Chemistry notes

Dr. Ezihie

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Amino Acids

• building blocks of proteins

• Consist of

• an asymmetric -carbon covalently bonded to:

Hydrogen atom

An amino (-NH2) group (basic group)

Carboxyl (-COOH) group (acidic)

Variable R group specific to each amino acid

Amino acids All are -amino acids - the amino and carboxyl

are connected to the same -Carbon

20 amino acids make-up proteins

are programmed by genetic code

Known as standard , primary, common or normal Several other amino acids and amino acid

derivatives are found in the body free or in combined states (i.e. not associated with peptides or proteins).

General structure for amino acids

C

Amino acids are tetrahedral structures

COO-

NH3 COO

Carboxyl group

H3N +

Amino group

R Side

chain

Acid part Base part

Basic structure of an Amino Acid  amino acids are distinguished from one another by the

substituted R-group on the alpha-carbon atom

This substituent group is called the side chain, with H as the fourth substituent except for proline

Proline, is a five-membered secondary amine, with N and the  C part of a five-membered ring

properties of a.a differ due to side chains/R groups

Variation of the side group accrues from

Shape, Size, Hydrogen bonding capacity, Charge density 5

6 6

Structure of amino Acids

1. Alanine Ala

2. Arginine Arg

3. Asparagine Asn

4. Aspartic acid Asp

5. Cysteine Cys

6. Glutamine Gln

7. Glutamic Acid Glu

8. Glycine Gly

9. Histidine His

10. Isoleucine Ile

11. Leucine Leu

12. Lysine Lys

13. Methionine Met

14. Phenylalanine Phe

15. Proline Pro

16. Serine Ser

17. Threonine Thr

18. Tryptophan Trp

19. Tyrosine Tyr

20. Valine Val

Abbreviations and Codes for 20 primary amino acids

1. Polarity of the R group

– 4-main groups

• Hydrophobic amino acids

– Nonpolar (hydrophobic) side chains- made of hydrocarbons

– Reside predominantly in the interior of protein structure.

– prefer to be buried within the interior of protein molecules to minimize exposure to water.

– not ionize nor participate in the formation of H-bonds.

– Include Aliphatic nonpolar amino acids such as • the hydrocarbons -Ala, Met, Val, Leu, and Ile •Aromatic nonpolar amino acids-Trp & Phe •Iminoacid- proline

•

Classification of amino acids

Aliphatic nonpolar amino acids: Ala, Met, Pro

C H

COO-

H3N +

CH3

Alanine

Ala

C H

COO-

H3N +

Methionine

Met

CH2

CH3

CH2

C H

COO-

H2N +

CH2

Proline

Pro

CH2

Aliphatic nonpolar amino acids: Val, Leu, and Ile

CH2

C H

COO-

H3N +

C H

COO-

H3N +

CH3 C

CH3 CH3

Leucine

Leu

Isoleucine

Ile

CH3

CH2

C H

COO-

H3N +

CH3 CH3

Valine

Val

Aromatic nonpolar amino acids: Tyr, Trp and Phe

C H

COO-

H3N +

Phenylalanine

Phe

CH2

C H

COO-

H3N +

Tryptophan

Trp

CH2

NH CH

C H

COO-

H3N +

CH2

OH Tyrosine

Tyr Most hydrophobic

Amphipathic- OH group

Amphipathic- polar group- NH- H- bonds

absorb light strongly in the near UV region of the spectrum. approximate absorption maxima in the region -Phe-257 nm; Tyr= 275 nm and Trp;=280 nm

Hydrophobic amino acids

progressively more nonpolar (hydrophobic) from Ala to Leu/Ile. differ substantially in shape and relative bulkiness. Glycine is the simplest has a achiral carbon and is both nonpolar and polar properties Ala has a chiral center. All amino acids structures are derivatives of Alanine except for Gly Met contains a sulfur atom or a thiol ether (R-S-R) in its side chain

which is much less polar than an oxy-ether. Proline- is an imino acid-its side chain bonds to the alpha amino

group in a ring structure which cannot twist around the bond between the alpha amino group and the alpha carbon- inflexible structure

Hydrophobic amino acids Phe ,Tyr and Trp- have an have a benzene ring structure- are aromatic

Phe -most hydrophobic amino acid.

Tyr and Trp, are amphipathic; they have both polar and nonpolar ends

The polar groups (–OH in Tyr and >NH in Trp) can engage in H- bonding.

like most conjugated compounds, these a.a absorb light strongly in the near ultraviolet region of the spectrum.

The approximate absorption maxima for each of these amino acids is

Phe-257 nm ,

Tyr -275 nm

Trp-280 nm

• 20 different amino acids make up proteins

O–

H3N + C C

O– H

CH3

H3N + C

O–

CH3 CH3

CH3

C C

O– H

H3N +

CH3

CH2

H3N +

CH3 CH3

CH2

H3N + C

CH3

CH2

C H3N +

O–

CH2

C H3N +

O–

CH2

O–

H3N + C

CH2

H2C

H2N C

CH2

Nonpolar

Glycine (Gly) Alanine (Ala) Valine (Val) Leucine (Leu) Isoleucine (Ile)

Methionine (Met) Phenylalanine (Phe)

O–

Tryptophan (Trp) Proline (Pro)

H3C

O–

Non polar or Hydrohobic amino acids

Hydrophilic (polar amino acids) tend to interact with the aqueous environment,

are often involved in the formation of H-bonds

predominantly found on the exterior surfaces proteins or in the reactive centers of enzymes.

• Polar, uncharged amino acids

– Have no net charge at physiolgical pH

– Contain R-groups that can form H- bonds with H2O

– Includes amino acids with

– alcohol R-groups (Ser, Thr, Tyr)

– Amide groups: Asn and Gln

– Sulfhydryl group: Cys . Cys can form a disulfide bond (2 cysteines can make one cystine trough a disulfide link)

– more soluble in water except Tyr (most insoluble at 0.453 g/L at 25 C)

Polar, uncharged amino acids

CH2

C H

COO-

H3N +

Serine

Ser

C H

COO-

H3N +

Threonine

Thr

C H

CH3

C H

COO-

H3N +

CH2

OH Tyrosine

Tyr

C H

COO-

H3N +

Glycine

Gly

Polarity of Ser, Thr & Tyr is due to the hydroxyl group

Polar, uncharged amino acids

CH2

C H

COO-

H3N +

Cysteine

Cys

R-SH R-S- + H+

Polar, uncharged amino acids

C H

COO-

H3N +

Asparagine

Asn

CH2

O NH2

C H

COO-

H3N +

CH2

Glutamine

Gln

CH2

O NH2

Polar, uncharged amino acids Polar a.a are important since they

1. provide chemical groups that can interact with water.

