Serkan SAYINER, DVM PhD. Assist. Prof.Near East University, Faculty of Veterinary Medicine, Department of Biochemistry

serkan.sayiner@neu.edu.tr

Amino Acids, Peptides

and Proteins

2

Proteins form a very important part of the living

organism.

Protein word means "leading" in Greek.

C, H, O and N are absolutely carried in the

constructions. They are extremely important for life.

Growth, proliferation and self-repair events, which are

the main characteristics of life, are closely related to

proteins and proteins that form very important

compounds.

Overview

3

The plants synthesize proteins from compounds such as

carbon dioxide, water and inorganic nitrogen. Animals

can not do that. Animals need plants for their protein

needs.

Proteins are large molecules.

Shapes can be fibrous, oval or spherical.

Building blocks are amino acids.• Proteins are amino acid polymers. The genetic code that

determines its synthesis.

• 20 amino acids are used in the synthesis of proteins.

Overview

4

While half of the amino acids used in protein synthesis

are synthesized as intermediate metabolic products,

some are required to be taken with nutrients.

Protein molecules than 45-55% C, H 6-8%, 20-25% O, N

can be between 15-17%.• N, It looks like a special element for protein.

◦ It is found in 16% of average molecules. Based on this determination of

the amount of nitrogen calculated the amount of total protein.

There are many different proteins in different tissues.• Each protein synthesized in the body is structurally and

functionally unique.

Overview

5

Cells, tissues, organs and organ systems are involved in

the processes that characterize the individuality

A cell contains thousands of proteins, each with a

different function. For example,◦ Enzymes; Every reaction in living cells requires enzymes...

◦ Transport proteins;. Lipoproteins, transferrin ....

◦ Bilirubin-binding proteins,

◦ Depot proteins; myoglobin...

◦ Defense proteins; Coagulation, immunoglobulins...

◦ Contractile proteins; Actin, myosin, troponin...

◦ Structural proteins;. Collagen, elastin...

Overview

6

Food percentage % Food %

Muscles 18 – 20 Lettuce 1,2

Blood plasma 6,5 – 7,5 Cabbage 1,6

Brain 8 Potato 2

Egg yolk 15 Almond 21

Egg white 12 Fruits 0,4 – 1,5

Cow milk 3,3 Haricot bean 18-22

Cheese 14 - 49 Soybean 37

Protein Rates in Some Tissues and Nutrients

7

D- and L-Forms, Classification, Essential Amino Acids, Modified Amino Acids, Non-

Protein Amino Acids, Physical and Chemical Properties

Amino Acids

8

Hydrolysis of Proteins

When proteins are hydrolyzed with acids, alkalis, or enzymes, they are cleaved into amino acids.Compounds having amino (-NH2) and carboxyl group (-

COOH) in their molecules are called amino acids. In the structure of amino acids, 4 different groups are

connected to 4 valences of carbon atom. 1. Carboxyl group2. Amine group3. Hydrogen4. Group R

9

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Hydrolysis of Proteins Amino acids are briefly indicated by a single letter or three letters.

• Glycine, for example; Gly or G

Only the simplest amino acid, glycine, is the R group H.• Glycine is the first isolated amino acid (1820). • The last identified and isolated is Treonine (1935).

The carbon atom nearest to the carboxyl group in the amino acids is called the α-carbon atom.

Since amino acids other than two amino acids are linked to the α-carbon atom of the NH2 group, these amino acids are called α-amino acids.

11

Hydrolysis of Proteins

Amino acids connect with each other by peptide bonds

which are acid amide bonds to form peptides and

proteins.

If the protein molecule gives only amino acids, after

hydrolysis, it is called a simple protein. If gives other

molecules together with amino acids, it is called a

conjugated protein or proteid.

12Source: W.H. Freeman and Company 2012. Biochemistry, 7th ed.

13

Amino acids are classified in various forms according to their properties. The most common classification is neutral amino acids, acidic amino acids and basic amino acids.

Another classification refers to the number of amino and carboxyl groups presented. • Mono-amino monocarboxylic acids,

• Mono-amino di-carboxylic acids,

• Di-amino monocarboxylic acids,

• Di-amino di-carboxylic acids.

Classification of Amino Acids

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Amino acids are also classified on the basis of chains

and ring structures in their molecules. Aliphatic amino

acids, aromatic amino acids, heterocyclic amino acids.

In classification of amino acids;• According to the chain and ring structures, they are classified

as aliphatic, aromatic and heterocyclic amino acids. Each class

is then sub-classified as acidic, basic and neutral amino acids,

and whether they contain sulfur or not.

Classification of Amino Acids

15

Aliphatic Amino Acids• Neutral amino acids, Glycine, Alanine, Valine, Serine,

Threonine, Leucine, Isoleucine

• Acidic amino acids : Aspartic acid, Asparagine, Glutamic acid, Glutamine

• Basic amino acids : Arginine, Lysine

• Sulfur-bearing amino acids : Cysteine, Methionine

Classification of Amino Acids

Aromatic Amino Acids• Tyrosine

• Phenylalanine

• Tyroptophan

Heterocyclic Amino Acids▫ Proline

▫ Histidine

16

In another classification;• Hydrophobic Amino Acids

◦ Aliphatic side chain

◦ Aromatic side chain

◦ Branched-chain amino acids

• Hydrophilic Amino Acid◦ Uncharged side chain

◦ Charged side chain; Basic and Acidic amino acids..

• Neither Hydrophilic nor Hydrophobic Amino Acid

Classification of Amino Acids

17

The side chains of hydrophobic amino acids do not interact well with the water and usually located inside the proteins. They consist largely of hydrocarbons. • Except the sulfur atoms in the structure of methionine and

cysteine and the nitrogen atom in the structure oftryptophan,

-SH group in the cysteine, that allow it to dimerize through the disulfide bond (-S-S-). The amino acid oftenly exists in the oxidized form in protein. This oxidized form is cystine.

Hydrophobic Amino Acids

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The different regions of the cystine polypeptide chains are covalently linked. So it balances proteins and makes denaturation more resistant

Proline is generally considered to be hydrophobic and is found frequently in the regions of folded polypeptide chains and is particularly found in collagen.

