MC2-3-Cell-Water-and-Buffers.pdf

Cell, Water, and Buffers MC 2 - Biochemistry  Cell Landmarks in the study of a cell Soon after Anton Van Leeuwenhoek invented the microscope, Robert Hooke in 1665 observed a piece of cork under the microscope and found it to be made of small compartments which he called “cells” In...

Cell, Water, and Buffers MC 2 - Biochemistry  Cell Landmarks in the study of a cell Soon after Anton Van Leeuwenhoek invented the microscope, Robert Hooke in 1665 observed a piece of cork under the microscope and found it to be made of small compartments which he called “cells” In Latin, cells means small room. In 1672, Leeuwenhoek observed bacteria, sperms and red blood corpuscles, all of which were cells. Much later, in 1831, Robert Brown, an Englishman observed that all cells had a centrally positioned body which he termed the nucleus. Cell Cell Theory In 1838, M.J. Schleiden and Theodore Schwann formulated the “cell theory.” Which maintains that: ✓ all organisms are composed of cells. ✓ cell is the structural and functional unit of life, and ✓ cells arise from pre-existing cells.  Cell What is Cell? A cell may be defined as a unit of protoplasm bound by a plasma or cell membrane and possessing a nucleus. Protoplasm is the life giving substance and includes the cytoplasm and the nucleus. The cytoplasm has in it organelles such as ribosomes, mitochondria, golgi bodies, plastids, lysosomes and endoplasmic reticulum. Plant cells have in their cytoplasm, large vacuoles containing non-living inclusions like crystals, and pigments. Cell What is Cell? The bacteria have neither defined cell organelles nor a well formed nucleus. But every cell has three major components: plasma membrane cytoplasm DNA (naked in bacteria) and enclosed by a nuclear membrane in all other organisms  Types of Cell Two basic types of Cell Cytologists recognize two basic types of cells: ✓ Organisms which do not possess a well formed nucleus are prokaryotes such as the bacteria. ✓ All others possess a well defined nucleus, covered by a nuclear membrane. They are eukaryotes. All cells, whether they are prokaryotic or eukaryotic, have some common features. Prokaryotic Cell ❑ Prokaryotic Cell Prokaryotic cell or prokaryotes are simple single-celled organisms with simple organelles necessary for life processes. The term “prokaryote” is derived from the Greek word “pro” which means “before” and “karyon” which means “kernel” or “nut”, thus “prokaryote” literally means “before the nucleus” There are two large groups of prokaryotes: archaebacteria (Greek arche-, “origin”) and eubacteria (again, from Greek eu means “true”). Prokaryotic Cell ❑ Archaebacteria Archaebacteria mostly inhabits extreme environments such as salt lakes, hot springs, highly acidic bogs and ocean depths. ❑ Eubacteria also called “true bacteria” are single celled prokaryotic microorganisms , have a wide array of characteristics and are found dwelling on surface waters, soils and tissues of other living or decaying organisms. The most common and the well studied eubacteria is the Escherichia coli. Prokaryotic vs Eukaryotic Cell Organelles Organelles are structures that enable the cell to live, grow and reproduce. Organelles ❑ Cell Membrane – “outer layer of the cell” A semi-permeable membrane that envelopes the cytoplasm, containing proteins, carbohydrates, and phospholipid bilayer. The phospholipid bilayer is described by the “Fluid Mosaic Model”. Function - Allow selective passage and facilitate transport of molecule which is either passive or active transport Organelles ❑ Cell Wall Only found in plants and fungi which is significantly thicker than a cell membrane. Rigid structure outside the cell membrane with three layers – middle lamella, primary wall, and secondary wall Function - Responsible for the shape of the cell, facilitate cell-to cell interaction, and shield the cell from harm, undesirable molecules and pathogens. Organelles ❑ Endoplasmic Reticulum - “Roadways of the cell” A system of small, tubular structures, with two parts – luminal (inside ER) and extra-luminal (cytoplasm) Rough ER is studded with ribosomes The only organelle that is directly connected with the nucleus and the nuclear membrane Function - Provides mechanical support to the cytoplasmic matrix. Organelles ❑ Golgi Complex/Apparatus a flat disc-shaped structures called cisternae arranged in parallel in parallel and