PHYSICAL BIOCHEMISTRY

BIOCHEMISTRY

1. PHYSICAL BIOCHEMISTRY Dr. Urender Singh P.G -1 S.G.T Medical college Gurgaon Haryana

2. 1. water

3. WATER Water is the most abundant fluid on earth. IT is justifiably regarded as the solvent of life. As much as 70% of a typical cell is composed of water .

4. One oxygen atom Two hydrogen atoms H 2 O Not symmetrical Electrons spend more time near the oxygen and less near the hydrogen Water molecule is polar Molecular Structure of Water O H H + + -

5. Hydrogen Bonds Between H 2 0 Molecules

6. Water as a Solvent Many important molecules in living things , especially sugars , have regions of partial positive and negative charge. These molecules attract water molecules and also dissolve in water . Such molecules are called HYDROPHILIC. Molecules that do not have polar regions , such as fats , tend to be insoluble. They are said to be HYDROPHOBIC

7. Properties of Water Density of water is 1g/ml. Boiling point --- 100 °C Freezing point ---0 °C Water is self-ionizing, that is ,there is a tendency for a hydrogen atom to jump from one oxygen atom to another forming OH - and H 3 O + in any volume of water there is a constant number of molecules that are ionized.

8. ICE In most liquids the density of the liquid increases as the temperature decrees. However ,ice is less dense than water. This is because at 4 ° C the water molecules are so close that they are all able to form hydrogen bonds with four other molecules. Because of the shape of the molecule, it requires the molecules to spread out and form an open latticework structure.

9. Resistance to Temperature Change The high specific heat of water is caused by hydrogen bonding. Because so much energy is required to break the hydrogen bonds , water resists temperature change. This is important because a number of biological processes can occur only within a narrow temperature change.

10. Physical States Vapor

11. BIOMEDICAL IMPORTANCE Water provides the aqueous medium to the organism which is essential for the various biochemical reactions to occur. Water directly participates as a reactant in several metabolic reactions . It serves as a vehicle for transport of solutes. Water is closely associated with the regulation of body temperature.

12. 2. Acids & Bases

13. Terms Acid Any substance that can donate a hydrogen ion (H + ) when dissolved in water Release of proton or H + Base Substance that can donate hydroxyl ions (OH - ) Accept protons or H +

14. Acids & Bases Acids: Arrhenius concept : Any substance that, when dissolved in water, increases the concentration of hydrogen ion (H + ) Bronsted -Lowry concept : A proton (H + ) donor Lewis concept : An electron acceptor Bases: Arrhenius concept : Any substance that, when dissolved in water, increases the concentration of hydroxide ion (OH - ) Bronsted -Lowery concept : A proton acceptor Lewis concept : An electron donor

15. Identifying Acids and Bases Acids have a pH from 0-7 , Lower pH value indicates a stronger acid. Bases have a pH from 7-14 , Higher pH value indicates a stronger base. Ampholytes: The substances which can function both as acids and bases are referred to as ampholytes. Example : water Alkalies ; The metallic hydroxides such as NaOH & KOH are commonly referred to as alkalies . These component do not directly satisfy the criteria of bases. However, they dissociate to from metallic ion and OH- ion. The latter, being a base accepts H+ ions.

16. Examples of Acids & Bases Acids HCL H 2 CO 3 H 2 PO 4 NH 4 + H 2 O Bases H + + CL _ H + +HCO 3 _ H + +HPO 4 _ H + +NH 3 H + +OH _

17. Acid-Base Balance Function Maintains pH homeostasis Maintenance of H + concentration Potential Problems of Acid-Base balance Increased H + concentration yields decreased pH Decreased H + concentration yields increased pH

18. 3. pH

19. pH pH is commonly expressed as –log[H + ] Pure water has [H + ]=10 -7 and thus pH=7. Acids have a high [H + ] and thus a low pH. Bases have a low [H + ] and thus a high pH.