Proline can be formed by cyclization of glutamate.

Hydrophobic Amino Acids

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Methionine

(Met or M)

Cysteine

(Cys or C)

Alanine

(Ala or A) Proline

(Pro or P)

(Imino acid)

Hydrophobic, Aliphatic Side

Chain Amino Acids

20

Cystine

Cysteine

(Cys or C)

Cysteine

(Cys or C)

21

The aromatic amino acids (AAA) category includes phenylalanine, tyrosine, and tryptophan.

The group R in the,• Phenylalanine contains a benzene ring,

• Tyrosine contains a phenol,

• Tryptophane contains a heterocyclic structure known as an indole.

These three amino acid in the aromatic moieties are linked to α-carbon through a methyl (-CH2-) bridge.

Hydrophobic Amino Acids

22

Aromatic amino acids are important hepatic

metabolites.

Although hydrophobic, phenol (-OH) group tyrosine and -

NH group of indole group of tryptophan allows them to

interact with water, thereby rendering a bit uncertain

properties.

Hydrophobic Amino Acids

23

Phenylalanine

(Phe) Tyrosine

(Tyr or Y)

Tryptophane

(Trp or W)

Hydrophobic, Aromatic Side

Chain Amino Acids

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Valine, Leucine and Isoleucine are branched chain

amino acids (BCAA).

They carry a aliphatic side chain which includes methyl

group (as a substituent).

They should be taken with diet in highly organizes

animals. In other words they are essential. There are no

enzymes necessary for synthesis in these organisms.

Although present in many tissues, BCAAs are

catabolized by muscle tissue, especially for energy

purposes in the final stage of hunger.

Hydrophobic Amino Acids

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Leucine

(Leu or L)Isoleucine

(Ile or I)

Valine

(Val or V)

Hydrophobic, Branched Side

Chain Amino Acids (BCAA)

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Amino acids with positively charged side chains include

basic amino acids such as histidine, lysine and arginine.

Histidine contains weak charge at only physiological pH.

In contrast, amino acids including negatively charged

side chains are acidic and they are aspartic acid and

glutamic acid (available as glutamate and aspartate).

Hydrophilic Amino Acids

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Glutamate

(Glu or E)

Aspartate

(Asp or D)

Hydrophilic, Charged

Side chain

Acidic Amino Acids

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Histidine

(His or H)Lysine

(Lys or K)Arginine

(Arg or R)

Hydrophilic, Charged

Side chain

Basic Amino Acids

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A hydroxylated version of the alanine which is serine

and threonine contains a non-charged side chain, and a

-OH group. For this reason, strong interactions with

water occur as a result of the formation of hydrogen

bonds.

They are much more hydrophilic compared to other non-

hydroxylated closely related amino acids (alanine and

valine).

Hydrophilic Amino Acids

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The uncharged side chains of asparagine and glutamine

have amide groups with greater hydrogen bonding

capacity.

Nonetheless, asparagine and glutamine are readily

hydrolyzed with acid or base to form non-amides

(aspartic acid and glutamic acid).

All nine of these amino acids are hydrophilic and

therefore interact with water. Side chains are oftenly

found on the surface of the protein to which they are

exposed to an aqueous medium.

Hydrophilic Amino Acids

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Asparagine

(Asn or N)

Glutamine

(Gln or Q)

Serin

(Ser or S)

Treonin

(Thr or T)

Hydrophilic, Amino Acids

with Uncharged

Side chain

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It is neither a hydrophilic nor a hydrophobic amino acid.

Glycine having only one hydrogen atom in the side chain

is known as the simplest amino acid.

Due to the simple structure, it can fit many gaps in the

polypeptide chains of protein molecules and therefore is

located on both the inner side surfaces.

Glycine

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Neither Hydrophilic

nor Hydrophobic

Amino acid

Glycine

(Gly or G)

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Proteins taken into the organism must be broken up into

amino acids in order to be absorbed through the intestines.

Amino acids absorbed from the intestine are transfered to

the tissues through the blood and are used for protein

synthesis.

Plants, microorganisms and fungi are autotrophs. They do

not need amino acids to feed. They can synthesize

themselves.

Advanced organisms are heterotrophic. They have to take

some amino acids from the outside. These organisms can

perform protein synthesis from amino acids.

Essential Amino Acids

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Essential amino acids or exogenous amino acids are

amino acids that are not synthesized by the organism

and are required to be taken externally with foods.

An amino acids which is capable to produce it’s α-

ketoacid are not essential and can be synthesized during

metabolism.

Dry Beans contain more protein than meat, but not as

valuable as meat. Why?• Because; it does not contain essential amino acids as mush as

to meet animals’ need.

Essential Amino Acids

36

If there is an inadequacy of any essential amino acid, the remaining 19 amino acids can not be used for protein synthesis. However, they become catabolized and negative nitrogen balance arises.

Essential amino acids show differences between animals. Amino acids that are essential for some species may not be essential in some species

It also varies with age.

Essential Amino Acids

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Lysine

HUMAN DOG

RAT

PIG CHICKEN

Triptofan

Phenylalanine

Leucine

Isolösin

Treonin

Methionine

Valin

Histidine

Arginine

Glycine

Glutamic acid

Essential Amino Acids

In young

people

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BCAAs are constantly oxidized by muscle tissues.

Phenylalanine is required for tyrosine biosynthesis. It is

necessary to synthesize catecholamines and thyroid

hormones.

Methionine is required for cysteine formation.

Tryptophan is required for serotonin and melatonin

biosynthesis.

The non-essentials are mostly obtained by conversion

with enzymes (aminotransferases) during metabolism.

Essential Amino Acids

39

There are some modified amino acids in the protein structure.Methylhistidine and methyllysine are methyl derivative

of histidine and lysine. They are located in the structure of some proteins.Hydroxyproline is a derivative of proline. It is found in

collagen structure in the form of -Proline-Hydroxyproline-Glycine- (tripeptide). • Collagen is a fibrous connective tissue protein.

• It is found in blood vessels, tendons, cartilage and bone.

• It is the most abundant protein in mammalians.

