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Bt202 aug 24, 2011new Bt202 aug 24, 2011new Presentation Transcript

  • BT-202 Netaji Subhas Institute of Technology, Dwarka, New Delhi. Dr. Amita Pandey Aug 24, 2011
  • Syllabus for mid-term Properties of water ✔ Acids and bases and buffers ✔ Covalent bond ✔ Non-covalent interactions in biological system ✔ Carbohydrates ✔ Proteins Enzymes Lipids Nucleic acids Vitamins and Co-enzymes Separation technique for biomolecules
  • Learning check!
    • What is the molecular formula for glucose?
    • C6 H12 O6
    • CO2
    • C12 H22 O11
    • C12 H22 O11
    • Which of the following is a simple sugar or monosaccharide?
    • galactose
    • Sucrose
    • maltose
    • lactose
    • Maltose is composed of which two simple sugars?
    • Two fructose
    • Glucose and galactose
    • Glucose and fructose
    • Glucose and glucose
    • What kind of sugars are found in the disaccharide sucrose?
    • Fructose and galactose
    • Two glucose
    • Glucose and fructose
    • two fructose
  • Polysaccharides
    • Polysaccharides are polymers of monosaccharides also called glycans.
    • Polysaccharides differ from each other
    • -identity of the recurring monosaccharide units.
    • -length of their chains.
    • -degree of branching.
  •  
    • Starch in plants and glycogen in animals are the most important storage polysaccharide.
    • Both occur as intracellular granules or large clusters.
    • Heavily hydrated since they have many exposed hydroxyl groups.
    Homopolysaccharides as stored fuel
  • Starch and Glycogen
  • Starch
    • Contains amylose and amylopectin which are glucose polymers.
    • Amylose has long chains of D-glucose. residues connected by ( α1 4) linkage.
    • Amylopectin is branched ( α1 6). Branching occur every 24-30 residues.
    • Most abundantly found in tubers in plants.
    • Glycogen is more extensively branched (branch on average at every 8-12 residues). More compact than starch.
    • Liver (hepatocytes), skeletal muscles.
    • Dextran: used in Size exclusion chromatography.
    Glycogen
  • Homoploysaccharides as structural biomolecules
    • Cellulose
    • Cellulose is fibrous, tough, and water insoluble substance found in cell walls of plant cells.
    • It is linear, unbranched, consisting of D-glucose monomers with ( β 1 4)-linkage.
  • Cellulose
    • Termites, bacteria, ruminants, cattles, and wood fungus can break down
    • cellulose.
    • Why animals cannot use cellulose as a fuel?
  • Chitin
    • Composed of N -acetylglucosamine residues in ( β 1 4)-linkage.
  • Heteropolysaccharides as structural components
    • Glycosaminoglycans
    • repeating disaccharide units
    • -either N-acetylglucosamnie or N- acetylgalactosamine.
    • -D-glucuronic acid or L-iduronic acid.
    • esterified sulfate groups.
    • Present in ECM.
    • Hyaluronan
    • Chondritin sulfate
    • Keratan sulfate
    • Heparan sulfate
    • Peptidoglycan
    • Alternating
    • ( β 1 4)-linked
    • N -acetylglucosamine and
    • N -acetylmuramic acid units.
  • Polysaccharides as information carriers
    • Communication between cell and extracellular components.
    • Transportation of proteins.
    • Recognition sites for extracellular signal molecules.
    • Glycocalyx serves is made up of oligosaccharides.
  • Glycoconjugates
    • Proteoglycans
    • Glycoproteins
    • Glycolipids
  • Glycoproteins
    • Carbohydrate protein conjugates.
  • Glycoproteins
    • Eg. Mucins, glycophorin A, immunoglobins, and certain harmones, collagen.
    Oligosaccharide chains are attached either posttranslation or cotranslation modification.
    • Glycomics
    • systematic characterization of all the carbohydrate component of the cell or tissue, including those attached to proteins and to lipids.
    • What is the biological advantage of adding oligosaccharides to proteins?
    • How Lysozyme kills bacteria?
    • What is mode of action of penicillin?