2. the H-bonding character is key in forming protein structures.

3. the ionic bonding character of charged polar a.a is also important for protein structures.

4. provide chemically reactive groups in proteins.

Polar, uncharged amino acids  Serine has a OH group that does not normally ionize

 not charged in proteins (it is neutral).  the smallest of the polar amino acids and very polar.  The OH group provides enzymes with a very good nucleophilic group for reactions  also important to form esters with phosphate making phosphoester proteins.- proteins and

enzymes phosphyorylation is very important in regulation of activity.

 Threonine- obtained by adding a carbon on to Ser,  This makes the hydroxyl group less accessible than in Ser.  Thr is more often have important structural roles in proteins  not as chemically active as Ser.  can form esters with phosphoric acid  often found in proteins.

 Tyrosine is a hydrophobic aromatic alcohol  has some polar character.  The hydroxyl of Tyr is similar to the OH- group on phenol,  It can ionise in basic pH ( if pH is high)  can also form phosphoesters like Ser and Thr.  It is important in proteins and in enzymes for regulation of the cell cycle.

Polar, uncharged amino acids s

Cysteine-

 is essentially a thiol-Ala.

The thiol (-SH) group of Cys can ionize at about pH 8 hence are usually protonated at biological pH.

Asparagine-

a small and polar amino acid.

 Amides are neutral and do not ionize nor do they accept protons.

Glutamine

 is larger than Asn because of the longer side chain.

Both of the amides are neutral derivatives of acidic amino acids (Asp and Glu).

Formation of cystine Cystine is formed by the oxidation of 2 cysteine residues (2 thiols groups)-form a disulfide  Occurs in the presence of oxygen or oxidizing conditions, the react to bond between them. Normally between 2 polypeptide chains forming a cross link between the two.  Since this is a redox reaction, the hydride ion released by each thiol is usually coupled to an electon acceptor reaction or in simple oxidation with oxygen, hydrogen peroxide is usually formed with further reduction to water. Cys-Cys bonds common in extracellular proteins but rare in cellular proteins due to the reducing cellular environment

Acidic Amino acids

• Also known as dicarboxylic acids

• R-group contains a carboxyl group

• Include Asp and Glu

• Polar with a net negative charge at physiological pH(negatively charged pH > 3)

– Negative charges play roles such as

• Metal-binding sites during enzyme catalysis

• Carboxyl groups may act as nucleophiles in enzymatic interactions

• Electrostatic bonding interactions

Charged polar (acidic) side chains

C H

COO-

H3N + C H

COO-

H3N +

CH2

Aspartic acid

Asp

Glutamic acid

Glu

CH2

O O

- C

CH2

O O

Basic amino acids – R-group have net positive charges at pH 7

– Include His, Lys, and Arg

– Lysine has a protonated alky amino group

– Arginine has a guanidinium

– Histidine has an imidazolium ionized group

– Lys and Arg are fully protonated at pH 7

• Participate in electrostatic interactions

– His has a side chain pKa of 6.0 and is only 10% protonated at pH 7

– Because His has a pKa near neutral, it plays important roles as a proton donor or acceptor in many enzymes.

– His containing peptides are important biological buffers

Charged polar (basic) side chains

C H

COO-

H3N + C H

COO-

H3N +

C H

COO-

H3N +

NH2 +

NH2 Lysine

Lys Arginine

Arg

Histidine

His

CH2 CH2

HC C

CH2

NH3 +

CH2

NH H+N NH

Histidine Contains an imidazole ring that is partially protonated in

neutral solution Only the pyridine-like, doubly bonded nitrogen in histidine is

basic. The pyrrole-like singly bonded nitrogen is nonbasic because

its lone pair of electrons is part of the 6  electron aromatic imidazole ring

O–

CH2

C C

H3N +

O–

H3N +

OH CH3

C C

H O–

CH2

H3N + C

O– H3N

+ C C

CH2

H H H

H3N +

NH2

CH2

C C

O–

NH2 O

CH2

C C H3N +

O–

Polar

Electrically charged

–O O

CH2

C C H3N +

O–

O– O

CH2

C C H3N +

O–

CH2

NH3 +

CH2

C C H3N +

O–

NH2

C NH2 +

CH2

C C H3N +

O–

CH2

NH+

CH2

C C H3N +

O–

Serine (Ser) Threonine (Thr) Cysteine

(Cys)

Tyrosine (Tyr)

Asparagine (Asn)

Glutamine (Gln)

Acidic Basic

Aspartic acid (Asp)

Glutamic acid (Glu)

Lysine (Lys) Arginine (Arg) Histidine (His)

Polar amino acids

29 29 29

Structure of the 20 Amino acids

• Grouping based on the nature of the R- group 1. Aliphatic Chain Amino Acids : Gly, Ala, Val, Leu, Ile 2. Non-Aromatic Amino Acids with –OH: Ser, Thr 3. Sulfur-containing R-Groups : Cys, Met 4. Acidic Amino Acids :Asp, Glu, 5. Basic Amino Acids :Arg, Lys, His 6. Amino Acids with Aromatic Rings : Phe, Trp, Try, Pro 7. Amines: Arg, His, Lysine, Tryptophan

8. Neutral Hydrocarbon Side Chains:

9. Amide containing group:Gln, Asn,

Amino acid classifications

Essential and Non essential Amino Acids All 20 of the amino acids are necessary for protein synthesis

Essential : a.a required or protein synthesize but not synthesised by the body

must be obtained exogenously from the diet

8 (or 9 or 10) are essential

Essential Non essential

– Isoleucine Leucine Glycine Alanine

– Valine Phenylalanine, Serine Threonine

– Tryptophan Threonine Glutamate Asarpate

– Lysine Methionine Glutamine Asparagine

– Histidine, Arginine Proline Cystine

• Histidine and arginine are partially essentially – cannot be synthesized in appreciable amounts by the body

Nonstandard amino acids

• Amino acid derivatives- derived from the primary amino acids

• Nonstandard amino acids are usually the result of modification of a standard amino acid after a polypeptide has been synthesized.