• ~ 30% of total body protein and ~ 6% of body weight is collagen.

Modified Amino Acids

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Methylhistidine Methyllysine Hydroxyproline

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Hydroxylisine is the 5-hydroxy derivative of lysine and

is found in the collagenous structure.

Desmosine and isodesmosine are formed by oxidation

and cross-linking of four lysines.• It is found in the structure of elastin which is a connective

tissue proten.

• It is found in tissues containing smooth muscle (such as blood

vessels) with collagen.

• Elastin has two-way elasticity.

• Arteries contain elastin much more than veins.

Modified Amino Acids

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HydroxylysineDesmosine

Isodesmosine

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Phosphothreonine, phosphotyrosine and

phosphoserine are occured by phosphorylation of the

hydroxyl groups of threonine, tyrosine and serine side

chains.• These three derived amino acids are particularly involved in

the construction of regulatory proteins.

γ-Carboxyglutamate is synthesized by carboxylation of glutamic acid in the liver.• This amino acid is involved in the structure of prothrombin (factor II),

one of the coagulation factors.

• Provides calcium chelating.

Modified Amino Acids

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Phosphothreonine Phosphotyrosine Phosphoserineγ-Carboxyglutamate

(Chelate with Ca++)

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There are amino acids other than the 20 amino acids

involved in the protein structure. These amino acids do not

participate in the protein structure.

They are important intermediate metabolites or

precursor molecules during metabolism.• β-Alanine is the building block of Pantothenic acid (B5 vitamin).

◦ This vitamin is also a building component of coenzyme A.

• Homocysteine and homoserine are important intermediates in

methionine metabolism.

• Ornithine and sitrulin take place in the hepatic urea cycle and

are important intermediates in the synthesis of arginine.

Non-protein Amino Acids

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• Taurine is formed by the oxidation of the sulphydryl group in the cysteine and subsequent carboxylation. ◦ In cats, because they can not synthesize as much as competence, they

have to take it with the diet. It is essential for the cat.

◦ Retinal degeneration, blindness, reproductive disturbances, growth retardation, and skeletal deformities are seen due to taurine failure in the cat.

• γ- Aminobutyric Acid (GABA) is a neurotransmitter inhibitor in the central nervous system (with glycine).◦ It is mainly located in substantia nigra, globus pallidus, and

hypothalamus.

◦ Alcohol, barbiturates, and benzodiazepines increase the GABA effect, especially in postsynaptic areas.

Non-protein Amino Acids

48

Non-protein Amino Acids

Homocysteine

Citrulline

Ornithine

Β-AlanineTaurine Homoserine γ- Aminobutyric

Acid (GABA)

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Solvents Water Acid Alkali Ethanol Ether

Solution States + + + ± -

Melting Points: Melts at 200 °C and sometimes at 300 °C.

Flavors

Tasteless Bitter Sweet

Leucine Isoleucine, Arginine Others

Physical Properties of Amino Acids

50

Amino acids are ampholytes.• Since amino acids carry the carboxyl group together with

the amino group, they are both acid and base.

• The α-carbon atom of amino acids is stable at physiologically pH;◦ The group R, H bonds to the unprotonated COO- and protonated NH3+.

◦ There is a difference in the prolin. It contains the imino group (NH2+)

instead of the amino group.

• The COOH group dissociates to the -COO- (carboxylate anion) and H+ ions by giving H ions in the alkaline environment.

• NH2 groups gain H+ in the acidic environment and converted to positively charged NH3

+ group (ammonium cation).

Physical Properties of Amino Acids

51

• At isoelectric points (at physiological

pH), H ions, passes through COOH groups

to NH2 groups. COOH group is a COO-

group carrying a negative charge

because it gives H ion and it is side by

side with the NH3+ group which is

positively charged.

• Ions that carry both positive and

negative charges are called

zwitterions. The substances that can

form zwitterion are also called

amphoteric/ampholyte.

Physical Properties of Amino Acids

Glycine

(Gly or G)

52

• Ionisation conditions vary depending on pH.

• In alkaline solutions (pH>7.4), the carboxylic group generally

remains in ionized form (-COO-). In contrast, the amino group

is anionic (-NH2).

• In acidic solutions (pH<7.4), the carboxyl group remains

anionic (-COOH) while the amino group protects its ionized

form (NH3+).

• The pK value of carboxyl or amino group of each amino acid

is different. This difference is related to some factors such as

temperature, ionic strength, and the presence of ionizable

group.

Physical Properties of Amino Acids

53

Cation(Acidic solution)

Anion(Alkali solution)

Zwitterion(Isoelectric point)

54

Optical Activities• All amino acids except glycine have α-carbon atoms and are

therefore optically active.

• There are mirror images which are L-isomer and D-isomer

(enantiomer).

• Amino acids similar to D-glyceraldehyde are D-amino acids,

Amino acids similar to L-glyceraldehyde are L-amino acids. It is

indicated by (+) if light turns to right and (-) if light turns to

left.

• While almost all carbohydrates of plants and animals are in

D series, amino acids in proteins are in L series.

Physical Properties of Amino Acids

55

• D-amino acids are usually found on cell walls of bacteria and

some antibiotics (D-glutamic acid etc.)

Physical Properties of Amino Acids

D-Form L-Form

If the functional group

is on the right

If the functional group is

on the left

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D-Glyceraldehyde

C OH

C

CH2OH

OHH

C OH

C

CH2OH

HHO

L-Glyceraldehyde

D-Amino acid

COOH

C

R

H2NH

L-Amino acid

COOH

C

R

HNH2

According to Fischer

projection;

• In D-amino acids - The NH3+

group is on the right side.

These amino acids are rarely

found in nature.

• In L-amino acids - The NH3+

group is on the left side.

These amino acids are

widely found in the nature.

57

MIRROR

L-Amino acid D-Amino acid

58

59

There are 3 types of functional groups in the amino

acid molecule. 1. Amino groups

2. Carboxyl groups

3. R groups

The chemical properties of amino acids depend on these

3 groups.

Chemical Properties of Amino Acids

60

Inactivation of the amino group with alcohol• In alcoholic environments, amino groups do not change to

ammonium cations (NH3+). This is known as the inactivation of

amino groups by alcohol.