  • Separation techniques for Carbohydrates
  • Chromatography
    • Mobile phase
      • Mixture dissolved in liquid or solid
    • Stationary phase
      • Porous solid matrix
    • Components of mixture pass through the column at different rates based on properties
  • Gel Filteration
    • Gel-filtration
      • Size/molecular exclusion chromatography
      • Stationary phase = gels with pores of particular size
      • Molecules separate based on size
        • Small molecules caught in pores
        • Large molecules pass through
  • Affinity chromatography
    • Affinity
      • Matrix chemically altered to include a molecule designed to bind a particular carbohydrate
      • Other carbohydrates pass through
  • Affinity Chromatography
    • Separation is based on highly specific interactions.
    • Lectin affinity chromatography is when lectin is used to separate components within a mixture. Lectin binds to carbodydrates. Eg. Concanavalin A
  • High Performance Liquid Chromatography (HPLC)
    • -Stationary phase = small uniform particles, large surface area
    • -a solvent is forced through under high pressures of up to 400 atmospheres.
    • -Adapt to separate based on polarity, size, etc.
  • Mass Spectrometery
    • Determines the mass-to-charge ratio of charged molecules.
    • loaded sample is vaporized.
    • ionization
      • Chemical ionization
      • Electrospray ionization
      • Matrix assisted laser desorption/ionization (MALDI)
      • Ions are separated by electromagnetic field
  • Oligosaccharides in a group of glycoproteins analyzed by MALDI MS
  • Learning check!
    • Heparin is routinely added to blood samples obtained for clinical analysis to
    • inhibit blood coagulation.
    • enhance blood coagulation.
    • activate thrombin.
    • enhance protease activity.
    • Which of the following are components of glycosaminoglycan?
    • N-acetylglucosamine
    • N-acetlygalactosamine
    • Sulfate
    • N-acetylmuramic acid
    • D-glucuronic acid
    • Hyaluronidase is an enzyme founf in pathogenic bacteria which hydrolysis
    • Chondritin sulfate
    • Keratan sulfate
    • Heparan sulfate
    • hyaluronan
  • Amino Acids, Peptides, and Proteins
  • Amino acids
    • The building blocks of proteins
      • Essential amino acids
    • 20 standard amino acids
    • Two functional groups:
      • carboxylic acid group
      • amino group on the alpha (  ) carbon
    • Have different side groups (R)
      • Properties dictate behavior of AAs
  • Zwitterions
    • Both the –NH 2 and the –COOH groups in an amino acid undergo ionization in water.
    • At physiological pH (7.4), a zwitterion forms
      • Both + and – charges
      • Overall neutral
      • Amphoteric
        • Amino group is protonated
        • Carboxyl group is deprotonated
  • Zwitterions
    • Soluble in polar solvents due to ionic character
    • Structure of R also influence solubility
  • Classification of Amino Acids
    • Classify by structure of R
      • Nonpolar
      • Polar
      • Aromatic
      • Acidic
      • Basic
  • Non-polar, aliphatic R groups
  • Polar, uncharged R group
  • Disulfide Bonds
  • Aromatic R groups
  • UV-Vis Spectroscopy
    • Absorbance used to monitor protein concentrations
  • Acidic and Basic Amino Acids
    • Acidic
      • R group = carboxylic acid
      • Negatively charged
    • Basic
      • R group = amine
      • Positively charged
      • His ionizes at pH 6.0
  • Uncommon amino acids
    • 4-hydroxyproline
    • 5-hydroxylysine
    • 6-N-methyllysine
    • Selenocysteine
    • Ornithine
    • Citrulline
  • Acid-base Properties
    • AAs also have multiple pK a ’s due to multiple ionizable groups
    pK 1 ~ 2.2 (protonated below 2.2) pK 2 ~ 9.4 (NH 3 + below 9.4) pK R (when applicable)
  • 3-letter and 1-letter abbreviations
  • pH and Ionization
  • Titration of Glycine
    • pK 1
      • [cation] = [zwitterion]
    • pK 2
      • [zwitterion] = [anion]
    • First equivalence point
      • Zwitterion
      • Molecule has no net charge
      • pH = pI (Isoelectric point)
      • pI = average of pK a ’s = ½ (pK 1 + pK 2 )
      • pI glycine = ½ (2.34 + 9.60) = 5.97
  • Titration curve of histidine -Aas with ionizable R groups have three pKa values
  • pI
    • For AAs with 3 pK a ’s, pI = average of two relevant pK a values
    • Consider lysine (pK 1 = 2.18, pK 2 = 8.95, pK R = 10.53):
    • Which species is the isoelectric form?
