Introduction, pH & Buffer PDF

INTRODUCTION, pH & Buffer Dr. Mahmood. Annajjar  Biochemistry Biochemistry is the Science concerned with the chemical composition of the living cells and their reactions. Aim of biochemistry Complete understanding at the molecular level, of all chemi...

INTRODUCTION, pH & Buffer Dr. Mahmood. Annajjar  Biochemistry Biochemistry is the Science concerned with the chemical composition of the living cells and their reactions. Aim of biochemistry Complete understanding at the molecular level, of all chemical processes in the living cells leads to : prevention or diagnosis and therapy of disease.  All diseases have chemical base Chemical, physical, biological, genetic, and nutritional agents lead to disturbance of the chemistry of the cell, leading to cell dysfunction which means a disease  Agents that may cause cell dysfunction Chemicals and drugs: lead, merury, alcohol⋯.. Physical : trauma, heat, radiation, pressure⋯⋯ Hypoxia: loss of blood supply due to blockage or thrombosis of arteries and veins. Poisoning of oxidative enzymes Microbial: bacteria, viruses, fungi, parasites Nutritional disorders Hormonal disturbance Gene defects Immunologic reactions  What dose the Biochemistry discuss? structure and function of cellular components - proteins, carbohydrates, lipids, nucleic acids and other biomolecules Metabolism and Regulation Gene expression DNA RNA Protein  The chief components of the human body are proteins, fat, carbohydrate, water, and minerals  Chemical composition of a normal man (weight 65 kg) Constituent Percent (%) Weight (kg) Water 61.6 40 Protein 17.0 11 Lipid 13.8 9 Carbohydrate 1.5 1 Minerals 6.1 4  Polymers and Monomers Each of these types of molecules are polymers (macromolecules) that are assembled from single units called monomers(micromolecules. Each type of micromolecule is an assemblage of different elements. Macromolecule (Polymers) Monomer Elements Carbohydrates Monosaccharide Lipids Not always polymers; Hydrocarbon chains Proteins Amino acids Nucleic acids Nucleotides Structural hierarchy in the molecular organization of cells  The human body is composed of a few elements that compine to form a great variety of molecules. C, H, O, N, Ca, P⋯are major elements iron, iodine, selenium ⋯⋯etc. are trace elements. Biomolecules are compounds of carbon with a variety of functional groups From Lehninger Principles Of Biochemistry  Chemical Bonds in Biochemistry 1. Covalent bonds. 2. Electrostatic bonds: a)Ionic bonds. b)Hydrogen bonds. c)Van der Waals bonds. d) the electrostatic attraction between two ionized groups of opposite charge, such as - + carboxyl (-COO ) and amino (-NH3 ). 3. Hydrophobic interactions.  Covalent Bonds Non-metals share one or more pairs of electrons Each pair makes one covalent bond.  Many non-metallic elements exist naturally as di- atomic-molecules,e.g.: - Hydrogen H2 single bond H H - Oxygen O2 Double bond - Nitrogen N2 O O triple bond N N - Carbon Dioxide two double bonds O C O  The C atom forms 4 covalent bond The N atom forms 3 covalent bond The O atom forms 2 covalent bond The H atom forms 1covalent bond  Non-covalent bonds and other weak forces Ionic bond: - Atoms lose or gain electrons to form charged particles (ions) - Ions are strongly attracted to each other by opposite charges, + ve and –ve (electrostatic).  Hydrogen Bonds Result from electrostatic attraction between an electronegative atom (O or N) & a hydrogen atom. Heat-labile bonds  e.g. water H-bond formed in H2O Bonding of water to other molecules. O H O C  Van der Waals Bonds are short charge attractive forces between chemical groups in contact. Caused by slight charge displacements  Hydrophobic Interactions non-polar groups such as hydrocarbon chains associate with each other in an aqueous environment.  Solutions The solution is formed of a solute and solvent. e.g when NaCl dissolved in water.  Concentration of a substance in a solution Is measured as a percentage ratio between the solute and the solvent As: weight per volume (mg % , g% ) , or molarity. ppm (part per million) 1:10 ,used for trace 6 elements (selenium ,zink,⋯⋯etc.)  