• They are not amphoteric in an alcoholic environment. They are

acids. Thus, they can be titrated

Sörensen Titration• It is a method of determining the carboxyl group in the amino

acid solution. It is utilized to determine the amino acid

content in a solution.

Reactions of Amino Groups

61

Bonding of the amino group with aromatic acids• The incorporation of amino acids into aromatic acids

eventually becomes insoluble.

• This ensures that harmful substances are expelled from the

organism. Ex. Hippuric acid (the most important nitrogenous

substance in urine after it is produced in grass eaters).

Reactions of Amino Groups

Betain Reaction• When the amino acids are in the zwitterion

state, betaines are formed with 3 CH3 groups passing instead of 3 H in the NH3

+ group.◦ Ex. Trimethylglycine is a betaine formed with glycine.

◦ Betaines are biologically involved in methylation reactions and detoxification of homocysteine.

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Carbomino acid reaction• Carbonic acids are formed on the amino groups by CO2

bonding.

• When it is heated, again CO2 and amino acid are decomposed. Hemoglobin brings carbamino acid with CO2 and it is important for transporting CO2 from tissues to the lungs.

Ninhydrin Reaction• All of the free amino groups give this reaction (amino acids,

peptides, proteins).

• Ninhydrin is an important qualitative assay for amino acids.Less than 1 gram is even susceptible. Practical importance is great.

Reactions of Amino Groups

63

Nitric acid reaction• When amino acids react with nitric acid, the nitrogen of the amino

groups passes into the free element, such as nitric acid nitrogen.

• On the other side the amino group is replaced by the -OH group.

• At the reaction, a molecule corresponding to each amino group is

released to nitrogen.

• For this reason, nitric acid reassignment can be used to determine the

amount of free amino groups.

• Nitric acid reaction; Also called Van Slyke method.

Reactions of Amino Groups

64

Uramine Acid Reaction• If a urea molecule is added to the amino groups, the uramine

acids will form.

• It is used in the identification of amino acids.

Diketopiperazine formation• The ethyl esters of amino acids are condensed to form

anhydride ring structures bearing two amino acid groups,

resulting in the formation of diketopiperazine.

Reactions of Amino Groups

65

Chelating amino acids with metal ions• Amino groups form complex chelates with many heavy metal

ions such as Cu++, Co++, Mn++, Fe++.

Reactions of Amino Groups

Kaynak: Ası T. Tablolarla Biyokimya Cilt I

Copper diglycerateCysteine-cobalt complex

66

Salt formation• Amino acids can form salts with heavy metals and easily

crystallize.

• Especially the salts formed with Cu are easily crystallized. This

property is used to obtain pure amino acids.

Esterification• If they react with alcohol in the presence of anhydrous

hydrochloric acid, they will bring their esters.

• The esters of amino acids are utilized in their functional

distillation.

Reactions of Carboxyl Groups

67

**Decarboxylation of amino acids• It is the name given to the separation of carbon dioxide from

the amino acid molecule. So the amines are occured.

• In the tissues, the amino acids decarboxylate to the amines.

• Ex; histidine to histamine, lysine to cadaverine, ornithine to

putressin, tyrosine to tyramine, tryptophan to tryptamine.◦ They are also converted by anaerobic microorganisms.

◦ That's the spoiled gut. Cadaverine and putressin are amines found in

stinky meats. Poisonous effects are low. The toxins are the poisons of

the microorganisms that constitute them.

Reactions of Carboxyl Groups

68

Decarboxylation of amino acids

Source: Ası T. Tablolarla Biyokimya Cilt I

Histidine

Decarboxylase

Histidine Histamine

Amino acid Amine

69

Reactions of R groups

The R groups of amino acids are composed of multiple

variable groups.

Due to the differences in the groups, different reactions

occur.

Especially they give their unique color reactions. These

color reactions allow amino acids to be separated and

recognized.

70

R-Groups Amino acid Reaction Color CreatedReaction specific

name

PhenylTyrosine,

Phenylalanine

Heating with Hg(NO3)2 and concantrated

HNO3

Red MillonReaction

Phenyl

Imidazole

Tyrosine,

Phenylalanine,

Histidine

In alkaline environment, treatment with

sulfanilic acid and NaClORed Pauly Reaction

Guanidine Argininen an alkaline medium, α-naphthol and

treated with NaClORed Sakaguchi Reaction

Free SH CysteineTreatment with dilute NH4OH solution of

sodium nitroprussideRed

Nitroprussid

Reaction

Indole TryptophanTreatment with H2SO4 solution of p-

dimethylaminobenzaldehydePink Ehrlich Reaction

Cyclic amino

acids

The phenyl

alanine,

tryptophan

Dense with HNO3

When heated

With the addition of alkali

White residue

Yellow

orange

Xanthoproteic

Reaction

Indole TryptophanIf after treatment with glyoxylic acid is

layered with dense sulfuric acidViolet ring

Hopkins Cole

Reaction

71

Peptide Bonds, Oligo- and Polypeptides, Some Important Di-, Oligo- and Polypeptides,

Seperation of Amino Acids from Proteins and Quantitative Determinations

Peptides

72

Amino acids polymerize with acid amide bond/peptide

bond to form proteins.

The number of amino acids added together by peptide

bonds is important when the peptide is named.• 2 is dipeptide, 3 is tripeptide

• 4-10 is oligopeptide

• Between 10-100, the polypeptide

• >100, the macro peptide

Peptide Bonds and Oligo-Polypeptides

73

When the peptide chain is formed, the COOH group of one of the amino acids joins with the NH2 group of the other amino acid. So that an NH2 group with a COOH group will eventually remain freeterminal ends.• The tips are on paper; NH2 at

the left end. N-terminus or terminal/C-terminus orterminal

The free NH2 group is called the free amino group.

Peptide Bonds and Oligo-Polypeptides

74

Amino acids Features

Carnoline β-alanine histidine Dipetid. It is found in the muscles

Anterin β-alanine N-methyl histidine Dipetid. It is found in the muscles.