20. pH: -Acidity The measurement of the H+ ions found in that particular substance The scale goes from 0 to 14 7 is neutral Below 7 is acidic Above 7 is alkaline (or basic) One pH unit represents a ten-fold change in H+ concentration

21. Terms Acidosis pH less than 7.35 Alkalosis pH greater than 7.45 Note: Normal pH is 7.35-7.45

22. Ways to measure pH pH meter Electrode measures H + concentration Must standardize (calibrate) before using. pH paper stripe

23. BIOMEDICAL IMPORTANCE pH affects solubility of many substances. pH affects structure and function of most proteins - including enzymes. Many cells and organisms (esp. plants and aquatic animals) can only survive in a specific pH environment.

24. 4. Buffers

25. Buffers Definition . : Buffers are defined as the solutions which resist change in pH by the addition of small amounts of acids or bases. A buffer usually consists of a weak acid and its salt (e.g. acetic acid and sodium acetate) or a weak base and its salt (e.g. ammonium hydroxide and ammonium chloride ).

26. Buffers A solution that resists change in pH Typically a mixture of the acid and base form of a chemical Can be adjusted to a particular pH value Blood : pH = 7.35-7.45 Too acidic? Increase respiration rate expelling CO 2 , driving reaction to the left and reducing H+ concentration. Excretory system – excrete more or less bicarbonate

27. How buffers work Equilibrium between acid and base. Example: Acetate buffer ---CH 3 COOH  CH 3 COO - + H + If more H + is added to this solution, it simply shifts the equilibrium to the left, absorbing H + , so the [H + ] remains unchanged. If H + is removed (e.g. by adding OH-) then the equilibrium shifts to the right, releasing H + to keep the pH constant

28. Step 1 : 1)CH 3 COONa (aq) ————> CH 3 COO - (aq) + Na + (aq) 2) CH 3 COOH (acetic acid) B)After Hcl addition CH 3 COO - ( aq ) + H + ( aq ) ⇌ CH 3 COOH Mechanism of buffer action

29. Step 2: 1)CH 3 COONa (aq) ————-> CH 3 COO - (aq) + Na + (aq) 2) CH 3 COOH (acetic acid) B)After NaOH addition 1)CH 3 COOH (aq) ————-> CH 3 COO - (aq) + H + (aq) 2) H + + OH - ---------------> H 2 O Mechanism of buffer action

30. Limits to the working range of a buffer Consider the previous example: CH 3 COOH  CH 3 COO - + H + If too much H + is added, the equilibrium is shifted all the way to the left, and there is no longer any more C H 3 COO - to “absorb” H + . At that point the solution no longer resists change in pH; it is useless as a buffer. A similar argument applies to the upper end of the working range.

31. Buffering Capacity The efficiency of a buffer in maintaining a constant pH on the addition of acid or base is referred to as buffering capacity .

32. BIOMEDICAL IMPORTANCE pH control is important, as many enzymes have a narrow range in which they function optimally. Buffering capability is essential for the well-being of organisms, to protect them from unwelcome changes in pH. For example, your stomach is about pH 1, yet the adjacent portion of your intestine is near pH 7—think about (or look up) how that might happen [Hint: what is one function of the pancreas?] Many compounds and macromolecules in addition to bicarbonate can serve a buffering function—proteins comprise one of the major classes.

33. 5. Solutions

34. Solutions Solutions are single phase homogeneous mixtures of two or more pure substances. In a solution, the solute is dispersed uniformly throughout the solvent .

35. Solutions The intermolecular forces between solute and solvent particles must be strong enough to compete with those between solute particles and those between solvent particles.

36. How Does a Solution Form? As a solution forms, the solvent pulls solute particles apart and surrounds, or solvates , them.

37. Classes of Solution True Solution – homogeneous mixture of two or more substances of ions or molecules. E.g. NaCl ( aq ) Suspensions- Heterogenous mix. of two or more then two substances ,filterable (>1000nm) E.g. mud, freshly squeezed orange juice. Colloid – appears to be a homogeneous mixture, but particles are much bigger, but not filterable .(1-1000nm) E.g.Proteins Fog, smoke, whipped cream, mayonnaise, etc.

38. Types of Solutions Saturated Solvent holds as much solute as is possible at that temperature. Dissolved solute is in dynamic equilibrium with solid solute particles.