Molarity: - The number of moles of solute per liter of solution. One mole: it is the weight of substance in gram when equal to molecular weight example: What is the molarity of 5 8.44 grams of NaCl dissolved in 1liter? = 1 molar - How many moles in 720 gram of glucose 720 / 18 0 = 4 moles - 10 g of milk was found to have 15 μg of selenium, what is the concentration of Se 15 μg /10 g = 1.5 ppm  Dilution Required when: The concentration of a substance is high, eg high blood glucose in a diabetic patient. -- Dilution factor= total final volume/original volume For estimation of serum protein, take 0.2 ml of serum+ 3.8 ml normal saline Dilution factor = 0.2 + 3.8 / 0.2 = 20 times  To maintain the health, the constancy of the internal environment of the body must be kept within relatively narrow limits. This applies to: Water Electrolytes PH  Electrolytes Electrolytes are substances that readily react with water and ionized (carrying an electric charge, -ve or + ve). - Anions: have a -ve charge and move toward anode (HCO- 3 , Cl- ) - Cations: have a + ve charge and move to cathode (Na+ , K + ).  The major anions found in the body fluids are: Cl : major extracellular anion. - HCO 3 : extracellular. - HPO 4 : intracellular, & present in the skeleton and - teeth in form of calcium phosphate. COO -  The major cations found in the body fluids are: Na + major extracellular cation. K + major intracellular cation. Ca ++ extracellular cation, & in the skeleton and teeth,. Mg ++ intracellular cation. H + NH4 +  Electrolyte function Hormonal stimulation ( Ca⋯ Nerve conduction: myocardial rhythm and contractility, and neuromuscular excitability ( Ca, Mg, K ) Osmotic pressure maintenance (Na, Cl , K) Contribute to the buffer system (HCO3 ) Co-factors in enzyme activation(Mg, Ca, Zn, Se,⋯) Production and utilization of ATP (energy), (PO4, Mg.) Regulation of ATPase ion pump (Mg) Blood coagulation (Ca, Mg), Hem synthesis ( Fe+ + )  Water Water is the main component of the body. Total body water varies with age and sex. The Range ~ 5 0- 70% of body weight.(less in females and obese). fat tissues contain 20% water Body water: - 2/3 ICF( intra cellular fluid) of body water. - 1/3 ECF of body water (E: extra , out side the cells) - Of the ECF approximately 25% in plasma and 3/4 in interstitial fluid. ( normal plasma 93% )  Function of water Transport nutrients to cells, and remove waste products Temperature regulation (water has high heat capacity : resist change in temperature), when temperature increase water acting by cooling through sweating ( perspiration) body fluids: urine, joint lubricants, saliva, bile, amniotic fluids. Water is the solvent for processes in the body Water is the medium for reactions. Water is a reactant or a product in many metabolic reactions  Water intake= water output=28 00 ml/day Men need 15 cups of water Women need 11cups Increase with fever, diarrhea, vomiting, sweating, and exercise.  Low hydration Thirst, loss of appetite, increasing hem concentration, emotional instability, flushed skin, tingling in arms, hands, and feet, fatigue, headache, dizziness, nausea. Increased body temperature, pulse, and respiratory rate, muscle cramp. In sever cases, kidney dysfunction, risk of death.  Structure of water H2O 6 ēs in the outer orbital of O2 2 ēs of O2 are involved in covalent bonds with H2 The other 4 ēs exist in non-bonded pairs (excellent H-bond acceptors).  Properties of water Water is a dipolar molecule. It has electrical charges unequally distributed about its structure  Each water molecule can bind to four molecules of water Liquid water is denser than its solid form.  In ice, H-bonds become most regular and clearly defined. Water serves as the universal solvent. This ability arises from the two properties of water (tendency to form H bonds & dipolarity). High boiling point. Low viscousity. Amphoteric , can act as an acid and as a base.  Mo lec ules in r es pec t to water 1-Hydrophilic: Water readily dissolves hydroxyl compounds, amines, esters, ketons, and ionic compounds 2- Hydrophobic: (hydrocarbon) 3-Amphipathic: (have hydrophilic head and hydrophobic tail eg: fatty acids)  Amphipathic substance in water, form: - Monolayer on the water surface with only the head groups immersed. - Micelles spherical structures formed by a single layer of molecules. Bilayer vesicles (hydrocarbon chains are in roughly parallel arrays).  