Glutathione Glutamyl cysteine glycine

Tripeptide. It is involved in oxidoreduction. It is

found in the structure of some enzymes. Controls the

absorption of Fe.

Ophthalmic acid Glutamyl aminobutyryl glycine Tripeptide. Located in the eye lens.

Non-Ophthalmic acid The glutamyl alanine glycine Tripeptide. Located in the eye lens.

Oxytocin 8 amino acids Octapeptide. It's the hormone of the pituitary gland.

Vasopressin 8 amino acids Octapeptide. It's the hormone of the pituitary gland.

ACTH 39 amino acids Pituitary anterior lobe hormone.

Insulin 51 amino acidsPancreas hormone. It plays a role in glucose

metabolism.

Glucagon 29 amino acids Pancreatic hormone.

Some important di-, oligo-, and polypeptides

75

Insulin It is secreted by the β-cells of

the langerhans islets of the

pancreas.

It is synthesized as proinsulin;

contains A-chain of 21 amino

acids, the B-chain of 30 amino

acids and the C-peptide chain

of 31 amino acids.

There is no C-peptide chain in

the active form.

76

77

Separation of Amino Acids in Proteins and Quantification

Tyrosine• They can be easily crystallized and separated at the isoelectric

point.

Arginine, Lysine, Histidine• Since these amino acids can be precipitated with the

phosphotungstic acid from the protein solution, their total amounts can be easily detected.

Tyrosine, Tryptophan• Since these amino acids can give color reactions, quantities can

easily be detected by colorimetric methods.

Monoamino acids • Since these amino acids can be converted into ethyl esters, they

can be subsequently removed from the reaction mixture by fractional distillation.

78

Methods Principle

Chromatographic

Adsorption Method- Paper chromatography

- Ion exchange chromatography

- Thin layer chromatography

- HPLC

Adsorption of various amino acids by different adsorbents at

different levels. Acid character amino acids are trapped by the

basic adsorbents, basic amino acids are trapped by the acid

adsorbents.

Isotope Dilution Method

It is a phenomenon in which an unknown amino acid mixture is

added to a known amount of a specific amino acid marked with

an isotope atom, and then it is determined from this mixture

how much it is diluted after the same amino acid is obtained in

pure form.

Microbiological Method

It is based on the principle that certain amino acids must be

present in a certain amount in the medium environment for the

reproduction of some microorganisms.

Separation of Amino Acids in Proteins and Quantification

79

Structure, General Properties, Classification (Structural: Simple-Conjugate-Derivative Proteins, Functional: Catalytic Proteins, Transport Proteins, Nutrient and

Depot Proteins, Contractile Proteins, Defense Proteins, Physiological Regulatory Proteins), Nucleoproteins (DNA, RNA, ATP ...), Chromoproteins (Porphyrins, Bile

colored substances), Icterus (Jaundice), Main Proteins in the Liquid and Tissues of the Organism

Proteins

80

Proteins are composed of peptide chains. It is in the

polypeptide structure.

If there are n-amino acids in the protein structure,

there are n-1 number of peptide bonds.

A complete protein molecule represents four

complementary substructures.1. Primary Structure

2. Secondary Structure

3. Tertiary Structure

4. Quaternary Structure

Structure of a Protein Molecule

81

Structure of a Protein Molecule

1. Primary Structure• A certain number of amino acids refers to the sequence of

chains that are ordered by a particular sequence.

2. Secondary Structure• α-Helix: It occurs when the peptide chain is twisted in the

form of a helix around an axis.

• β-Sheet: It is a structure that occurs when two or more

polypeptide chains are joined together by hydrogen bonds.

82

Structure of a Protein Molecule

3. Tertiary Structure• Helix structures folded with -S-S bridges to become globular

and ellipsoidal.

4. Quaternary Structure• It is formed by the combination of primary, secondary, and

tertiary formations.

83Source: McDarby M.

84Source: McDarby M.

85

General Properties of Proteins Flavors of Proteins• Pure proteins are usually sweet. Hydrolysis products are mostly

bitter.

Odor• Pure proteins are odorless.

Homogeneity• Most of the proteins are not pure but have sub-fractions.

Solubility• Soluble in water and neutral saline solutions.

• Proteins that are precipitated in an equal amount of ammonium sulfate solution are called globulin, if not it is called albumin.

• Non-soluble proteins in water and neutral salt solutions are called scleroproteins.

86

Denaturation of proteins• Denaturation is the deterioration of the natural structure of

proteins.

• In the deterioration of the natural structure, the protein molecule has to be exposed to a number of factors.

• The opening of the folds of the protein molecule, and the folding in a different way, i.e. changes in the secondary and tertiarystructures, alter the properties of the protein molecule.

• For denaturation the dissolution of peptide bonds is not necessary. In the case of deterioration of the tertiary structure, where there is a returnable denaturation, in the case of the deterioration of the secondary structure, the resulting denaturation is not reversible.

General Properties of Proteins

87

• The proteins that undergo denaturation change their

solubility, some color reactions are exacerbated, and the

biological activity of enzymes and hormones is lost.

• Treatment with acid salts, alkaline retention, dissolution

in ethanol, heat, X-rays, UV irradiation, strong agitation,

SDS (sodium dodecyl sulfate), treatment with urea salts,

freeze-thawing and high pressure are some of the reasons

for denaturation.

General Properties of Proteins

88

Normal

Protein

Denaturated

Protein

Denaturation

Renaturation

Factors: pH, Temperature, Ionic strenght, Solubility, UV rays, SDS ...

Loss of biological

activity

Recovery of

biological activity

89

Protein Structure and Denaturation - A Level Biology

90

Molecular weight• They are substances with a high molecular weight.

• Molecular weights are increased by the formation of the

quarternay structure, and by the separation of the monomers

is decreased. Therefore, the molecular weight varies

according to the conditions.

General Properties of Proteins

Protein MW (Dalton)

Insulin 12.000

Myoglobin 17.000

Hemoglobin 68.000

Albumin 69.000

Thyroglobulin 660.000

Fibrinojen 450.000

91

Molecular shape of proteins• Spherical shape to the yarn shows the changing forms.

• Most of the easy solids are spherical.

