Proteins

PROTEINSProteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Proteins are the basis for the major structural components of animal and human tissue.

Classification of Some Proteins and their FunctionsClass of Protein Function in the body Examples

Structural Provide structural components

CollagenKeratin

Contractile Move muscles MyosinActin

Transport Carry essential substances throughout the body

HemoglobinLipoprotein

Storage Store nutrients CaseinFerritin

Hormone Regulate body metabolism and nervous system

InsulinGrowth hormone

Enzyme Catalyze biochemical reactions in the cells

SucraseTrypsin

Protection Recognize and destroy foreign substances

immunoglobulins

Question• Classify each of the following proteins according to its

function

____________1. hemoglobin(oxygen carrier in the blood)

____________2. collagen(a major component of tendons and cartilage

____________3. keratin( a protein found in hair)

____________4. amylase( an enzyme that hydrolyze starch

____________5. casein(milk protein)

AMINO ACIDS Overview

• Amino Acids are the chemical units or "building blocks" of the body that make up proteins. Protein substances make up the muscles, tendons, organs, glands, nails, and hair. Growth, repair and maintenance of all cells are dependent upon them. Next to water, protein makes up the greatest portion of our body weight.

Amino acids are the basic structural units of proteins.

An alpha-amino acid consists of an amino group, a carboxyl group, a hydrogen atom, and a distinctive R group bonded to a carbon atom, which is called the alpha-carbon because it is adjacent to the carboxyl (acidic) group. An R group is referred to as a side chain.(

• Contain 2 functional groups:

amino (-NH2) group

carboxylic acid (-COOH) group.

R group = amino acid side chain. This is what distinguishes one amino acid from another.• If the amino group, carboxylic group, R-group, and

hydrogen are bonded to a central carbon atom (α-carbon), they are termed α-amino acids.

• Only 20 different amino acids are present in the human Their names are abbreviated with 3 letters.

• All of the α-amino acids (except glycine) are chiral because the a-carbon is attached to 4 different groups.

Chiral Carbon• A carbon atom that is bonded to four different atoms or

groups of atoms.• The α carbon is chiral or asymmetric ( 4 different groups

are attached to the carbon; exception is glycine.)

CLASSIFICATION OF AMINO ACIDS

I. BASED ON THE NATURE OF R

A. non-polar or hydrophobic R

1. R is an aliphatic radical e.g. ala, val, ile, leu

2. R is aromatic e.g. phe, trp

B. polar R but uncharged

1. R is aromatic e.g. tyr, trp, phe

2. R has an –OH group e.g. ser, thr

3. R has an –S-group, e.g. cys, met

4. R has an amide group, e.g. asn, gln

5. R is an H, e.g. gly

C. R is positively charged, e.g. lys, arg, his

D. R is negatively charged, e.g. asp, glu

E. an imino acids, e.g. pro

II. ACCORDING TO ACID-BASE PROPERTIES

A. neutral amino acids, e.g. ala, val

B. acidic amino acids, e.g. glu, asp

C. basic amino acids, e.g. arg, lys

NOTE: acidic amino acids are negatively charged because they have two carboxylic groups(-COOH) and one amino group (-NH2). Basic amino acids are positively charged because they have two amino groups (-NH2 ) and only one carboxylic radical (-COOH). Neutral amino acids have one amino group and one carboxylic group.

PROLINE• Proline shares many properties with the aliphatic group.• Proline is formally NOT an amino acid, but an imino

acid(is any molecule that contains both imino (>C=NH) and carboxyl (-C(=O)-OH) functional groups)

• Nonetheless, it is called an amino acid. The primary amine on the α carbon of glutamate semialdehyde forms a Schiff base with the aldehyde which is then reduced, yielding proline.

• In addition to amino acids found in proteins, some amino acids are known not to occur in proteins. Examples of non-protein amino acids are: beta alanine (a building block of the vitamin, pantothenic acid); ornithine, an intermediate of the urea cycle; and gamma-aminobutyric acid (GABA), a chemical agent for the transmission of nerve impulses.

