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