• amino acids derivatives are components of proteins • Nonstandard amino acids play a variety of roles: – structural,- maintain the protein – antibiotics – signals – hormones – neurotransmitters – intermediates in metabolic cycles, etc.

Amino acid derivatives Structural roles

4-hydroxyproline and 5-hydroxylysine: structural components of collagen a structural protein

N-formyl methionine : the N-terminal residue of all prokaryotic proteins but is usually removed as the protein matures

Gamma-carbocyglutamate is part of proteins involved in blood clotting

Methylated and acetylated amino acids: are important parts of ribosomal proteins and chromosomal proteins called histones (important for chromatin formation in eukaryotes)

Amino acid derivatives Signal and neurotransmitters

1. Gama aminobutyric acid (GABA): a glutamate decarboxylation product of dopamine and glycine used as neurotransmitters

2. Histamine : is a mediator of allergic reactions

3. Thyroxine :is an iodine-containing hormone that stimulates metabolism

Intermediates in metabolic processes

1. Citrulline and ornithine : are important in urea biosynthesis

2. Homocysteine : an intermediate in amino acid metabolism

3. S-adnosylmethionine: is methylating agent (adds methyl groups to other compounds)

4. Beta-cyanoalanine : is an intermediate in cyanide production in plants.

5. Azaserine: is an antibiotic

Nonstandard amino acids

Properties of Amino Acids • Ionisation: Amino acid have atleast 2 ionizable groups--COOH and -NH2 the acidic and basic R-groups of the amino acids can also ionize. The charge properties are depended on the solution pH Neutral solution, the(around 7.4) the carboxyl (COOH )group will

be unprotonated and the amino (NH2 ) group will be protonated.

The resulting structures have “+” and “-” charges ie no ionizable R- group and is electrically neutral at this pH.

 This species is a dipolar ion, and is termed a zwitterion

 generally the amino terminal has a pKa~9.4 and carboxy-terminal is at pKa~2.0

Ionic forms of amino acids

COO -

H3N +

COO - H2N

COOH H3N

R Zwitterion

pH 7 Net charge 0

pH 1 Net charge +1 pH 13 Net charge -1

H+ H+

PROPERTIES OF AMINO ACIDS

Charge properties are important in separation of amino acid mixtures 38

Ionisation The net charge on any amino acid, peptide or protein

the algebraic sum of all the charged groups present

depend upon the pH of the surrounding aqueous environment.

the net charge changes as the pH changes

Easily observed in the titration of any amino acid or protein.

Isoelectric Points

In acidic solution, the carboxylate and amine are in their conjugate acid forms, an overall cation

In basic solution, the groups are in their base forms, an overall anion In neutral solution cation and anion forms are present and the overall

charge is zero.

 Isoelectric oint is the pH when an amino acid species has a net zero charge

pI= the arithmathetical mean of 2 Pka ie

(pKa1+pKa2)/2

pI depends on side chain

The 15 amino acids thiol, hydroxyl groups or pure hydrocarbon side chains have pI = 5.0 to 6.5 (av. of the pKa’s)

Aspartate and glutamate have acidic side chains and a lower pI Lysine , Arginine and histidine have basic side chains and higher pI

Titration Curves of Amino Acids

• If pKa values for an amino acid are known the fractions of each protonation state can be calculated (Henderson-Hasselbach Equation)

• pH = pKa – log [A -]/[HA]

• This permits a titration curve to be calculated or pKa to be determined from a titration curve

Separation of amino acid mixtures: Electrophoresis

Proteins have an overall pI that depends on the net acidity/basicity of the side chains

The differences in pI can be used for separating proteins on a solid phase permeated with liquid

Different amino acids migrate at different rates, depending on their isoelectric points and on the pH of the aqueous buffer

Properties of amino acids Chirality – stereospecificity/ Optical Activity

• the 20 amino acids except glycine are chiral - Central carbon is an asymmetric

• Exhibit optical activity -amino acids can be D or L

– L amino acids found in food

– D amino acids found in bacteria cell walls • Proteins are derived exclusively ference for amino acids is the Fischer

projection of L-serine

Acid base properties

In solution, Amino acids have two weak acid groups - COOH and -NH3+

both are able to lose a H+ to contribute to the [H+] of solution

At physiologic pH, a.a exist as zwitterions, a -COO- and a NH3+ with out a net charge

They can accept or give off an H+ as needed

Hence amino acids can act as pH buffers

Peptides • Amino acids can join via peptide bonds to form peptides • Polymerization occurs btwn the amino (-NH3

+) and carboxyl (-COO-) groups • Amino acids react in a head-to-tail fashion

– Elimination of a water molecules – Formation of covalent amide linkage (peptide bond) – Peptide bond formation is thermodynamically unfavorable-so reaction is

coupled • Peptides

– 2 amino acid (aa) residues - dipeptide – 3 aa residues - tripeptide – A few aa residues - oligopeptide – Many aa residues - polypeptide

• Proteins are molecules that consist of one or more polypeptide chains – Polypeptides range from 40 to 33,000 amino acids (most about 1500 aa)

• The Peptide Bond – Peptide Bond formed by condensing amino acids together to

form a peptide – They are called peptides because when the carboxyl group of one

amino acid joins to the amino group of another, a peptide bond is formed.

– Chemically it is an amide bond but when it occurs in proteins – peptide bond

– The partial double bond nature of the peptide bond means that there is not free rotation about the C -- N bond.

– The most stable conformationof the peptide bond is is planar and trans

Condensation of two amino acids to form peptide bond

Amino Acid Polymers

• Amino acids are linked by peptide bonds

DESMOSOMES

DESMOSOMES DESMOSOMES

CH2

H O

H OH OH

Peptide bond

H H

N N

SH Side chains

O O

O O O

H2O

CH2 CH2

CH2 CH2 CH2

C C C C C C

C C C C

Peptide bond

Amino end (N-terminus)

Backbone

(a)

Figure 5.18 (b) Carboxyl end (C-terminus)

Peptides and Proteins • Peptide chain (a.k.a. polypeptide) has direction. – N-Asparagine-Glutamate-Glycine-C – There is no set length of a polypeptide (how

long is a piece of string) although most polypeptides in nature are between 50 and 2000 residues long.