39. Types of Solutions Unsaturated Less than the maximum amount of solute for that temperature is dissolved in the solvent.

40. Types of Solutions Supersaturated Solvent holds more solute than is normally possible at that temperature. These solutions are unstable; crystallization can usually be stimulated by adding a “seed crystal” or scratching the side of the flask.

41. Ways of Expressing Concentrations of Solutions

42. Mass Percentage mass of A solution Mass % of A =-------------------------------X 100 Total mass of solution A- solute eg . 9% saline (9g/100ml solution)

43. Parts per Million ppm = mass of A in solution total mass of solution  10 6 Parts per Million (ppm)

44. moles of A total moles in solution X A = Mole Fraction ( X ) In some applications, one needs the mole fraction of solvent , not solute — make sure you find the quantity you need!

45. NORMALITY (N) No. of gram equivalaents of A N= --------------------------------------------- volume of solution in LT. UNIT- GRAM EQ/L

46. mol of solute Per Lt. of solution M = Molarity ( M )

47. Molality No of moles of solute per 1000g of solvent. One molal solution can be prepared by dissolving 1 mole of solute in 1000g of solvent. moll Solute Molality (m) = ------------------------ mass of Solvent(kg)

48. Colligative Properties Changes in colligative properties depend only on the number of solute particles present, not on the identity of the solute particles. Among colligative properties are Vapor pressure lowering Boiling point elevation Melting point depression Osmotic pressure

49. Energy Changes and the Solution Process Intermolecular forces are also important in determining the solubility of a substance. “like” intermolecular forces for solute and solvent will make the solute soluble in the solvent.  H soln is sometimes negative and sometimes positive. Solvent – solvent interactions : energy required to break weak bonds between solvent molecules. Solute – solute interactions : energy required to break intermolecular bonds between the solute molecules. Solute – solvent interactions :  H is negative since bonds are formed between them. Solvent Solvent – solvent Solute Solute – solute + Solution Solute – solvent

50. 6 Colloids

51. Colloids Particles are too small to see but are larger than molecules. Colloids are mixtures of a solvent and suspended particles. Due to their small size they do not settle out of solution .

52. Colloidal State Colloidal stat is the particle have size of between true solution(less than 1nm) and visible particle (more then 100nm) Example: Dispersion of starch, fine fat droplets

53. Types of colloid: foam (liquid + gas, e.g. whipped cream), emulsion (liquid + liquid, e.g. milk), sol (liquid + solid, e.g. paint), solid foam (solid + gas, e.g. marshmallow), solid emulsion (solid + liquid, e.g. butter), solid sol (solid + solid, e.g. pearl, o pal). aerosol (gas + liquid or solid, e.g. fog and smoke),

54. Type of colloids Hydrophilic colloids: colloid particales are hydrophilic spread thoughout water (agar gel + proteins) Hydrophobic colloids: colloid system when the colloid partical are hydrophobic polymer. Protective colloids: small amount of hydrophilic colloid added to hydrophobic colloid increse its stability.

55. Removal of Colloid Particles Colloid particles are too small to be separated by physical means (e.g. filtration). Colloid particles are coagulated (enlarged) until they can be removed by filtration. Methods of coagulation: heating (colloid particles move and are attracted to each other when they collide); adding an electrolyte (neutralize the surface charges on the colloid particles). Dialysis: using a semipermeable membranes separate ions from colloidal particles.

56. Hydrophilic and Hydrophobic Colloids Most dirt stains on people and clothing are oil-based. Soaps are molecules with long hydrophobic tails and hydrophilic heads that remove dirt by stabilizing the colloid in water. Bile excretes substances like sodium stereate that forms an emulsion with fats in our small intestine. Emulsifying agents help form an emulsion.

57. Hydrophilic and Hydrophobic Colloids Sodium stearate has a long hydrophobic tail (CH 3 (CH 2 ) 16 -) and a small hydrophobic head (-CO 2 -Na+). The hydrophobic tail can be absorbed into the oil drop, leaving the hydrophilic head on the surface. The hydrophilic heads then interact with the water and the oil drop is stabilized in water.