    • So, pI = ½ (pK 2 + pK R )
    • = ½ (8.95 + 10.53) = 9.74
  • Stereochemistry of AAs
    • All amino acids (except glycine) are optically active
    • Fischer projections:
  • D and L Configurations
    • d = dextrorotatory
    • l = levorotatory
    • D, L = relative to glyceraldehyde
  • Importance of Stereochemistry
    • All AA ’s found in proteins are L geometry
      • S enantiomer for all except cysteine
    • D-AA ’s are found in bacteria
    • Geometry of proteins affects reactivity (e.g binding of substrates in enzymes)
      • Thalidomide
  • Learning Check!
    • Glycine side chain is –CH3
    • Alanine side chain is CH2-phenyl
    • Phenylalanine side chain is -H
    • Cysteine side chain is –CH2-SH
    • Proline is the only one of the 20 standard amino acids that lacks a primary amine.
    • The side chain of histidine has pK close to neutrality. (T/F)
    • Give the single letter notation for any one of the polar neutral amino acids.
    • S, T, C, N, Q
  • The Peptide Bond
  • Polypeptides
    • Linear polymers (no branches)
    • Terminal residues:
      • Free amino group (N-terminus)
        • Draw on left
      • Free carboxylate group (C-terminus)
        • Draw on right
    • pK a values of AAs in polypeptides differ slightly from pK a values of free AAs
  • Naming Peptides
    • Name from the free amine (NH 3 + )
    • Use - yl endings for the names of the amino acids
    • The last amino acid with the free carboxyl group (COO - ) uses its amino acid name
  • Learning Check! Ser-Gly-Tyr-Ala-Leu serylglycyltyrosylalanylleucine
  • Calculate number of AAs present in a protein
    • For a simple protein
    • Molecular weight / 110
  • Separation and purification
    • Determine a source (tissue)
    • Extract protein
      • Suspend cell source in buffer
      • Homogenize
        • Break into fine pieces
        • Cells disrupted
        • Soluble contents mix with buffer
        • Centrifuge to separate soluble and insoluble
    • Separate protein of interest
      • Based on solubility, size, charge, or binding ability
  • Concentration of salts
    • Salting out
      • Continue to increase [salt] decreases [protein]
    • Different proteins salt out at different [salt]
    • Ammonium sulfate is commonly used salt.
  • Types of Chromatography
    • Paper
      • Stationary phase = filter paper
      • Same theory as thin layer chromatography (TLC)
      • Components separate based on polarity
    • High-performance liquid (HPLC)
      • Stationary phase = small uniform particles, large surface area
      • Adapt to separate based on polarity, size, etc.
  • Types of Chromatography
    • Ion-exchange chromatography
    • Gel filtration / size-exclusion chromatography
    • Affinity chromatography
  • Electrophoresis Separation of proteins based on their charge in an electric field.
    • -binds to proteins and gives them
    • a similar charge-to-mass ratio.
    • -Unfolds the proteins so that
    • all assume similar shape.
  • Isoelectric focusing
    • -determining isoelectric point of a protein.
    • -Gradient gel is prepared by ampholytes.
    • -2D electrophoresis
    • -separation of proteins
    • With identical molecular
    • Weight but different pI
    • E-mail address of one student required.
    • Electronic submission of assignments.
  • Agar
    • Present in cell walls of marine algae and sea weeds.
    • D-galactose and an L-galactose derivative ether linked between C3 and C6
    • Agarose solid support to grow bacterial colonies. Capsules in which some vitamins and drugs are packaged.
  •  
  •  
  • Ion Exchange Chromatography
    • Ion-exchange
      • Stationary phase = chemically modified to include charged groups
      • Separate based on net charge of carbohydrates
      • Anion exchangers
        • Cation groups (protonated amines) bind anions
      • Cation exchangers
        • Anion groups (carboxylates) bind cations
  • Ion Exchange Chromatography
  • Affinity Chromatography