Peptide Linkages

• Two dipeptides can result from reaction between two a.as eg Ala and Ser, depending on which COOH reacts with which NH2 we get AS or SA

• The long, repetitive sequence of NCHCO atoms that make up a continuous chain is called the protein’s backbone

• Peptides are always written with the N-terminal amino acid (the one with the free NH2 group) on the left and the C-terminal amino acid (the one with the free CO2H group) on the right

• Alanylserine is abbreviated Ala-Ser (or A-S), and serylalanine is abbreviated Ser-Ala (or S-A)

Covalent Bonding in Peptides • The amino acids are lined by peptide bonds

• Amide nitrogens are nonbasic because their unshared electron pair is delocalized by interaction with the carbonyl group.

• This overlap of the nitrogen p orbital with the π orbitals of the carbonyl group imparts a certain amount of double-bond character to the C–N bond and restricts rotation around it.

• The amide bond is therefore planar, and the N–H is oriented 180° to the C=O.

Disulfides

• Thiols in adjacent chains can form a disulfide RS–SR through spontaneous oxidation

• A disulfide bond between cysteine residues in different peptide chains links the otherwise separate chains together, while a disulfide bond between cysteine residues in the same chain forms a loop

Structure Determination of Peptides: Amino Acid Analysis

• The sequence of amino acids in a pure protein is specified genetically

• If a protein is isolated it can be analyzed for its sequence- order of a.as

• The composition of amino acids can be obtained by automated chromatography and quantitative measurement of eluted materials using a reaction with ninhydrin that produces an intense purple color

• Prior to sequencing peptides it is necessary to eliminate disulfide bonds within peptides and between peptides – Done using 2-mercaptoethanol – reduces the disulfide

linkages

• To determine N-terminus – Sanger Agent: 2,4-dinitrofluorobenzene (DNF) detected by

yellow pigment observed via SDS-PAGE

– Dansyl Chloride: Like Sanger however detected via Fluorescence.

– Edman Degradation: sequential removal of amino terminal amino acid using phenylisothiocyanate.

– Edman now automated.

Structure Determination of Peptides: Amino Acid Analysis

• Due to the limitations of the Edman degradation technique,

peptides longer than around 50 residues can not be sequenced

completely!

– Trypsin: cuts carboxyl terminal of LYS, ARG

except Pro

– Chymotrpsin: cuts carboxyl terminal of Aromatic,

except Pro

– Carboxypeptidase A: not specific; cuts carboxyl

terminal of almost aas, except Lys & Arg, or if Pro

is terminal residue

– Carboxypeptidase B: not specific cuts carboxyl

terminal of Lys & Arg

Structure Determination of Peptides: Amino Acid Analysis

–Other techniques available: – Cyanogen bromide (CNBr): This reagent causes specific cleavage at

the C-terminal side of Met residues. • The number of peptide fragments that result from CNBr cleavage is equivalent to

one more than the number of Met residues in a protein. – The most reliable chemical technique for C-terminal residue

identification is hydrazinolysis.

– A peptide is treated with hydrazine, NH2-NH2, at high temperature (90oC) for an extended length of time (20-100hr).

– This treatment cleaves all of the peptide bonds yielding amino-acyl hydrazides of all the amino acids excluding the C-terminal residue which can be identified chromatographically compared to amino acid standards.

Structure Determination of Peptides: Amino Acid Analysis

Amino Acid Analysis Chromatogram

Peptide Sequencing: The Edman Degradation

• The Edman degradation cleaves amino acids one at a time from the N-terminus and forms a detectable, separable derivative for each amino acid

Peptide Sequencing: C-Terminal Residue Determination

• Carboxypeptidase enzymes cleave the C-terminal amide bond

• Analysis determines the appearance of the first free amino acid, which must be at the carboxy terminus of the peptide

Peptide Synthesis

• Peptide synthesis requires that different amide bonds must be formed in a desired sequence

• The growing chain is protected at the carboxyl terminal and added amino acids are N-protected

• After peptide bond formation, N-protection is removed

Carboxyl Protecting Groups • Usually converted into methyl or benzyl esters

• Removed by mild hydrolysis with aqueous NaOH

• Benzyl esters are cleaved by catalytic hydrogenolysis of the weak benzylic C–O bond

Amino Group Protection

• An amide that is less stable than the protein amide is formed and then removed

• The tert-butoxycarbonyl amide (BOC) protecting group is introduced with di-tert-butyl dicarbonate

• Removed by brief treatment with trifluoroacetic acid

Proteins

• Structure:

• Polypeptide chains of amino acids linked by peptide bonds

• The sequence of amino acids in a polypeptide is genetically determined

• Proteins consist of peptide bonds between 20 possible amino acid monomers

• They have a 3 dimensional globular shape

Functions of Proteins

Enzymes: accelerate specific chemical reactions up to 10 billion times faster than they would spontaneously occur.

Structure: Structural materials, including keratin (the protein found in hair and nails) and collagen (the protein found in connective tissue).

Antibodies: Specific binding, such as antibodies that bind specifically to foreign substances to identify them to the body's immune system.

Transport: Specific carriers, including membrane transport proteins that move substances across cell membranes, and blood proteins, such as hemoglobin, that carry oxygen, iron, and other substances through the body.

Movement : Contraction, such as actin and myosin fibers that interact in muscle tissue.

 Hormones and Receptors: Signaling, including hormones such as insulin that regulate sugar levels in blood.

•

PROPERTIES OF PROTEINS

A. Solubility Proteins vary greatly in their solubility in water Most globular proteins are soluble -many structural proteins are highly insoluble due to high cross-linkages Solubility of a protein is enhanced by the formation of weak ionic interaction eg H-bonds between solute molecules and water.  Protein solubility influenced by 1.Salt concentrations,  Addition of a small amount of neutral salt to a protein solution increases its solubility. the added ions causes changes in the ionisation of amino acids chains  interferes with interaction between protein molecules. This increases interactions btw protein solute and solvent to cause an increase in solubility.

Phenomenon known as salting in depends the ionic strength At very high salt conc., the abundance of interactions between the added ions and water decrease the possibilities for' protein water interactions. results in increased protein- protein interactions decreasing the solubility leading to protein precipitation. This phenomenal is known as salting out.

PH:-  influences the ionisation pattern on a protein the pattern of charge carried by the ionisable side chains changes to cause an alteration of the 3D structure of protein leading to protein denaturation under extreme pH the hydrophobic side groups of amino acids t are exposed to the aqueous environment decreasing the solubility considerably Solubility also decreases at an isoelectric pH- absence of a net change  at PI no repulsion between the protein molecules to prevent the formation insoluble aggregates. precipitated by treatment with acids such as trichloroacetic perchloric, picric - form acid-insoluble salts with protein cations. Ppt by acids used to remove proteins from solutions prior to analysis.