α - Amino Acids   • All amino acids isolated from proteins, with the exception

of proline, have this general structure.Lost proton- carboxylategroup

Gained proton- protonatedAmino group

Carboxylate group• a carboxyl group that has lost a proton; conjugate base

form (-COO-)

Protonated amino group• An amino group that has gained a proton; conjugate acid

form (NH+3)

zwitterion• The dipolar form of an amino acids and has a net charge of zero. Two

isomeric forms of an amino acid. The isomer on the right is a zwitterion.

Amino acid Zwitterion (dipolar ion)

ZWITTERION• Amino acid physical properties indicate a "salt-like"

behavior. Amino acids are crystalline solids with relatively high melting points, and most are quite soluble in water and insoluble in non-polar solvents. In solution, the amino acid molecule appears to have a charge which changes with pH.

ZWITTERION• An intramolecular neutralization reaction leads to a salt-

like ion called a zwitterion. The accepted practice is to show the amino acids in the zwitterion form.

• (1) The carboxyl group can lose a hydrogen ion to become negatively charged.

• (2) The amine group can accept a hydrogen ion to become positively charged.

ZWITTERION• At pH of 7, amino group is protonated (-NH3+) and

carboxyl group is ionized (COO-). The amino acid is called a zwitterion.

• At the pH of most bodily fluids, the carboxyl group will loose H+ and the amino groups accepts an H+

• This is a dipolar (DIPOLE ION)form of the amino acid

ISOELECTRIC POINT• At a certain pH called the

isoelectric point (pI), the positive and negative charges on the amino acid are equal and the overall charge is zero.  • In a solution that is more basic

than pI, the -NH3+ group will

loose H+, and the overall charge will be negative.

• In a solution that is more acidic than pI, the -COO- accepts an H+, and the overall charge will be positive.

Acid - Base Properties of Amino Acids:

• Acidic Side Chains:

• If the side chain contains an acid functional group, the whole amino acid produces an acidic solution. Normally, an amino acid produces a nearly neutral solution since the acid group and the basic amine group on the root amino acid neutralize each other in the zwitterion. If the amino acid structure contains two acid groups and one amine group, there is a net acid producing effect. The two acidic amino acids are aspartic and glutamic.

• Basic Side Chains:

• If the side chain contains an amine functional group, the amino acid produces a basic solution because the extra amine group is not neutralized by the acid group. Amino acids which have basic side chains include: lysine, arginine, and histidine.

• Amino acids with an amide on the side chain do not produce basic solutions i.e. asparagine and glutamine.

AMINO ACID AS ACID

Zwitterions for acidic amino acids typically exist at pH values of about 3 because the carboxyl group in their side chain must pick up H+

AMINO ACID AS BASE

Zwitterions for basic amino acids typically exit at pH 7.6-10.8

POLAR AND NONPOLAR AMINO ACIDS

Zwitterions for polar and nonpolar amino acids typically exit at pH values of 5.0-6.0

NEUTRAL SIDE CHAINS

• Since an amino acid has both an amine and acid group which have been neutralized in the zwitterion, the amino acid is neutral unless there is an extra acid or base on the side chain. If neither is present then then the whole amino acid is neutral.

• Amino acids with an amide on the side chain do not produce basic solutions i.e. asparagine and glutamine. You need to look at the functional groups carefully because an amide starts out looking like an amine, but has the carbon double bond oxygen which changes the property. Amides are not basic.

• Even though tryptophan has an amine group as part of a five member ring, the electron withdrawing effects of the two ring systems do not allow nitrogen to act as a base by attracting hydrogen ions.

•

PRINCIPLES OF POLARITY

• The greater the electronegativity difference between atoms in a bond, the more polar the bond. Partial negative charges are found on the most electronegative atoms, the others are partially positive

Non-Polar Side Chains:• Side chains which have pure hydrocarbon alkyl groups (alkane

branches) or aromatic (benzene rings) are non-polar.

• The number of alkyl groups also influences the polarity. The more alkyl groups present, the more non-polar the amino acid will be. This effect makes valine more non-polar than alanine; leucine is more non-polar than valine

NONPOLAR SIDE CHAINS• The non-polar amino acids include: alanine, cysteine,

glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.