concentrically near the nucleus. The cisternae are arranged in parallel and concentric near the nucleus with two faces – cis face and trans face. Function - Organelle responsible for packaging material within the cell, protein modification, and formation of glycolipids (carbohydrates + lipids) and glycoproteins (carbohydrates + proteins) Organelles ❑ Ribosomes - “Factories of the cell” Composed of ribonucleic acid and proteins and bound by membrane. For eukaryotic cell – 80S type with 60S (large subunit), and 40S (small subunit) Function - Major site of protein and polypeptide synthesis Organelles ❑ Mitochondria Membrane-bound organelle, also known as the as “powerhouse” of the cell. Contains RNA , DNA, needed for protein synthesis Function - Primary site for ATP synthesis (ATP or adenosine triphosphate is the energy currency of the cell,), and regulates cell metabolism. Organelles ❑ Lysosomes a membrane bound vesicles formed in the golgi apparatus and also called “suicidal bags” of the cell as they rich in hydrolytic enzymes like lipases, proteases, amylases, etc. Function - Lipids, proteins, carbohydrates, and nucleic acids are digested by this organelle. Organelles ❑ The Nucleus a double membrane structure, considered as the main organelle of the cell that contains all the genetic information, thus also known as the “Brain” of the cell. Found in all eukaryotic cell except in human RBCs and sieve cells of the plants. Function - stores genetic information (in the form of DNA) necessary for development and reproduction; contains all information needed for protein synthesis and cellular functions. Organelles ❑ Cytoskeleton network of filaments and tubules present in the cytoplasm of a cell. Comprised of three types of filaments that are elongated chains of proteins - microfilaments, intermediate filaments and microfilament. Function - It provides cell with mechanical support, give the cell its shape, organize the cell interior, and allow for cell movement. It also takes part in molecule transport. cell division, and cell signaling. Organelles ❑ Centriole are cylindrical organelles containing tube- shaped molecules known as microtubules that help separate chromosomes and move them during cell division. Present in animal cells but not in plant cells. Function - Involved in the formation of spindle apparatus, which function during cell division, the absence of which will cause errors and delays in the mitotic process; basal bodies direct the formation of cilia and flagella. Organelles ❑ Cilia and Flagella both responsible for the movement of a cell. Cilia is a short, hair-like structures that cover the entire surface of the cell. Flagella is a long structure that are present at one end of the cell. Cilia is responsible for the rowing movement of the cell, while flagella facilitates the up and down movement of the cell. Function - movement of the cell Organelles ❑ Plastids a double membrane organelle found in plants and other photosynthetic eukaryotes and contain three types of pigments – chloroplasts, chromoplasts, and leukoplasts. Function - involved in photosynthesis, synthesis of amino acids, and lipids as well as storage of various materials. Organelles ❑ Peroxisomes membrane-bound organelle that contain enzymes that oxidize bioorganic molecules producing hydrogen peroxide (giving the name peroxisome). contains catalase converting the toxic hydrogen peroxide into water and oxygen. Function - play important role in the oxidation of specific biomolecules, and contribute to the biosynthesis of membrane lipids. Organelles ❑ Vacuole a space inside the cell that does not contain cytoplasm. It is a membrane -bound organelle filled with fluid and are found in the cells of plants (including algae and fungi) and some protists and bacteria. Vacuole Vacuoles are acidic and share some characteristics with the lysosomes in animals, though fewer in number, but take 80-90 % of the entire cell volume Function - Storage of various molecules such as enzymes, waste products of the cell, water, and food material depending on the type of cell. Also involved in the exportation of cell waste thus protecting the cell from toxicity. Organelles ❑ Vesicle a spherical compartment separated from the cytosol by at least one lipid bilayer, that are formed by the Golgi apparatus, endoplasmic reticulum, or cell membrane by endocytosis. They are made of phospholipids that