PROPERTIES OF PROTEINS • Organic solvents:-

Lowers the dielectric constant of water

Results in increase of the attractive forces btn groups of opposite charge within a protein molecule

this diminishes their linkages with the surrounding water molecules.

The techniques involving varying pH, salt concentration and organic solvent are used to separate mixtures of proteins by differential precipitation.

• Temperature:-

solubility of proteins increases with the temperature up to about 40°C - 50°C.

Above, thermal agitation disrupt tertiary structure leading to denaturation and sharp decrease in solubility.

2. Acid base properties an acid is a proton donor while a base is a proton acceptor proteins have large numbers of ionisable groups that contribute to the acid-base properties. Include the N- and C- termini, amino acid side chains and some other attached groups. At low pH there are more positively charged groups than negatively charged, the protein is cationic and migrates towards the cathode in an electric field. At high pH, negatively charged groups predominate and the proteins is anionic. At isoelectric pH, there are equal numbers of positive and negative changes is a protein &hence immobile in an electric field. A protein with a relatively high content of basic amino acids will have a high isoelectric point, one with a high content of acidic amino acids will have a low isoelectric pH. protein ionisation and isoelectric pH is used to resolve the mixture of protein by an electrophoretic and ion exchange chromatography techniques.

PROPERTIES OF PROTEINS

• 3. Osmotic pressure

 Proteins are large sized molecules and do not pass through semi-permeable membranes e.g. the capillaries blood vessels.

 Proteins contained within such membranes exert an osmotic pressure,

4. Hydrolysis

 Proteins may' be hydrolysed by heating with acids or alkali or by action of protedytic enzymes

 The products of hydrolysis are peptides and amino acids

Protein Conformation and Function

• Conformation is the 3D arrangement of atoms in space

• A protein’s specific conformation determines how it functions

• There are four Levels of Protein Structure that determines function

Primary Structure • Linear amino acid sequence, the order of amino acids and the

location of disufide bonds if available in a protein • Describes the covalent connections within the protein • Determined genetically • Unique sequence of amino acids in a polypeptide • Slight change in primary structure can alter function • Condensation synthesis reactions form the peptide bonds between

amino acids

+H3N Amino end

Carboxyl end

Secondary Structure

• Repeated folding or coiling of the polypeptide into a repeating configuration

• Folding of the polypetide backbone without reference to the R- groups

• stabilized by H bonds between peptide linkages in the protein’s backbone

• 2 types, alpha helix, beta pleated sheets

Alpha-helix: •The formation of the a-helix is spontaneous • stabilized by H-bonding between amide nitrogen's and carbonyl carbons of peptide bonds spaced four residues apart. •disrupted by proline

Beta pleated sheets: •are composed of 2 or more different regions of stretches of at least 5-10 amino acids. •The folding and alignment of stretches of the polypeptide backbone aside one another to form beta-sheets •stabilized by H-bonding between amide nitrogens and carbonyl carbons beta-Sheets are either parallel or antiparallel. •parallel sheets adjacent peptide chains proceed in the same direction (i.e. the direction of N-terminal to C-terminal ends is the same), •antiparallel sheets adjacent chains are aligned in opposite directions. •There are also Super-secondary Structures (helix-turn-helix, helix-loop-helix and zinc finger domains of eukaryotic transcription factors);

http://web.mit.edu/Local Settings/Temp/Animation 5.4.3 Secondary St.MOV

O C  helix

 pleated sheet

Amino acid subunits N C

C N

O H

C N

O H

R C

O H

H H

N H

C R

N H

O C N

C H R

N H

O C

C H

N H

O C

C R

N H

O C

C R

N H

O C

C H

Tertiary Structure • Overall folding of o the protein consisting both the backbone and

the R-groups (overall three-dimensional shape of a polypeptide)

• Results from interactions between amino acids backbone and the R groups

• 3-D structure maintained by weak bonds including: – H bonding between polar side chains

– ionic bonding between charged side chains

– hydrophobic

– van der Waals interactions

• Strong bonds: – disulfide bridges form strong covalent linkages

Interaction between amino acid residues Hydrophobic amino acids inside Hydrophilic amino acids on the surface Also present could be disulfide bonds. tertiary structure also describes the relationship of different domains to one another within a protein

Tertiary Structure

CH2 CH

O H

C HO

CH2

CH2 NH3 + C -O CH2

CH2 S S CH2

CH3

H3C

Hydrophobic interactions and van der Waals interactions

Polypeptide backbone

Hyrdogen bond

Ionic bond

CH2

Disulfide bridge

Quaternary Structure • The degree of associations of two or more polypeptide chains

– 2 or more different polypeptide chains that are held in association by the same non- covalent forces that stabilize the tertiary structures of proteins.

– The intrachain disulfide bond is the one covalent bond involved in maintenance.

– The interchain disulfide bond of the tertiary structure can also stabilize quaternary structure.

– Results in domains, subunits, monomers

– Dimers or tetramers

– Homo- dimers, heterodimers

– Possible for oligomeric proteins

Polypeptide chain

Collagen

 Chains

 Chains

Hemoglobin

Iron

Heme

• The four levels of protein structure

+H3N

Amino end

Amino acid subunits

helix

Factors That Determine Protein Conformation

Occurs during protein synthesis within cell

Depends on environmental physical conditions such as

pH, temperature, salinity, etc.

Change in environment may lead to denaturation of protein

Denatured protein is biologically inactive

Can renature if primary structure is not lost

Sickle-Cell Disease:

– A Simple Change in Primary Structure

– Results from a single amino acid substitution in the protein hemoglobin

Hemoglobin structure and sickle-cell disease

Fibers of abnormal hemoglobin deform cell into sickle shape.

Primary structure

Secondary and tertiary structures

Quaternary structure

Function

Red blood cell shape

Hemoglobin A

Molecules do not associate with one another, each carries oxygen.