• Non polar side chains consist mainly of hydrocarbon. Any functional groups they contain are uncharged at physiological pH and are incapable of participating in hydrogen bonding

POLAR SIDE CHAINS

• Side chains which have various functional groups such as acids, amides, alcohols, and amines will impart a more polar character to the amino acid. The ranking of polarity will depend on the relative ranking of polarity for various functional groups as determined in functional groups. In addition, the number of carbon-hydrogens in the alkane or aromatic portion of the side chain should be considered along with the functional group.

• Example: Aspartic acid is more polar than serine because an acid functional group is more polar than an alcohol group.

• Example: Serine is more polar than threonine since threonine has one more methyl group than serine. The methyl group gives a little more non-polar character to threonine.

• Example: Serine is more polar than tyrosine, since tyrosine has the hydrocarbon benzene ring.

Structures of Amino Acids

R = any number carbons in a hydrocarbon chain *CHIME plug-in required to view these images.

Amino Acid Name

A b r e v.

A b r e v.

Structure

of R group (red) Comments

Alanine ala A

Neutral Non-polar

Arginine arg R

Basic Polar

Asparagine asn N

Neutral Polar

Aspartic Acid asp D

Acidic Polar

Cysteine cys C

Neutral Slightly Polar

Glutamic Acid glu E

Acidic Polar

Glutamine gln Q

Neutral Polar

Glycine gly G

Neutral Non-polar

Histidine his H

Basic Polar

Isoleucine ile I

Neutral Non-polar

Leucine leu L

Neutral Non-polar

Lysine lys K

Basic Polar

Methionine met M

Neutral Non-polar

Phenyl- alanine

phe F

Neutral Non-polar

Proline pro P

Neutral Non-polar

Serine ser S

Neutral Polar

Threonine thr T

Neutral Polar

Trypto- phan

trp W

Neutral Slightly

polar

Tyrosine tyr Y

Neutral Polar

Valine Val V

Neutral Non-polar

ISOELECTRIC POINT• Solid amino acids have a very high melting points

because the zwitterion has the properties of a salt. The ionic charges of the amino acids make them more soluble in water, but not in organic solvents

PROPERTIES OF AMINO ACIDS

1. Protein are soluble in water, insoluble in organic solvents like benzene and ether. They have high meting points (above 200 ◦Celsius) and low vapor pressure. They have large dipole moments and high dielectric constants. These properties indicate the amino acids are polar. Polar forms of an amino acid are called zwitterions.

PROTEIN SOLUBILITY

Protein solubility depends on several factors.

1. It is observed that at low concentration of the salt, solubility of the proteins usually increases slightly. This is termed Salting in. But at high concentrations of salt, the solubility of the proteins drops sharply. This is termed Salting out and the proteins precipitate out.

2. Precipitation by changing the pH of the protein solution. This effect is due to the different functional groups on a protein. There will be some pH, known as the isoeletric point where the net charge on the protein is zero. This is different for different proteins.

3. You can also precipitate proteins by the addition of a non-ionic polymer or metal ions. (ionic strength)

PROTEIN PRECIPITATION• Precipitation is widely used for product recovery of

biomolecules especially proteins. The most common type of precipitation for proteins is salt induced precipitation. Precipitation is usually induced by

1. addition of a salt or an organic solvent

2. by changing the pH to alter the nature of the solution.

• Why Is Protein Precipitation Special?• Proteins are different than most other molecules. The

physical structure is just as important as the chemical structure. If the structure of the protein changes, the protein does not have the activity you want. This could even be harmful

PROTEIN DENATURATION• Denaturation of a protein occurs when there is a disruption in any

of the bonds that stabilize the secondary, tertiary, or quaternary structures.  The primary structure is not affected.  Proteins can be denatured by:

• ·        Heat = disrupts hydrogen bonds and hydrophobic attractions between R groups

• ·        Acids and bases = disrupt hydrogen bonds and salt bridges• ·        Organic compounds = disrupt hydrophobic interactions

between R groups• ·        Heavy metal ions = disrupt disulfide bonds• ·        Agitation = disrupts hydrogen bonds and hydrophobic

interactions between R groups by stretching the polypeptide chain.

Proteins Can Be Denatured

Both temperature and pH can change polypeptide shape.

a. Examples: heating egg white causes albumin to congeal; adding acid to milk causes curdling. When such proteins lose their normal configuration, the protein is denatured.

b. Once a protein loses its normal shape, it cannot perform its usual function.