can break off or fuse with other membranous materials. \ Examples of vesicles include secretory vesicles, transport vesicles, and synaptic vesicles. Function - Vesicles store and transport materials with the cell; - involved in buoyancy control and temporary storage of food and enzymes; - isolating materials from the cell particularly those that contain waste products; - ingest and destruct invading bacteria, and in Characteristics of Water Water is a major chemical component of the earth’s surface. It is indispensable to life. The only liquid that most organisms ever encounter. Typically, organisms are constituted 70% to 90% water. Characteristics of Water ❑ Water as a Molecule Water is a bent molecule with a H-O-H bond angle of 104.5, composed of an atom of oxygen and two (2) atoms of hydrogen. Oxygen being the more electronegative atom, there is an unequal sharing of electrons between H and O producing a polar covalent bond. These partial charges are collectively known as dipole. Characteristics of Water ❑ Properties of Water has high heat of vaporization (40.71 kJ/mol at 250 °C) has high boiling point (100 °C), which allows it to be liquid at Earth’s surface temperatures has a relatively large heat capacity (4.184 J/g K) in its liquid phase is more dense than its solid phase has high surface tension (71.97 dyne/cm at 250 °C) has high dielectric constant (80.1 at 250 °C) Hydrogen bonding of water is key to its properties ❑ The Water Molecule is Polar A molecule has a positive and negative regions are called polar molecules Important properties of water arise from its angled shape Angle of 104.5 between two covalent bonds Polar O-H bonds due to uneven distribution of charge Angled arrangement of polar bonds creates a permanent dipole for a water molecule Dipole has a net charge separation within a molecule Hydrogen bonding of water is key to its properties ❑ Water as a Solvent Water is a very powerful solvent. This solvent property stems from its polar nature. Salts or Ionic compounds (like NaCl, and KCl) and polar organic compounds (like ethanol, and isopropyl alcohol) are all soluble in water. The solubility of many substances is governed by the principle of attraction between unlike charges (positive charges are attracted to negative charges and vice versa) Hydrogen bonding of water is key to its properties ❑ Water as a Solvent Ion–dipole and dipole–dipole interactions help ionic and polar compounds dissolve in water. Hydrogen bonding of water is key to its properties ❑ Water as a Solvent Dissolution of NaCl in water showing water molecules forming hydration shells/spheres around Na+ and Cl- Hydrogen bonding of water is key to its properties ❑ A molecule may have both polar (hydrophilic) and nonpolar (hydrophobic) part and are called amphipathic. ❑ Amphipathic substances in the presence of water tends to form micelles. Amphipathic substances Micelles Hydrogen bonding of water is key to its properties ❑ Hydrophobic – “hates water” ❑ Hydrophilic – “loves water” Acid-Base Equilibria ❑ Acids are H+ donor, while bases are H + acceptor ❑ The strength of an acid depends on its acid dissociation constant (Ka), and ❑ The strength of a base depends on its base dissociation constant (Kb). The greater the K value, the stronger is the acid/base. Acid-Base Equilibria ❑ Water is an ampholyte (can function as acid and base) , and can react to itself. We can define the ion product constant for water as: ❑ The pH scale can be derived from this expression and by using the definition of a p-function: Acid-Base Equilibria ❑ Most body fluids have pH values in the range of 6.5-8.0, often referred as the physiological pH. Most biological reactions occur in this pH range. Buffers ❑ A buffer is something that resist change, and is consists of a mixture of a weak acid and its conjugate base. ❑ Generally, buffers has two types (acidic and basic buffers) Buffers ❑ Acidic Buffers – is a ❑ Basic Buffers - is a combination of weak acid combination of weak base and its salt with a strong and its salt with a strong base (conjugate base). acid (conjugate acid). Buffers Equation ❑ The Henderson-Hasselbach equation can be used to qualitatively calculate the pH of buffers or any other solutions containing a weak acid and its conjugate base. ❑ These equation is based on the concept of conjugate acid- base pairs as defined by the Bronsted-Lowry definition of acids and bases. For example, for a hypothetical weak acid HA; Buffers Equation ❑ Sample Problem: Calculate the pH of a buffer solution containing 0.03 moles/liter of acetic acid (CH3COOH) and 0.1 moles/liter of sodium acetate (NaCH3COO-), pKa for CH3COOH IS 4.57. ❑ Solution: (0.1) = 4.57 + 𝑙𝑜𝑔 (0.03) = 4.57 + 0.52 𝒑𝑯 = 𝟓. 