Normal cells are full of individual hemoglobin molecules, each carrying oxygen







10 m 10 m





 

Primary structure

Secondary and tertiary structures

Quaternary structure

Function

Red blood cell shape

Hemoglobin S

Molecules interact with one another to crystallize into a fiber, capacity to carry oxygen is greatly reduced.

 subunit  subunit

1 2 3 4 5 6 7 3 4 5 6 7 2 1

Normal hemoglobin Sickle-cell hemoglobin

. . . . . . Exposed hydrophobic region

Val Thr His Leu Pro Glul Glu Val His Leu Thr Pro Val Glu

Protein Classification

• Simple proteins yield only amino acids on hydrolysis

• Fibrous proteins consist of polypeptide chains arranged side by side in long filaments

– Fibrous Proteins: keratin (hair, feathers, claws, wool, skin); silk

– repeating gly-ser-gly-ala-gly-ala

• Globular proteins are coiled into compact, roughly spherical shapes

– hydrophobic inside and hydrophilic outside

– Globular Proteins: insulin, plasma albumins, globulins, hemoglobin

– Most enzymes are globular proteins

Protein Classification

• Simple proteins- yields amino acids only on hydrolysis

• generally have structural roles- Example-

• Fibrous proteins

• polypeptide chains arranged side by side in long filaments

– keratin (hair, feathers, claws, wool, skin); silk

– repeating gly-ser-gly-ala-gly-ala

Conjugated proteins, proteins conjugated to a non-protein component yield other cpds eg carbohydrates,fats, or nucleic acids in addition to a.a on hydrolysis  classified in regard to the conjugated group- phosphoproteins, glycoproteins, lipoproteins much more common than simple proteins, Globular proteins are coiled into compact, roughly spherical shapes hydrophobic inside and hydrophilic outside Example: insulin, plasma albumins, globulins, hemoglobin, most enzymes

Some Common Fibrous and Globular Proteins

-Keratin • A fibrous structural protein coiled into a right-handed helical secondary

structure,

• -helix stabilized by H-bonds between amide N–H groups and C=O groups four residues away a-helical segments in their chains

Fibroin

• Fibroin has a secondary structure called a b-pleated sheet • the polypeptide chains line up in a parallel arrangement held

together by hydrogen bonds between chains

Myoglobin

• Myoglobin is a small globular protein containing 153 amino acid residues in a single chain

• 8 helical segments connected by bends to form a compact, nearly spherical, tertiary structure

100

• Collagen:

– Triple helix of 25% glycine, 25% proline and hydroxyproline

• Proline is inflexible because it is an imino acid with side chain attached to backbone in two locations

• Therefore it is a long extended helix

• It can’t bend as much as a chain of amino acids

– skin, cartilage, bone, intercellular glue

101

– Refers o the partial or complete unfolding of the polypeptide

– Is when a protein unravels and loses its native conformation

– Only affects the secondary, tertiary and quartenary stucture

– Digestion affects also the primary structure

Denaturation

Renaturation

Denatured protein Normal protein

Protein denaturation

102

Alteration in pH:- . changes the ionic states of ionisable group of the protein (-NH2, COO - & some side groups) this breaks the hydrogen bonds & other weak forces to creat regions of charge repulsions Also disrupts the ionic pairs affects proteins, 2,3 & 4, leading to protein denaturation. large net positive or large negative charges on the protein leads to the repulsion among the ionasable groups this places the molecules under strain leads to denaturation

AGENTS OF PROTEIN DENATURATION

103

Temperature •High temp increase the thermal energy •leads to an increase in vibrational and rotational energy •this upsets the delicate balance of weak interactions of the 2, 3 & 4 quaternary structure • destabilies the functional folded conformation. •high temperature can cause irreversible inactivation through covalent changes e.g. deammination of asparagines or glutamine residues. •Heat denaturation usually results in eventual protein precipitation due to the destruction of 2 •advantage in sterilization of surgical instruments canning and cooking of foods

104

Chaotropic agents: Cpds such as urea and quanidine hydrochloride Often used in high concentrations e.g. 4M or 8M. Allows water molecule to penetrate into the interior of the proteins and solvate the non-polar side chains to disrupt the secondary tertiary and quartenary structures hence disrupts the hydrophobic interaction that normally stabilise the native conformation. weaken the hydrophobic interactions in the proteins They also form a competing hydrogen bond with the amino acids residues of the peptide to destabilizing the internal hydrogen bonding that stabilizes the native structure. Such denaturation is usually reversed when the concentration of the competitive hydrogen bonding agent is lowered by dialysis (sieving) or dilution.

105

Detergents •Example Sodium dodecyl sulphate (SDS) • detergents have hydrophobic tails, •The hydrophibic tails penetrate into the hydrophobic interior of proteins and disrupts the hydrophobic interior and thereby denaturing the proteins. •Detergents also disrupts association between polypeptide chains

106

Thiol reagents •Examples dithiothreitol (DTT) and beta-mercaptoethanol ( SH CH2 CH20H). •They cleave the desulphide bonds in addition to disruptions of hydrophobic interactions and hydrogen bonds. •These bonds are reduced to sulphurhydryl groups SH while also disrupting the thiol reagent Miscible organic solvents. competes with proteins for the H2O of hydration Hence lowers the dielectric constant of the protein. cause protein- protein interaction causing precipitation. Examples are alcohols, phenol or acetone. Heavy metal ions. Eg Ag+ Hg+ Pb 2+ involves reactions of sulfurhydryl side groups of cysteine either on the same polypeptide chain or different chains The tiol groups willreact with heavy metal ions to alter the protein 2 , 3and 3 structures and cause protein precipitation

107

The Protein-Folding Problem

• Most proteins – Probably go through several intermediate

states on their way to a stable conformation • Chaperonins – Are protein molecules that assist in the proper

folding of other proteins

X-ray crystallography- Is used to determine a protein’s three-dimensional structure

108

Hollow cylinder

Cap

Chaperonin (fully assembled) Steps of Chaperonin

Action: An unfolded poly- peptide enters the cylinder from one end.

The cap attaches, causing the cylinder to change shape in such a way that it creates a hydrophilic environment for the folding of the polypeptide.

The cap comes off, and the properly folded protein is released.

Correctly folded protein Polypeptide

Figure 5.23

109

Lipids heterogenous group of hydrophobic molecules

made of C,H,O

Greasy or oily

No general formula.

Fats - solid at room temperature.

Oils - liquid at room temperature

110

Functions:

 Energy storage

 membrane structure

 Protecting against desiccation (drying out).

 Insulating against cold.

 Absorbing shocks.

 Regulating cell activities by hormone actions.

