Transcript
BT-202Netaji 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 ✔ProteinsEnzymesLipidsNucleic acidsVitamins and Co-enzymesSeparation 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 (β1 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 (β1 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(β1 4)-linkedN-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.
1. What is the biological advantage of adding oligosaccharides to proteins?
2. How Lysozyme kills bacteria?
1. 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 –NH2 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 pKa’s due to multiple ionizable groups
pK1 ~ 2.2(protonated below 2.2)
pK2 ~ 9.4(NH3
+ below 9.4)
pKR
(when applicable)
3-letter and 1-letter abbreviations
pH and Ionization
Titration of Glycine• pK1
– [cation] = [zwitterion]
• pK2
– [zwitterion] = [anion]
• First equivalence point
– Zwitterion
– Molecule has no net charge
– pH = pI (Isoelectric point)
– pI = average of pKa’s = ½
(pK1 + pK2)
– pIglycine = ½ (2.34 + 9.60) =
5.97
Titration curve of histidine
-Aas with ionizable Rgroups have three pKa values
pI
• For AAs with 3 pKa’s, pI = average of two relevant pKa
values
• Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):
• Which species is the isoelectric form?
• So, pI = ½ (pK2 + pKR)
= 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 –CH3Alanine side chain is CH2-phenylPhenylalanine side chain is -HCysteine 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
• pKa values of AAs in polypeptides differ slightly from pKa values of free AAs
Naming Peptides
• Name from the free amine (NH3+)
• 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 inan 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 molecularWeight 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
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