Overhydration Water intoxication is defined as an increase in total body water Common causes are excessive water consumption (rare). impaired renal water excretion as a result of excess anti diuretic hormone( ADH) secretion Increased infusion of intravenous solutions heart failure, cirrhosis  Clinical manifestations - Hyponatremia appears -Abdominal cramps, nausea, vomiting, dizziness, and lethargy -It can potentially lead to convulsions and coma  Io nizatio n o f water One water molecule can transfer a proton to + another to yield a hydronium ion (H3O ) and a - hydroxyl ion (OH ). So water acts as both: a proton donor (acid) and a proton acceptor (base). i.e amphoteric H2O + H2O H3O+ + OH- At simple form + - H2O H + OH  Where K is the dissociation constant. [ H ] concentration of hydrogen ions. + [ OH ] concentration of hydroxyl ions. - [ H2O] concentration of undissociated water molecules.  What is the probabelity of presence of H ion or OH ion in one liter of water  The probability of hydrogen atom in pure -9 water existing as free ion is ~ 1.8 x10 in one mole 1mol of water weights 18 g One liter (1000g) of water contains 1000 ÷18 = 5 5.5 6 mol. [ H ] or [ OH ] = 5 5.5 6 x (1.8 x10 ) = 1.0 x 10 + - -9 -7 mol Kw =[ H ][ OH ] =(1.0 x 10 )x(1.0 x 10 )= + - -7 -7 1.0 x 10-14  pH concept: The pH is the negative log of the hydrogen ion concentration. PH = - log [ H ] + o -7 at 25 C, pure water PH= -log 10 = -(-7) = 7.0 -7 POH = -log 10 = -(-7) = 7.0 + - -14 Kw = [ H ] [ OH ] = 10 -14 PKw = -log10 PK w =14  The pH range starts from (0) up to (14). PH of 1mole of H-ion = 0 PH of 1.0 x 10 mole of H- ion= 14 -14 low pH values correspond to high concentration + of H (acid), and high pH values correspond to low + concentration of H (base). In lab., for neutral solution pH = 7. If the pH < 7 ⋯.. acidic solution. If the pH > 7 ⋯.. basic solution. In the blood, physiological pH= 7.40 ± 0.05 If the pH < 7.35 ⋯.. acidosis. If the pH > 7.45 ⋯.. alkalosis.  The impo r tanc e o f Ph Each enzyme has a certain optimum pH at which it acts at maximum level. Anti bacterial Increase in pH causes alkalosis. Decrease in pH causes acidosis  Changes in pH affect body functions: e.g. enzyme catalysis, O2 transport. In sever cases, may lead to coma and death.  pH of some body fluids Fluid PH Blood plasma 7.4 Intercellular fluid Cytosol (liver) 6.9 Lysosmal matrix 5.5-6.5 Gastric juice 1.5 –3.0 Pancreatic juice 7.8 –8.0 Human milk 7.4 saliva 6.4 –7.0 urine 5.0 –8.0 Example: What is the pH of a solution whose −4 hydrogen ion concentration is 3.2 x 10 mol/L? pH = –log [H+] = –log (3.2 x 10-4) = –log (3.2) –log (10 ) -4 = –0.5 + 4.0 = 3.5 Example: What is the pH of a solution whose −4 hydroxide ion concentration is 4.0 X 10 mol/L? + - -14 Kw = [H ][OH ] = 10 pKw =14 = pH + pOH [OH-] = 4.0 x10-4 pOH = –log [OH ] - = –log (4.0 x 10-4) = –log (4.0) –log (10-4) = –0.60 + 4.0 = 3.4 pH = 14 –pOH = 14 –3.4 = 10.6  Acid and base Acid: Is a proton donor. Weak acids: Acids that have only a slight tendency to give up protons to water CH3COOH CH3COO- + H+ Strong acids: Acids that readily give up their protons HCl → - Cl + H +  Base: Is a proton acceptor NaOH → Na+ + OH- Salt: is a substance which contains cations rather + - than (H ) or anions rather than (OH ). eg. NaCl, MgSO4 When acid and base react together, they neutralize each other forming salt and water. NaOH + HCl → H2O + NaCl  POLYPROTIC ACID: Some acids are capable of loosing more than one proton. eg. Phosphoric acid (H3PO4 ) and carbonic acid (H2CO3)  Phosphoric acid (H3PO4) H 3P O4 ↔ H 2P O4- + H+ H 2P O4- ↔ H P O4-2 + H+ Carbonic acid (H2CO3) HH2CO -2 P O3 4↔ ↔ P - - 3 ++ H + O HCO3 4+ H HCO3- ↔ CO3 -2 + H+  DIS S OCIATION OF ACID: HA H+ + A- The equilibrium constant for dissociation of an acid (often called the dissociation constant). pKa = -log Ka  Ka and pKa: K a is the dissociation constant of an acid. The larger Ka, the greater tendency of the acid to dissociate (the stronger the acid). pK a: is the negative logarithm of Ka - Small values of pKa correspond to a strong acid. - Large values of pKa correspond to a weak acid.  + pKa1 4.76 H3PO4 H2PO4 + H 2 H2PO4 HPO4 + H+ pKa2 7.2 2 3 + HPO4 PO4 + H pKa3 10.2  Buffers Buffer solution is a solution which resists a change in pH, when small amounts of an acid or a base are added to it. It is formed from a weak acid and its conjugated base, or a weak base and its conjugated acid. Thus buffer is a conjugate acid /base pair. CH3COOH H+ + CH3COO- acid base  + - CH3COOH H + CH3COO acid base If an acid is added the H are “ + mopped up”by acetate ions as the reverse reaction. If an alkali is added, the forward reaction + - producing H to react with OH. Titration curve shows that the titration curve of each acid has a flat zone extending about 1.0 pH unit on both sides of its midpoint. in this zone the pH of the system changes little when small increment of + - H or OH are added. The buffering power is maximum at the pH of the exact midpoint of the titration curve, at which the concentration of the proton acceptor equals that of the proton donor and pH = pK.  Henderson Hasselbalch equation,(relation between PH and pka. it is used in predicting the best buffer solution. − pH=pKa + log [A ] [HA]. At equilibrium, [A−] = [HA]. Therefore, at neutralization, pH = pKa.  Sources of H-ions 1- complete oxidation of glucose and fatty acids CO2 + H2O + ATP 2 Fixed acid production catabolism(oxidation) of sulfur containing amino acids giving sulfuric acid and phospholipids and phosphoproteins giving phosphoric acid 3- Unusual conditions : - Heavy exercise (lactic acid) - Diabetic ketoacidosis - Ingestion of acidifying salts (ammonium chloride, calcium chloride)  Body buffers: Bicarbonate system. Blood plasma buffer consists of carbonic acid (H2CO3) as proton donor and bicarbonate - (HCO 3) as proton acceptor: H2CO3 HCO3 + H+ Kidney H2CO3 HO2+CO2 Lung It is effective physiological buffer in the blood near pH 7.4  This system is one of the most effective buffer in the body because : Ø it is present in highe concentration; Øthe amount of dissolved CO2 is controlled by respiration (carbonic acid is easily formed from CO2 by carbonic anhydrase and easily excreted through lungs as CO2); Øplasma bicarbonate is regulated by kidney.  The base /acid ratio for this buffer system is 20/1at pH 7.4 it is a good buffer when it is being acidified, but very poor if the blood is alkalinated  Plasma proteins In the blood plasma, proteins are effective buffers because their free carboxyl and amino groups dissociate. This ionization enables to consume [H+] or [OH-] when an acid or a base is added to the system. When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or zwitterion. A zwitter ion can act as either an acid or abase  Hemoglobin Another buffer system is provided by the dissociation of the imidazol groups of the histidine residues in hemoglobin hemoglobin molecule contains 38 histidine residues.  Hemoglobin in blood has 6 times the buffering capacity of the plasma proteins. These buffers are responsible for buffering of the most CO2 added to the blood by tissues (CO2 bind to the N-terminal of Hb forming carbaminohemoglobin)  phosphate buffer system It is effective buffer in biological fluids; including, extracellular fluids and most cytoplasmic compartments at a pH range of 6.9 to 7.4.  Glucose-6- phosphate and ATP Nucleotides such as ATP, GTP glucose-6-phosphate, also act as buffering system  Physiological regulation of blood pH lungs, kidneys and liver play an important role in pH regulation:- Lungs function to reduce the pCO2 in the blood, - thus increasing the [HCO 3]/[ H2CO3] ratio. Kidneys serve to retain as much HCO 3 to the - blood as necessary, and to generate more by - + converting H2CO3 to HCO 3 and H. Processing of the carboxyl group of the amino - acid produces HCO 3 Processing of the amino group produces ammonium  Glutamine which is synthesized in kidney or released from the liver to the kidney is converted to bicarbonate and NH3. NH3 + H + NH 4- excreted HCO 3 + H - + H2CO3 HO2+CO2 exhaled Kidney can use phosphate to excrete about 40mmol of H+ per day.  Liver can regulate the pH by synthesis of neutral urea from the alkaline ammonia and synthesis of glycogen from lactic acid.  Disturbances of acid-base balance: Acid –base disorders fall into two main categories: metabolic and respiratory Acidosis and Alkalosis. Acidosis:- ↑[ H ], i.e ↓PH + Alkalosis: ↓[ H ], i.e ↑PH + [ ]= concentration - Metabolic: disturbance in [HCO3 ] R espiratory: disturbance in pCO2  Acid-Base Abnormalities Main pathology Compensated by pH pCO2 HCO3- pCO2 HCO3- Metabolic ↓ ↓ Respiratory ↓ acidosis alkalosis Metabolic ↑ ↑ Respiratory ↑ alkalosis acidosis Respiratory ↓ ↑ Metabolic ↑ acidosis alkalosis Respiratory ↑ ↓ Metabolic ↓ alkalosis acidosis  Metabolic acidosis HCO3- ↓, compensated by pCO2 ↓ most common causes are: 1. Formation of excess quantities of metabolic acids normally formed in the body such as keton bodies (DM, starvation), lactic acid (heavy exercise). 