• Those in the form of yarn are insoluble proteins.

General Properties of Proteins

Shape Features

GlobularThey are easy to dissolve.

Solutions reduce viscosity.

Basil likeIt greatly increases the viscosity of the

solutions.

Fibrous Insoluble.

92

Precipitation of proteins• Proteins are precipitated by certain acids, heavy metals and

specific antibodies.

• Heavy metals; Hg, Pb, Ag, Cu, Fe, Cd, Zn.

• When the protein is given to the organism by an extrinsic

pathway, it is regarded as a foreign substance and forms

antibodies.

• Each protein is precipitated by its specific antibody, which is

formed against its own protein.

General Properties of Proteins

93

Heavy metal salts• In metal poisonings, antidote proteins are used.

Negative ions• It is used for the precipitation of blood proteins with the

purpose of analysis.

Specific antibody proteins• It is used to obtain antigen.

General Properties of Proteins

94

Hydration of proteins• They bind water. The polar groups in the peptide chain

include amino, carboxyl, hydroxyl, imino, and etc.

• Polar groups have the ability to bind with water.

Viscosity of proteins• It is directly related to the molecular shape.

• If the molecule is long, the viscosity is higher. Viscosity is

also high in large-molecule proteins.

Crystallization of proteins• Enzyme proteins such as pepsin, trypsin and urease have

been obtained in crystal form.

General Properties of Proteins

95

Classification of Proteins

Two types of classification are possible.

1.Classification according to their

Structures

2.Classification according to their

Biological Functions (Functional

Classification of Proteins)

96

Simple Proteins• Simple proteins only consist of amino acids. They are found in

the structure of polypeptide chains that only give amino acids when hydrolyzed. Simple proteins are divided into subgroups according to their different qualities; Globular and FibrillarProteins.

Conjugated Proteins (Proteids/Combined Proteins)• When it is hydrolyzed, it is proteins that give different

chemical substances together with amino acids.

Derivative Proteins• They are proteins that are formed as a result of alteration of

proteins belongs to simple or conjugated proteins.

Classification according to their Structures

97

Simple proteins are made up only of amino acids. They

are separated into two subgroups; Globular and Fibrillar

Proteins.

Globular Proteins• Proteins in which the molecule is in the form of rotational

ellipsoids of the three-dimensional shape.

• Globular proteins are divided into subgroups such as albumin,

globulins, globins, glutelines, prolamins, protamines and

histones.

Simple Proteins

98

• Albumins◦ Soluble in water and aqueous solutions.

◦ They coagulate with heat.

◦ MW<100,000.

◦ Glycine-poor proteins.

◦ Eg.: Ovalbumin, serum albumin, lactalbumin, legumelin

• Globulins◦ Not soluble in water. It dissolves in neutral saline solutions.

◦ Glycine-rich proteins.

◦ MW>100,000.

◦ Eg.: ovoglobulin, lactoglobulin, α-, β-, γ-globulins, legumin, faseolin

Simple Proteins

99

• Globin◦ They are usually found as compound, mainly in the hemoglobin

structure.

• Glutelines◦ Plant origin.

◦ Not soluble in water. Soluble in dilute acid and alkalines.

◦ They become denatured by heat.

◦ Eg.: Wheat glutenin, barley hordenin, orizenin in rice.

• Histones◦ Water soluble.

◦ It is rich in basic amino acids. It's rich in arginine. Eg.: Timothyton(gland tissue), skombron (mackerel)

Simple Proteins

100

• Prolamins◦ Plant origin.

◦ Not soluble in water. Soluble in 70-80% ethanol .

◦ They contain less Lysine and Cystine but, rich in Prolin.

◦ They are especially found in grain plants. Eg: Gliadin (wheat), zein(maize).

◦ When wheat flour dough loses its starch under running water, a very elastic material remains. This substance, which is called gluten, is a mixture of gliadin and glutenin.– Coeliac Disease (Gluten Enteropathy): Coeliac disease, also spelled celiac

disease, is a long-term autoimmune disorder primarily affecting the small intestine that occurs in people who are genetically predisposed. Coeliac disease is caused by a reaction to gluten, which are various proteins found in wheat and in other grains such as barley, and rye. Classic symptoms include gastrointestinal problems such as chronic diarrhoea, abdominal distention, malabsorption, loss of appetite, and among children failure to grow normally

Simple Proteins

101

• Protamines◦ It was obtained from fish semen.

◦ Soluble in water, dilute acids and alkalis, dilute ammonium hydroxide

solutions.

◦ It does not include tryptophan, tyrosine and sulphated amino acids.

◦ They are rich in arginine.

◦ They display strong basic characters.

◦ It is the shortest chain of proteins (MW 1000-5000).

◦ They are particularly associated with nucleic acids in tissues.

◦ Ex: Skombrin (mackerel), salmin (soma), lupine (herring).

Simple Proteins

102

Fibrillar Proteins• They are proteins in the form of three-dimensional multi-

stretched ellipsoids.

• Scleroproteins◦ It is of animal origin.

◦ They are insoluble in water, neutral salt solutions, dilute acids and

alkalis, and pure alcohol.

◦ They are resistant to enzymes such as pepsin and trypsin.

◦ Keratin (horn, hair) is not digested, collagen (connective tissue,

bone, fracture, tendo) is digested. An another example is elastin

(ligament). Keratin is very rich in sulphurous amino acids.

Simple Proteins

103

• Fibrinogen◦ It is found as dissolved protein in the blood plasma.

◦ It is the acute phase reactant involved in the coagulation of blood

(coagulation).

◦ MW is 340,000. Consists of two subunits.

◦ It has important functions in tissue repair and wound healing.

◦ During blood clotting, fibrinogen is converted to fibrin by thrombin.

◦ It is synthesized by hepatocytes in the liver.

• Myosin◦ It is found in the muscle tissue.

◦ It is involved in muscle contraction.