58. Biological importance Biological fluids as colloids : these include blood, milk and cerebrospinal fluid. Biological compounds as colloidal particles: the complex molecules of life, the high molecular weight proteins, complex lipids and polysaccharides exist in colloidal state. Blood coagulation : when blood clotting occurs , the sol is converted finally into the gel. Fat digestion and absorption : the formation of emulsions, facilitated by the emulsifying agents bile salts, promotes fat digestion and absorption in the intestional tract.

59. 7. Diffusion

60. Diffusion Diffusion is the movement of solute molecules from a higher concentration to a lower concentration . Factors that affect the rate of diffusion: size of molecules , size of pores in membrane, temperature, pressure, and concentration .

61. BIOMEDICAL IMPORTANCE Exchange of O 2 and CO 2 in lungs and in tissues. Certain nutrients are absorbed by diffusion in the gastrointestinal tract e.g. pentoses , minerals, water soluble vitamins. Passage of the waste products namely ammonia, in the renal tubules occurs due to diffusion. Increased thickness of inflamed alveolar membrane reduces the diffusion of O 2 in pneumonia. Water, ion and small molecules pass largely by diffusion through plasma membrane.

62. 8. Osmosis

63. Osmosis The diffusion of water across a selectively permeable membrane . Osmosis is a type of Passive Transport. Osmosis requires NO ENERGY .

64. Osmosis – A Special kind of Diffusion Diffusion of water across a selectively permeable membrane (a barrier that allows some substances to pass but not others). The cell membrane is such a barrier. Small molecules pass through – ex: water Large molecules can’t pass through – ex: proteins and complex carbohydrates

65. Osmosis Osmosis is the movement of WATER across a semi-permeable membrane At first the concentration of solute is very high on the left. But over time, the water moves across the semi-permeable membrane and dilutes the particles.

66. Similarity Molecules move around to create equilibrium OSMOSIS Molecules go through a semipermeable membrane Just water DIFFUSION Molecules spread out over a large area Everything but water

67. Osmosis Hypertonic - a solution that causes a cell to shrink because of Osmosis. Hypotonic - a solution that causes a cell to swell because of Osmosis. Isotonic - a solution that causes no change in the size of the cell

68. Hypotonic – The solution on one side of a membrane where the solute concentration is less than on the other side. Hypotonic Solutions contain a low concentration of solute relative to another solution. Hypertonic – The solution on one side of a membrane where the solute concentration is greater than on the other side. Hypertonic Solutions contain a high concentration of solute relative to another solution.

69. Over time molecules will move across the membrane until the concentration of solutes is equal on both sides. This type of solution is called ISOTONIC .

70. Cytoplasm is a solution of water and solids (solutes dissolved in the water). Water moves into and out of cells because of the different concentrations of the solutes. Different kinds of cells react differently depending on the solution they are in. Below are examples of red blood cells in different types of solutions and shows what happened to the red blood cells.

71. PASSIVE TRANSPORT Passive transport occurs without expenditure of energy. Molecules move using their own kinetic energy . Diffusion and osmosis are examples of passive transport. Passive transport allows cells to get water, oxygen and other small molecules that they need. It also allows the cell to get rid of waste such as carbon dioxide. DIFFUSION OSMOSI S

72. Osmotic Pressure Osmotic pressure : the amount of pressure necessary to stop Osmosis. Small molecules such as water can move through certain types of materials (membranes). The tendency for this to occur is related to the molarity of the solution, is also a function of the temperature and is measured with a device called a Thistle tube. Osmol is the unit of osmotic pressure. Osmotic pressure is measured by Osmometer .

73. Reverse Osmosis Application of a pressure to the solution (that is equal to or greater than the Osmotic pressure) and the solvent flows from the more concentrated side to the other one. This process is used to obtain pure water from salt water.

74. Summary Diffusion is the movement of molecules from an area of higher concentration to an area of lesser concentration. Osmosis is the movement of water through a semi permeable membrane. Active transport requires energy . (molecules move from an area of lesser to higher concentration) Passive transport needs NO ENERGY ! (molecules move from an area of higher to lesser concentration).