The sequence of amino acids, therefore, forecasts the protein's final shape.

Color Reactions of Proteins • A color test for a protein tests for a particular functional

group or structural component present in a protein or in a particular amino acid found in proteins.

(a) Biuret reaction.

(b) Xanthoproteic reaction

The color is produced as the result of the formation of nitro-derivatives of the compounds which contain a benzene ring, for example, tyrosine.

(c) Million’s reaction.

The reaction serves as a test for the presence of tyrosine.

(d) Sulphur reaction

The precipitate, which is lead sulphide, is formed as the result of the decomposition of the cysteine by the alkali.

(e) Hopskins Cole Reaction

The color produced is due to the formation of a compound from the glyoxylic acid in the reagent and the tryptophan in the protein

Name of the Test Reagent Used Positive results Remarks

Biuret Test NaOH, dilute CuSO4

violet + results with polypeptides and proteins

Xanthroproteic Test

Conc. H2SO4 AA with benzene ring (yellow)

Proteins with trp, tyr, phe

Million’s Test Hg(NO3) and Hg(NO2)2

Tyrosine (red) + phenolic compounts

Sulphur Test Lead acetate, dissolved withNaOH

Gray or black precipitate

+ lead sulphide, formed as the result of the decomposition of the cysteine by the alkali.

Hopkins Cole Test Glyoxylic acid, sulfuric acid

Tryptophan (violet ring)

+ with any compound with indole ring

Ninhydrin Test ninhydrin Free-NH2 group (blue)

+ results given by NH3, primary amines, amino acids, peptides, and proteins

Indole

-an aromatic heterocyclic organic compound.It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. Indole is a popular component of fragrances and the precursor to many pharmaceuticals. Compounds that contain an indole ring are called indoles. The indolic amino acid tryptophan is the precursor of the neurotransmitter serotonin.

PROPERTIES OF AMINO ACIDS

2. Except for glycine, the alpha carbon is asymmetric or chiral and therefore capable of optical isomerism. The number of possible isomers is given by the formula 2n where n is the number of chiral carbons

3. Aromatic amino acids absorb light in the ultraviolet region of the EMR. This property is used for estimating protein content. E.g. phe absorbs at 260 nm, trp at 280 nm, tyr at 275 nm

4. Naturally occurring amino acids have an L-configuration. By convention, L-amino acids are written with NH3 to the left of the alpha carbon and the –COO on top.

5. Amino acids may be acidic, basic, or neutral.

Stereoisomers

Stereochemistry is the study of the 3-dimensional structure of molecules. Isomers are molecules with the same chemical formula and often with the same kinds of bonds between atoms, but in which the spatial arrangement of atoms differs. Isomers are grouped into two broad classes. (Most of the non-substituted cycloalkanes have conformational isomers, or diastereomers also known as conformers.)

Two enantiomers of a generic amino acid

If you orient the molecule so that you look along it from the COO− group to the NH3

+ group, the

methyl (R) group can extend out to the left, forming L-alanine(shown below on the left) or to the right, forming D-alanine (on the right).

• L amino acids are used exclusively for protein synthesis by all life on our planet.

• Does it really matter? Yes.

• The function of a protein is determined by its shape. • A protein with a D amino acid instead of L will have its

R group sticking out in the wrong direction. • Many other kinds of organic molecules exist as

enantiomers. Usually only one form is active in biological systems. For example, if one form binds to a receptor protein on the surface of a cell, the other probably cannot.

QUESTIONS

I. Write the zwitterion of each of the following amino acids

1. Glycine

2. Cysteine

3. Serine

4. Alanine

5. Phenylalanine

II. Write the Fischer projection for

6. L-serine

7. L-cysteine

III. List all amino acids with non-polar side chains.

IV. Rank the following according to increasing non-polarity i.e. 1 = least non-polar, 4 = most non-polar.

A. leu

B. phe

C. val

D. ala

V. List all amino acids by abbreviation which are considered

somewhat polar.

VI. Rank the following amino acids by increasing polarity. i.e.1 = more non-polar.

A. ser

B. glu

C. asp

D. lys

E. ala

F. gln

VII. Which amino acid is most insoluble in water: isoleucine or alanine ? Explain.

VIII. Which amino acid is most soluble in water: lys or ser?Explain.