Simple Proteins

104

When it is hydrolyzed, it is proteins that give different chemical substances together with amino acids. It is formed by binding of polypeptide chains formed

from amino acids to the so-called prosthetic group.• Phosphoproteins

• Glycoproteins

• Proteoglycans

• Lipoproteins

• Metalloproteins

• Nucleoproteins

• Chromoproteins

Conjugated Proteins

105

• Phosphoproteins◦ Proteins that carry phosphoric acid (phosphate) as a prosthetic

group.

◦ Phosphoric acid is attached to the -OH groups of serine, threonine and

tyrosine in the protein molecule; so it is esterified.

◦ Casein in milk; vitelline, livetin and phosvitin in eggs; ihtulin in fish

eggs (haviar) are some examples.

• Glycoproteins ◦ Proteins that carry carbohydrates as a prosthetic group.

◦ They range from 1 to 80% carbohydrates.

◦ If the carbohydrate ratio is <4%, it is called glycoprotein.

Conjugated Proteins

106

◦ If the ratio is between 10-20%, it is called mucoprotein, if carbohydrate ratio is very high and protein ratio is low, it called mucoid.

◦ Some carrier proteins (seruloplazmin, transferrin) and immunoglobulins from blood plasma proteins; osseomucoprotein in bones, tendonmucoprotein in tendons, cartilago mucoprotein in cartilaginous tissue, and mucin in saliva are glycoproteins.

• Proteoglycans◦ Conjugated proteins that contain very high amount of carbohydrates

such as 80-95%.

◦ It consists of a core protein with a glycosaminoglycan (GAG) chain attached with one or more covalent bonds.– Heparin, chondroitin sulphate can be given as an example.

Conjugated Proteins

107

• Lipoproteins (Proteolipids)◦ Proteins that carry lipids such as

phospholipids, triglycerides, and

cholesterol as prosthetic groups.

◦ Easily soluble in water.

◦ Because lipids are not soluble in water,

they are bound to proteins and

transported as lipoproteins.

◦ Examples include chylomicron,

Lipovitellin, VLDL, IDL, LDL and HDL.

Conjugated Proteins

© McGraw-Hill Companies Inc.

108

Conjugated Proteins

Metalloproteins• Proteins that carry metals (Fe, Cu, Zn ...) as prostatic

groups.

• Fe carriers (ferritin, hemoglobin, cytochromes), Cu carriers (ceruloplazmin, SOD), Zn carriers (SOD), Se carriers (GPx, IDs).

Chromoproteins• They are colored proteins formed by the presence of a

metallic element in the prosthetic groups. They are formed by metal-porphyrin complex systems.

• Hemoglobin, myoglobin, cytochromes are examples.

109

• Nucleoproteins◦ Proteins that result in the binding of nucleic acids to protamines,

histones, and other simple proteins.

◦ Nucleoprotamines are the simplest nucleoproteins. The nucleic acid

and proteins are linked by arginine-phosphate linkage. They are

abundant in fish sperm.

◦ Nucleohistones which the nucleic acid and proteins are linked by

arginine-phosphate linkage are found in fish sperm and bird

erythrocytes.

◦ High nucleoproteins are found in ribosomes, covalently linked RNA-

protein, DNA-protein complex.

Conjugated Proteins

110

They are proteins that are formed as a result of alteration of proteins belongs to simple or conjugated proteins. There are two groups in this type of proteins; Primary and Secondary Derivative proteins.• Primary Derivative Proteins

◦ Peptide ligands are formed by degenerating agents without destroying them.

◦ They are also called denatured type proteins.

◦ These water-insoluble proteins are called proteans by the action of dilute acids and enzymes.

◦ Metaprotein is known to be formed by continuous effects of acids and alkalis.

◦ Boiling, agitation, UV rays and ethanol effect are called coagulation or coagulation proteins.

Derivative Proteins

111

• Secondary Derivative Proteins◦ They are formed by the action of acids or enzymes that partially

cleave peptide bonds.

◦ Protein molecules divide into smaller fragments.

◦ The big fragment is called proteose (albinoin); small pieces are called

peptones.

◦ The smaller chains are also polypeptides and peptides.

◦ These derivative proteins do not precipitate with boiling or coagulate

with heat.

Derivative Proteins

112

Catalytic Proteins

Carrier proteins (Transport proteins)

Nutrient and Storage Proteins

Contractile Proteins

Structural Proteins

Defense Proteins

Physiological Regulatory Proteins

Functional Classification of Proteins

113

Enzymes which catalyze biochemical reactions, are highly specialized proteins.

Enzymes are defined as biocatalytic substances that allow biochemical reactions to take place rapidly under normal conditions and constitute the basic character of living tissue.

Amylase, pepsin, lipase are examples of important catalytic proteins or enzymes.

Catalytic Proteins

114

Proteins that bind specific molecules or ions and transports them from one organ to another or from one side of the cell membrane to the other side.• Serum albumin, the most well known carrier protein; bilirubin,

calcium, fatty acids and many drugs are transported by binding to serum albumin.

• Hemoglobin,oxygen-carrier.• Lipoproteins, lipid-carrier.• Transferrin, iron-carrier.

The carrier proteins found in the plasma membranes and intracellular membranes of all organisms bind to glucose, amino acids and other substances; they carry them from one side of the membrane to the other side.

Transport Proteins

115

They are proteins that serve as biological reserves for

metal ions and amino acids used by the organism.• The main protein of the egg flock which is ovalbumin and the

essential protein of the milk which is casein are nutritional

proteins; amino acid stores.

• Many plant seeds also store nutrient proteins necessary for

germinating seed growth. Gluten the best-known storage

protein in wheat.

• Ferritin is iron storage protein.

Nutrient and Storage Proteins

116

They are proteins that can contract or spontaneously

move.

Myosin and actin function in the contractile system of

skeletal muscles and at the same time in many non-

muscle cells.

Tubulin is the microtubule forming protein.• The microtubules in the cells act together with the dynein

protein in the whip and eyelashes to carry the cells.

Contractile Proteins

117

The main structure of tendons and cartilage is collagen

with very high tensile strength.• Leather, almost pure collagen.

• The ligaments contain elastin, a structural protein capable of

stretching in two dimensions.

• Hair, nails and fur contain keratin.

• The main component of silk fibers and spider webs is fibroin.

The wing axes of some insects are made from the resilin.