75. Biological importance of osmosis Fluid balance - fluid balance of the different compartments of the body is maintained due to osmosis. Blood volume- osmosis significantly contributes to the regulation of blood volume and urine excretion Osmotic fragility test – In human blood, RBC begin to hemolyse in 0.45% NACl and the hemolysis is almost complete in 0.33% NACl . Increasd fragility of RBC is observed in hemolytic jaundice while it is decreased in certain anemia. Transfusion - isotonic solution of NACl or glucose are commonly used in I/V transfusion in hospitals for the treatment of dehydration, burns etc. Action of purgatives – Epson(MgSO4 7H2O) or Glauber,s (Na2SO4 10H2O) salts withdraw water from the body ,besides preventing the intestinal water absorption.

76. Biological importance of osmosis Osmotic diuresis – the high blood glucose concentration causes osmotic diuresis resulting in the loss of water,electrolytes and glucose in urine.This is the basis of polyuria observed in diabetes mellitus.diuresis can be produced by administering compounds( eg.mannitol ) which are filtered but not reabsorbed by renal tubules. Edema due to hypoalbuminemia – Disorders such as kwashiorkor and glomerulonephritis are associated with lowered plasma albumin concentration and edema.Edema is caused by reduced oncotic presssure of plasma,leading to the accumulation of excess fluid in tissue spaces. Cerebral edema – Hypertonic solutions of salts(NaCl,MgSO4) are in use to reduce the volume of the brain or the pressure of cerebrospinal fluid . Irrigation of wounds – Isotonic solutions are used for washing wounds

77. 9 Dialysis

78. Dialysis The disperse phase of a colloidal system have such large particle as cannot diffuse through the pores of a membrane made of cellophane, parchment, collodion , or inert cellulose ester like cellulose nitrate or acetate. However, such membranes are freely permeable to water and small molecules or ion in true solution .

79. Electrodialysis This is the another from of dialysis. Here the semipermeability and migration of electrolyte ion in an electric field are utilised in separating colloid particles from electrolyte ions.

80. BIOMEDICAL IMPORTANCE OF DIALYSIS Separation of proteins from small solutes : Dialysis is used to seprate proteins in pure form from the mixture with salts. It is used to separate out macromolecules cell extract .Dialysis can also be used for stopping enzymatic or metabolic reactions by removing small cofactor molecules from the cell extract. Biological ultrafiltrates –Many extracellular fluids like interstitial fluids, CSF, glomerular filtrate of kidney are formed by ultra filtration .Proteins do not appear in ultrafiltrate . Dialysis by artificial kidney – In patient with acute kidney failure and uremia ,blood is dialyzed in artificial kidneys to eliminate nonelectrolytes waste products as well as the excess of electrolytes.

81. 10. Donnan Membrane Equilibrium

82. DONNAN MEMBRANE EQUILIBRIUM When two solutions containing diffusible and non-diffusible ions are separated by a semipermeable membrane, the nondiffusible ions enhance the diffusion of oppositely charged diffusible ions. The diffusion takes place towards nondiffusible ion containing side. This also reduces the diffusion of like charged ions to that side. As a result , on the side which contains nondiffusible ions, diffusible counterions are more concentrated while the like charged diffusible ions concentrate more on the opposite side. This is called as GIBBS- DONNAN EFFECT.

83. Examples A B Na+[90] Na+[90] Na+[120] Na+[60] Pr-[90] Cl -[90] Pr-[90] Cl -[60] Cl -[30] Sodium NaCl Proteinate A B

84. SUMMARY The Donnan effect has brought the following changes in above example On the one side in which non-diffusible ion is present,there is accumulation of oppositely charged diffusible ions, i.e. Na+ . In the other side of the membrane ,the non-diffusible ions have made the accumulation of diffusible ions of the same, i.e. Cl - . The total concentration of all the ions will be greater in which the non-diffusible Pr- is present leading to osmotic imbalance between the two sides.