111

Fatty acids  Building blocks of lipids  A long hydrocarbon chain (12-36 C)  Have a hydrophilic group -COOH (acid) on one end and a –

hydrocarbon CH3 (fat) at the other.

112

Structure of Fatty Acids

 Functions of fatty acids • components of more complex membrane

lipids. • components of triacylglycerols a stored fat

• When they are part of lipids, the fatty acids resemble long flexible tails.

• They are known as acyl groups in lipids

113

Structure of Fatty Acids

• β α

H3C-(CH2)n - C – C – C-COOH

• 3 2 1

C 2 & 3 are often referred as  and  respectively.

They methyl carbon atom at the distal end of the chain is called the omega carbon.

Nomenclature of fatty acids

115

Saturated fatty acids

– Have the maximum number of hydrogen atoms possible – Have no double bonds but single C-C bonds in the

hydrocarbon tails – solid at room temp – most found in animal fats

• .

saturated Fat fatty acids

116

(a) Saturated fat and fatty acid

Stearic acid

Example of Fatty Acids: saturated Common

name

Systematic name Structure Abbreviation

Laurate CH 3 (CH

2 )10COO- 12:0

Myristate Tetradecanoate CH 3 (CH

2 )12COO- 14:0

Palmitate n-hexadecanoate CH 3 (CH

2 )14COO- 16:0

Stearate n-octadecanoate CH 3 (CH

2 )16COO- 18:0

Arachidate n-eicosanoate CH 3 (CH

2 )18COO- 20:0

Behenoate n-docosanoate CH 3 (CH

2 )20COO- 22:0

Lignoceate n-tetracosanoate CH 3 (CH

2 )22COO- 24:0

Carbon atoms are numbered from the carboxylate group. the carboxyl group is readily ionized,  have a negative charge at physiological pH.

117

Unsaturated fatty acids one or more C=C bonds in their structure. Vary in the length and number and locations of double bonds they contain D. bonds cause “kinks” in the in the hydophobic tails molecule’s shape. I.e d-b pushes the molecules apart, lowering the density, mp liquid at room temp most plant fats

118 (b) Unsaturated fat and fatty acid

cis double bond causes bending

Oleic acid

Palmitoleate

C is 9-hexadecenoate CH 3 (CH

2 ) CH=CH(CH

2 )7COO- 16:1

Oleate C is 9-octadecenoate CH 3 (CH

2 ) CH=CH(CH

2 )7COO- 18: 1

Vaccenate C is 11-Octadecenoate CH 3 (CH

2 )5CH=CH(CH

2 )9COO- 18:1 1

Linoleate Cis9,Cis12-Octa-

decadienoate

CH 3 (CH

2 )4CH=CHCH

2 CH =CH(CH

2 )7COO-

Unsaturated fatty acids

119

the "kink" or bend, in the H- tails, in the unsaturated fats prevents close packing together kinks in the structure make cell membranes flexible & permeable  makes the fatty acid stay fluid at r.t Some mono-unsaturated fats, eg olive oil will solidify when refrigerated due less number of unsaturations . Poly-unsaturated fats, stay fluid even when un refrigerated due to many kinks in their structures. kinks in the structure make cell membranes flexible & permeable and nutrients to enter the cell and waste products to leave.

Why trans-fats are so bad for us Naturally-occurring unsaturated vegetable oils have almost all cis bonds, Heat eg frying causes some of the cis bonds of an oil to convert to trans bonds. Only few bonds are involved like in the frying an egg and hence it is not bad Cis bonds are however converted to trans form when the oil is constantly and repeatedly used as in fast food French fry machines, more and more of the cis bonds are a significant numbers of fatty acids with trans bonds build up.

121

Hydrogenation bubbling hydrogen through polyunsaturated oils also creates"partially

hydrogenated" fats The chemical structure is the same.-same number of carbon, oxygen

and hydrogen atoms, the same COOH acid at the alpha end, and the double bond is in the same place—but now it's straight instead of kinked.

These are less vulnerable to becoming rancid than the original oils  have a longer shelf life. hydrogenation process also converts the bent "cis" form to a

straightened "trans" form fatty acids with trans bonds are carcinogenic, may lead to type2 diabetes

due to cells becoming resistant to insulin.

122

The body recognizes this chemical structure and tries to use it in the same places and for the same purposes that it uses the bent cis form. But the trans form stacks together just like saturated fats, which sabotages the flexible, porous functionality the body needs from unsaturates. Exposure to prolonged heat (as in deep fat frying) also creates trans fats by loosening the double bond and allowing it to "flip" into the straight form. Poly-unsaturated fats  poly-unsaturated fats have more than one H-pair is missing. The more pairs that are missing, the more kinks in the chain and the more fluid the oil.

123

Omega fatty acids Consists of Omega-3, omega-6 and omega-9 The acid end of the molecule is called the "alpha", and its opposite is the "omega" end. Position of first double bond from the omega carbon determines the type of omega fatty acid The first double-bond kink in an omega-3 fatty acid occurs between the third and fourth carbon atoms away from the omega end- omega -3.

linolenic acid (ALA, an omega-3)  an omega-6, between the sixth and seventh. an omega-9, between the ninth and tenth. Essential fatty acids

human body needs and cannot manufacture some fatty acids in sufficient amounts linolenic acid (ALA, an omega-3) and linoleic acid (an omega-6)—are the two essential fatty acids

Functions of fatty acids

• Fatty acids serve the following major roles in the body • Building blocks for phospholipids and glycolipids in biological

membranes • Target proteins to membranes • High energy source of fuel • Fatty acid derivatives are used as hormones and intracellular

messengers • Act as components of more complex membrane lipids. • Act as the major components of stored fat in the form of

triacylglycerols.

125

Classification of lipids

• Classification based on

• Solubility – Water soluble ( polar) Vs fat soluble (nonpolar)

• Water soluble –lipids

• Short chain fatty acids such as ethanoate, pronanoate, and butanoate

• have appreciable water solubility as do their derivatives the ketone bodies, acetoacetate and β-hychroxybutryate.

• The solubility of fatty acids in water decreases progressively as the hydrocarbon chain increases.