𝟎𝟗 Preparation of Buffers ❑ Selection of Buffer system: 1. pKa - ideal buffer solution must be near its maximum buffering capacity. Buffering capacity is at maximum when the pKa is within 1 unit away from the desired pH or nearer. 2. Solubility - since biological systems is in an aqueous environment, the selected buffer system must be very soluble in water. 3. Membrane permeability - to reduce the accumulation of buffer system molecules or ions in the subcellular tissue, best buffer system should not readily pass through membranes. 4. Salt effects - too much ions can cause problems in biological systems. For example, citrate and phosphate buffers must be avoided in calcium dependent reactions, and Tris buffer chelates calcium. Preparation of Buffers ❑ Selection of Buffer system: 5. Dissociation effects - – dissociation of buffers should not be affected by concentration, temperature and ionic strength. 6. Ionic strength - there are buffer system that can drastically concentration of ions in solution like phosphates, bearing in mind that physiological ionic strength should be between 100mM – 200mM of KCl or NaCl. 7. Cation interactions - best buffer should not form complexes, if the buffer form complexes, the complexes should be water soluble. 8. Stability - buffers should be chemically stable. Preparation of Buffers ❑ Selection of Buffer system: 9. Biochemical inertness - buffers should not influence nor participate in any biochemical reactions. 10. Optical absorbance- should not have an absorbance at a wavelength between 230 to 700 nm to prevent interference in spectrophotometric assays. 11. Ease of preparation and cost - easy to prepare from inexpensive materials. Buffer System in Biological System ❑ Bicarbonate Buffer. The maintenance of blood pH is regulated via the bicarbonate buffer. This system consists of carbonic acid (H₂CO₃) and bicarbonate ions (HCO₃⁻). When the blood pH drops into the acidic range, this buffer acts to form carbon dioxide gas. The lungs expel this gas out of the body during the process of respiration. During alkaline conditions, this buffer brings pH back to neutral by causing excretion of the bicarbonate ions through the urine. Buffer System in Biological System ❑ Phosphate Buffer. The phosphate buffer system, operates in the internal fluids of all cells, acts in a manner similar to the bicarbonate buffer, but has much stronger action. The internal environment of all cells contains this buffer comprising hydrogen phosphate ions and dihydrogen phosphate ions. Under conditions when excess hydrogen enters the cell, it reacts with the hydrogen phosphate ions, which accepts them. Under alkaline conditions, the dihydrogen phosphate ions accept the excess hydroxide ions that enter the cell. Buffer System in Biological System ❑ Protein Buffer. Proteins consist of amino acids held together by peptide bonds. The amino acids possess an amino group and a carboxylic acid group. At physiological pH, the carboxylic acid exists as the carboxylate ion (COO-) with a negative charge and the amino group exists as the NH3+ ion. When the pH becomes acidic, the carboxyl group takes up excess hydrogen ions to return back to the carboxylic acid form. If the blood pH becomes alkaline, there is a release of a proton from the NH3+ ion, which takes the NH2 form Buffer System in Biological System ❑ Hemoglobin Buffer. The respiratory pigment present in blood, hemoglobin, also has buffering action within tissues. It has an ability to bind with either protons or oxygen at a given point of time. Binding of one releases the other. In hemoglobin, the binding of protons occurs in the globin portion whereas oxygen binding occurs at the iron of the heme portion. At the time of exercise, protons are generated in excess. Hemoglobin helps in the buffering action by binding these protons, and simultaneously releasing molecular oxygen. References ❑Campbell, M.K. and Farrel, S.O. (2014) Biochemistry, 8th ed., Brooks Cole ❑Nelson, David L. and Michael M. Cox (2013). Lehninger PRINCIPLES OF BIOCHEMISTRY, 6th edition, W.H. Freeman and Company, New York.