85. BIOLOGICAL IMPORTANCE OF DONNAN MEMBRANE EQUILIBRIUM Difference in the ionic concentrations of biological fluids- The lymph and interstitial fluids have lower concentration of inorganic cations ( Na+,K +) and higher concentration of anions( Cl -)compared to plasma. This is attributed to the higher protein(Pr-) content in the plasma. Membrane hydrolysis – The relative strength of H+ and OH- ions and, therefore, the acidic or alkaline nature on either side of a membrane, is influenced by the presence of non-diffusible ions. This phenomenon is referred to as membrane hydrolysis . Donnan membrane equilibrium explains the greater concentration of H + ions in the gastric juice. Lower pH in RBC – The hemoglobin of RBC is negatively charged and , therefore causes the accumulation of positively charged ions including H+. Therefore , the pH of RBC is slightly lower(7.25) than that of plasma(7.4) Osmotic imbalance – Donnan membrane equilibrium-which results in the differential distribution of ions in different compartments of the body- partly explains the osmotic pressure differences.

86. 11. viscosity

87. Viscosity

88. Viscosity Defined as the internal resistance offered by a liquid or gas to flow . Viscous liquids move slower. The greater the intermolecular forces the more is the viscosity. Poise is the unit of viscosity .

89. Viscosity of Common Liquids Liquid Viscosity [N s/m 2 ] Gasoline 600 Water 1000 Milk 1200 Beer 1800 Olive Oil 40,000 #10 Motor Oil 500,000 Pancake Syrup 2,500,000 Molasses 7,300,000 Honey 10,000,000 Ketchup 50,000,000 Peanut Butter 250,000,000

90. Biological importance Viscosity of blood – blood is about 4 times more viscous than water . The viscosity of blood is mainly attributed to suspended blood cells and colloidal plasma proteins . as the blood flows through capillaries the viscosity decreases to facilitate free flow of blood .Blood viscosity is increased in polycythemia (elevation of RBC) , while tit reduced in anemia and nephritis . A more viscous blood increases cardiac work load. When dehydration occurs , the viscosity of the blood increases. Viscosity change in Muscle –Excitation of the muscle is associated with increase in the viscosity of the muscle fibers . This delays the change in the tension of the contracting muscle. Vitreous body – this is an amorphous viscous body located in the posterior chamber of the eye . It is rich in albumin and hyaluronic acid. Synovial fluid – it contains hyaluronic acid which imparts viscosity and helps in the lubricating function of joints.

91. 12. SURFACE TENSION

92. SURFACE TENSION

93. SURFACE TENSION

94. Surface Tension The surface of any liquid behaves as if it was a stretched membrane. This phenomenon is known as surface tension Surface tension is caused by intermolecular forces at the liquid’s interface with a gas or a solid. Surface tension depends on the nature of the liquid, the surrounding media and temperature. Liquids that have strong intermolecular forces will have higher values of surface tension than liquids that have weak intermolecular forces.

95. Surface tension Surface tension is expressed in dynes

96. BIOLOGICAL IMPORTANCE OF SURFACE TENSION Digestion and absorption of fat – bile salts reduce the surface tension,act as detergents and cause emulsification of fat , thereby allowing the formation of minute particles for effective digestion and absorption. Hay,s sulfur test - Hay,s test is based on the principle that bile salt in urine lowers surface tension which is responsible for sulfur to sink. Surfactant and lung function- surfactant ( dipalmitoyl lecithin) deficiency cause respiratory distress syndrome (due to increase in surface tension of alveoli) in the infants. Surface tension and adsorption -due to the coupled action of these two process , the formation of complexes of protein and lipid occurs in the biological system.

97. 13. Adsorption

98. Adsorption Adsorption is a surface phenomenon. It is the process of accumulation of a substance ( adsorbate ) on the surface of another substance (adsorbent). Adsorption is differs from absorpition , as the later involves the diffusion into the interior of the material.

99. Adsorption The capacity of an adsorbent depend on the surface area. Adsorption is a dynamic and reversible process which decreases with rise in temperature.

100. Biomedical Importance Formation of enzyme-substrate complex- For the catalysis to occur in biological system, formation of enzyme-substrate complex is a prerequisite. This happens by adsorption of substrate on the enzyme.. Action of drugs and poisons-On adsorption at the cell surface, drugs and poisons exert their action. Adsorption in analytical biochemistry-this technique use for separation and purification of compounds (enzymes , immunoglobulins )