126

Classification of lipids • Chemical reactions

– Saponifiable Vs nonsaponifiable lipids – Saponification is the hydrolysis of lipids using a base such as

sodium hydroxide. – Saponifiable lipids contain atleast on ester group, which must

undergo hydrolysis in the presence of acid, base or enzyme. – Hydrolysis process cleaves a saponifiable lipid into two or more

smaller molecules. – Saponifiable lipids are not derivatives of long chain fatty acids, and

are more soluble in organic solvents than in water. – Non-saponifiable lipids do not undergo hydrolytic cleavage into

smaller molecules in presence of acids or bases. – Common examples of saponifiable lipids include, sterols, terpenes,

leukotrienes and prostaglandins

127

Saponification • Saponification process can be used in the manufacture soaps and

detergents.

• Saponification glycerol by a base yields salts of the carboxylic acids and glycerol.

• The carboxylate salts formed have considerable high water solubility.

• This is important in biological systems since the body mustsolubilise compounds containing the COOH group.

• Such compounds, especially drugs are usually administered in the form of carboxylate salt instead of the carboxylate acid to achieve faster absorption into the body.

• The salts of carboxylic acids formed by a saponification process are known as soaps.Soaps are used for removing off greasy dirt from surfaces such as machinery clothing floors and other surfaces.

128

129

Saponifiable lipids

SAPONIFIABLE

SIMPLE LIPIDS

ACYLGLCEROLS

FATS OILS

WAXES

COMPOUND

PHOSPHOLIPIDS PLASMALOGENS

PHOSPHOGLYCERIDES

SPHINGOLIPIDS

GLYCOLIPIDS SPHINGOMYELIN

Classification of lipids

• Chemical compositions

– Simple lipids -Fatty acids or esters of fatty acids with alcohol groups

– Complex –compound-esters of fatty acids with alcohol groups and other groups

– Derived lipids-Derivatives of simple or complex lipids

130

Classification of lipids

Simple lipids Include : fatty acids, Fatty acid esters, and waxes Fatty acid esters  Known as Acylglycerols or neutral fats.  formed by the esterificaiton of fatty acids to a glycerol (alcohol ) backbone.  The esterified fatty acids known as the acyl groups.  f.a esters are found in plants and animals (adipose tissue)  Three types of acylglcerols.  Monoacylglcerols:- one acyl group esterified to a glycerol  Diacyglyerols :2-acyl groups esterified to OH of glycerol  Triacylacylglycerols:- 3 acyl groups esterified to 3-OH  TAGs are the common lipids in the body, MAG and DAG are derived from the

hdrolysis of TAGs

131

Triacylglycerols Also known as neutral Fats

Build from, 3 f.a esterified to to a glycerol backbone

Glycerol + 3 fatty acids

3 ester linkages are formed between a hydroxyl group of the glycerol and a carboxyl group of the fatty acid.

TAGs are the most common in the body Simple TAGs –similar R-groups Mixed TAGs -differ in fatty acids composition

Used for energy storage, cushions for organs, insulation.

TAGs have more C-H bonds - provide more energy per mass.

132

133

Monoacylglcerols They consist of an acyl group esterified to a glycerol backbone.

Diacyglyerols (diglycerides) Consist of two acyl groups esterified to two hydroxyl groups of a glycerol backbone.

Triacylglycerols Triacylglycerols consist of three acyl groups esterified to the three hydroxyl groups of glycerol backbone.

Acid Fat

134

Structure of Triacylglycerols

135

Waxes

• Esters of long chain alcohols and long chain fatty acids

• serve as coatings for plant parts and as animal coverings.

Complex/compound lipids esters of fatty acids containing other groups in addition to alcohol and fatty acid.  alcohol moiety is usually glycerol or sphingosine. Examples Phospho lipids  glycolipids.

 phospholipids lipids are major class of membrane lipids. Have only two fatty acids Have a phosphate group instead of a third acyl group They are two types

Phosphoglycerols phosphosphingolipids,

137

Glycerol based phosholipids – have glycerol as the alcohol backbone Phosphoglycerides have two acylgroups and a phosphate group esterified on the 3-OH to give a phosphatidate, A phosphatidate is key in the synthesis of other phospholipids The amino alcohol groups are often esterified to a phosphatidate to generate specific phospholipids

Examples of amino alcohols ethanolamine, serine,  choline,  inositol.

138

Similar to fats, but have only two fatty acids. Consists of a hydrophilic “head” and hydrophobic “tails”

The third -OH of glycerol is joined to a phosphate containing molecule. Structure: Glycerol + 2 fatty acids + phosphate group. Function: Main structural component of membranes, where they arrange in bilayers. Self-assembles into micelles or bilayers, an important part of cell membranes.

Phospholipid structure

139

CH2

P O O

CH2 CH CH2

O O

C O C O

Phosphate

Glycerol

(a) Structural formula (b) Space-filling model

Fatty acids

Hydrophilic head

Hydrophobic tails

–

CH2 Choline

Figure 5.13

N(CH3)3

140

141

Phosphatidylcholine (PC) lecithin –contain glycerol and fatty acids and choline esterified to a phosphate

Phosphatidylethanolamine (cephalins) They occur in tissue in association lecithin

Phosphatidylserine (PS) .

Phosphatidyl inositol (lipositol) acts as a second massager in a Ca2+. Dependent hormone action they are more acidic than other phospholipids

Phosphatidylglycerol (PG)

142

• The structure of phospholipids

– Results in a bilayer arrangement found in cell membranes

Hydrophilic head

WATER

Hydrophobic tail

143

Phospholipids in Water

144

Sphingosine based phospholipids

Sphingosine is the alcohol moeity composed of a polar head group and two nonpolar tails. are a component of all membranes and are abundant in the myelin sheath 1. sphingomyelins structural lipid components of nerve cell membranes.

the only sphingolipid that are phospholipids 2. glycosphingolipids

the cerebrosides, sulfatides, globosides and  gangliosides).. Ceramide: obtained by Amino acylation, with an acyl group, at carbon 2 of sphingosine.

Derived lipids • Derived from the hydrolysis of simple or compound lipids.

Examples glycerol, fatty acids,  steroids, sterols, fatty aldehydes,  ketone bodies, fatty soluble vitamins and  steroid hormones.

146

Steroids • Lipids characterized by a carbon skeleton consisting of four

fused rings

• Differ in the functional groups attached to the rings.

• Examples:

– cholesterol

– sex hormones

147

• Cholesterol • A steriod • Structure: Four carbon rings with no fatty acid tails • Functions: • Component of animal cell membranes • Modified to form sex hormones

CH3

H3C CH3

CH3

148