2. Large amount of acids in diet. - 3. Excessive loss of HCO 3 caused by diarrhea or using diuretic drug. + 4. Decreased excretion of H by kidneys caused by acute kidney failure. 5. Bacterial infection. 6. anoxia.  Metabo lic alkalo s is : HCO3- ↑, compensated by pCO2 ↑ Most common causes are: ¨ 1. Excess secretion of aldosterone (aldosterone stimulates the excretion of hydrogen ions). ¨ 2. Vomiting causes loss of the HCl secreted by stomach. ¨ 3. Using of alkaline drugs. Such as sodium bicarbonate for treatment of ulcer.  Res pir ato r y ac ido s is Due to hypoventilation, the pCO2 ↑(hypercapnia), producing a ↓in the - [ HCO3 ] / [ dissolved CO2] ratio.. Causes 1. chronic lung disease: asthma, emphysema. 2. Breathing air containing high CO2 3. depression of the respiratory centre by anaesthesia or morphine poisoning.  Respiratory alkalosis ¨ Hyperventilation for any reason results in a ↓ in pCO2 (hypocapnia), that decreases the availability of H ), producing ↑in the [ HCO3 ] + - / [ dissolved CO2] ratio... ¨ Causes: 1. Head injuries 2. influence of some drug 3. High respiratory rate because of fever, hysteria, anxiety, hot atmosphere, & high altitude.  The study of the compenents of the cell. Separation and purification of bio-molecules: 1. Sub cellular fractionation by - extraction - homogenization - centrifugation Ultra centrifugation 2. Cromatography 3.Electrophoresis 4.P CR (polymerase chain reaction) for sequencing and cloning DNA  Spectroscopy Atomic absorption X-ray crystallography  Cells Basic building blocks of life Smallest living unit of an organism A cell may be an entire organism (unicellular) or it may be one of billions of cells that make up the organism (multicellular). Grow, reproduce, use energy, adapt, respond to their environment Many cannot be seen with the naked eye - a typical cell size is 10µm; a typical cell mass is 1 nanogram.) Prokaryotes and Eukaryotes  Cells May be Prokaryotic or Eukaryotic Prokaryotes (Greek: pro-before; karyon- nucleus) include various bacteria - lack a nucleus or membrane-bound structures called organelles Eukaryotes (Greek: eu-true; karyon-nucleus) include most other cells (plants, fungi, & animals) - have a nucleus and membrane-bound organelles  Cell membrane & cell wall Cell Membrane Nucleoid region contains the DNA Nucleus Contain ribosomes (no membrane) Cytoplasm with organelles Character istic Bio-membr anes and Or ganelles Plasma Membran e-Cell’ s defin in g bo un dary Providing a barrier and containing transport and signaling systems. Enzyme marker : Na-K ATPase Nucleus – Cell’ s in fo rmatio n cen ter Double membrane surrounding the chromosomes and the nucleolus. The place where almost all DNA replication and RNA synthesis occur. The nucleolus is a site for synthesis of RNA making up the ribosome Mito cho n dria- the po wer gen erato rs Mitochondria : Surrounded by a double membrane with a series of folds called cristae. Contains its own DNA.Power House”of the cell Functions in energy production through metabolism. Food converted into energy(ATP) Consumes Oxygen, produces CO2 Endoplasmic reticulum (ER) – The transport network for molecules Rough endoplasmic reticulum (RER) Covered with ribosomes (causing the "rough" appearance) which are in the process of synthesizing proteins for secretion or localization in membranes. Ribosomes Protein and RNA complex responsible for protein synthesis Smooth endoplasmic reticulum (SER) A site for synthesis and metabolism of lipids.  Golgi apparatus -process and package the macromolecules. A series of stacked membranes. Vesicles carry materials from the RER to the Golgi apparatus. Vesicles move between the stacks while the proteins are "processed" to a mature form. Lysosomes-contain digestive enzyme A membrane bound organelle that is responsible for degrading proteins and membranes in the cell. Cytoplasm enclosed by the plasma membrane, liquid portion called cytosol and it houses the membranous organelles.