migrate down the column at different rates, depending on their interaction with the stationary phase. Course Lead: Christal Jean  5 Fractionation by Column 5 Chromatography Some proteins: “Stick” to the stationary phase, so they travel slowly “Don’t interact” with the stationary phase, so they travel quickly. The buffer that drips out of the column is collected in small increments called fractions. Proteins A, B, and C will be in separate fractions – they have been separated by chromatography. Course Lead: Christal Jean  5 Types of Column chromatography: 6 1. Ion Exchange Chromatography Can be anion exchange or cation exchange. Separates proteins based on differences in net charge at a given pH. 2. Size Exclusion Chromatography Separates proteins based on “size” (molecular weight and shape). 3. Affinity Chromatography Separates proteins based on binding specificity. Course Lead: Christal Jean  5 7 Course Lead: Christal Jean  5 8 Course Lead: Christal Jean  5 Types of Column Chromatography: 9 HPLC (High Performance Liquid Chromatography)- The solvent/buffer is pumped through the column under high pressure. Achieves better resolution of proteins than the solvent to dripping by gravity. HPLC columns can be types 1, 2, or 3. Analysis of proteins, drugs and explosives Separation of pharmaceutical drugs Separation of lipids, fatty acids and steroids Explanation: Elimination of undesirable substances from blood is done using affinity chromatography. All the other Course Lead: Christal Jean options are the application of high performance liquid  Cation Exchange Chromatography Stationary phase composed of microscopic polymer beads with negatively charged functional groups attached to the beads. Proteins with a net positive charge are attracted to the beads, they travel slowly down the column. Proteins with a net negative charge are not attracted to the stationary phase, they are carried quickly down the column by the buffer. The application of ion exchange chromatography is softening of hard water, demineralisation of water and the separation and determination of Course Lead: Christal Jean anions. Cation Exchange Chromatography “Resin” (Stationary phase material) Surface of bead covered with many negatively charged functional groups. Some counter-ions (Na+) are shown. If a positively charged protein comes down the column, it will exchange with a Na+ ion to interact with a negative functional group– hence the name “cation exchange” chromatography. Course Lead: Christal Jean Cation Exchange Chromatography What about the proteins with a large net positive charge that remain “stuck” to the column even with extensive buffer washing? These proteins interact strongly with the column’s negative functional groups. Must decrease the strength of their interaction with the stationary phase. Put a buffer of higher pH through the column to deprotonate some of the functional groups on the proteins, so that their net positive charge decreases and they don’t interact as strongly with the stationary phase. If the pH change is drastic enough, the proteins may even become negatively charged. Use the higher pH buffer until the proteins elute from the column. Course Lead: Christal Jean Anion Exchange Chromatography Bead surface covered with positively charged groups. Proteins with net negative charge are attracted to the positive functional groups and travel slowly down the column (“stick” to the column; they “exchange places” with the Cl- counter-ions). Proteins with net positive charge are not attracted to stationary phase and are carried quickly down the column by the buffer. The particular anion exchange chromatography resin represented here is called DEAE resin (di-ethyl amino ethyl). It is one of the most commonly used resins for protein purification. Course Lead: Christal Jean Size Exclusion Chromatography The stationary phase is composed of microscopic cross-linked polymer beads that have pores of a specific size range. Course Lead: Christal Jean Size Exclusion Chromatography Small proteins (red and green circles) will fit into the pores. Large proteins (blue circles) won’t be able to fit into the pores, so they only move between the beads and don’t meander in and out of the pores. Size exclusion chromatography beads can be purchased with pores of a specific size range in order to accommodate proteins of a certain size. Course Lead: Christal Jean Affinity Chromatography The stationary phase is composed of polymer beads with a ligand cross-linked to the beads. Any proteins that bind to the ligand will “stick” to the stationary phase and won’t elute from the column (green). Other proteins will be washed off the column quickly in the buffer (yellow & purple proteins). Course Lead: Christal Jean Affinity Chromatography In order to make the bound proteins elute from the column, a solution containing the ligand can be used. The ligand competes for the binding of the protein molecules. Proteins dissociate from the column-bound ligand and bind to free ligand in the solution, these proteins can be washed off the column. Elimination of undesirable substances from blood is done using affinity chromatography. Course Lead: Christal Jean  Virtual Lab Course Lead: Christal Jean