Structural Proteins

118

Organisms are proteins that defend against invasion by

other species and protect the organism from harm.

Immunoglobulins are specialized proteins synthesized by

the lymphocytes of the vertebrates.

Immunoglobulins can recognize foreign proteins

(antigens) of another species and can precipitate or

neutralize.• Eg. Bacteria, viruses, proteins of another species.

Defense Proteins

119

• Blood clotting proteins, such as fibrinogen and thrombin, help

prevent blood loss by closing the injured site with a blood clot

when the vascular system is injured.

• Toxic plant proteins such as snake poisons, bacterial toxins and

ricin appear to have defensive functions at the same time.

• Some of the defense proteins, including fibrinogen, thrombin

and some poisons, are also enzymes.

Defense Proteins

120

They are proteins that aid in cellulite regulation or

physiological activity.

Some hormones such as insulin, growth hormone,

regulatory proteins.• Insulin is effective in regulating sugar metabolism

• The growth hormone is effective in regulating the growth

• For many hormone signals, the cellular side is often a class of

GTP-binding proteins called G-proteins.

• Some regulatory proteins surround the DNA; Regulate the

biosynthesis of enzymes and RNA molecules.

Physiological Regulatory Proteins

121

Nucleoproteins

It forms a very important group of compound proteins.

Prosthetics are proteins that are nucleic acids.

NUCLEIC ACIDS• They are hereditary-carrying genes and key features in protein

biosynthesis.

• Every particular biological event, including cell division, is of

interest to nucleic acids.

122

Nitrogenous

base Pentose Phosphate

Nucleoside

Nucleotide

(Mononucleotide)n=Nucleic acid

There are three substances in the structure of nucleic acid

Nitrogenous base + Pentose + Phosphate

+ +

123

1

5

The pentose is located in

the middle of a nucleotide

structure. 1st carbon linked to

nitrogenous base.

5th carbon linked to

phosphate.

3th carbon linked to

phosphate group of another

nucleotide.3

124

A nucleotide phosphate and pentose bond is formed by

the post-esterification of the pentose with the

phosphate of the primary alcohol (CH2OH) group on the

5th carbon atom to form a mole H2O.

Pentose is connected via its first carbon to a nitrogenous

base.

Nucleoproteins

125

The conjugation of the mononucleotides occurs via

pentose and phosphate.

The first nucleotide is generated by the esterification of

the second nucleotide phosphate with the OH group on

the third carbon of the pentose.

According to this linkage, the number n of nucleotides

continues to join and nucleic acids form.

Nucleoproteins

126

Nitrogenous bases found in a nucleotide are purine and

pyrimidine bases. • There are also rare different bases. These bases are methyl-,

dimethyl-, and N-methyl derivatives.

Purine bases are adenine and guanine.

Pyrimidine bases are cytosine, uracil and thymine. • Cytosine is found in both DNA and RNA. Uracil is found in

only RNA. Thymine is found only in DNA.

Nucleoproteins

127

128

Cells can synthesize purine and pyrimidine bases.

Nucleic acids take DNA and RNA names from the pentose

of their structure.

If there is a deoxyribose, it is called DNA

(deoxyribonucleic acid).

If there is a Ribose, it is called RNA (ribonucleic acid).

Nucleoproteins

129

130

Biologically important free nucleotides• In addition to being found in the structures of nucleic acids,

nucleotides are also found in tissues as free nucleotides, which are in close proximity to the nucleotides.

• Most of these substances have catalytic functions and work together with enzyme systems (coenzymes).

• Nitrogenous base + pentose = Nucleoside (Adenine + ribose = Adenosine)

• Adenosine + Phosphate = The nucleotide called adenosine monophosphate (AMP) is formed. Two phosphates form adenosine diphosphate (ADP), three phosphates bind adenosine triphosphate (ATP).

• In other bases, phosphate-binding nucleotides are formed.

Nucleoproteins

131

AMPADP

ATP

132

Biologically important free nucleotidesAMP (Adenosine monophosphate = Adenilic acid): Adenine - Ribose– PO4

ADP ((Adenosine diphosphate): Adenine - Ribose– PO4 – PO4

ATP (Adenosine triphosphate): Adenine - Ribose– PO4 – PO4 – PO4

NAD (Nicotinamide Adenine Dinucleotide): Adenine - Ribose– PO4 – PO4 – Ribose - Nicotinamide

NADP (Nicotinamide Adenine Dinucleotide phosphate) : Adenine – Ribose (– PO4) – PO4 – PO4 – Ribose –

Nikotinamide

FMN (Flavin mononucleotide) : Flavin – Ribitol - PO4

FAD (Flavin adenine dinucleotide) : Flavin – Ribitol - PO4 - PO4 – Ribose - Adenine

UDP-G (Uridine diphosphate-Glucose) : Uracil – Ribose - PO4 - PO4 - Glucose

Koenzim A : Adenine – Ribose (– PO4) – PO4 – PO4 – Pantothenate thiolethylamine

133

Nitrogenous

baseNucleoside Nucleotide Symbol

Adenine Adenosine Adenilic acid AMP

Guanin Guanosine Guanilic acid GMP

Cytosine Sitidin Cytidylic acid CMP

Uracil Uridin Uridylic acid UMP

Tymine Thymidine Thymidic acid TMP

134

Both DNA and RNA are common in animal tissues.

DNA is found in the nucleus and also RNAs.

The major nucleic acid fraction of the cytoplasm is RNA.

In cytoplasm, RNA is usually found in ribosomes.

Structure and Functions of Nucleic Acids

135

Building blocks; Phosphate, deoxyribose and purine and

pyrimidine bases such as Adenine (A), Guanine (G),

Cytosine (C), Timin (T).

The ratio of (A + T):(G + C) is always changed. However,

adenine and thymine or guanine and cytosine are

equally distributed.

There are three structures in the DNA molecule.• Primary structure

◦ Certain species and a certain number of nucleotides form

polynucleotide chains according to one sequence; Chain structure.

Deoxyribonucleic Acid (DNA)

136

• Secondary structure◦ It occurs when two polynucleotide chains are mutually located.

◦ These two chains are connected to each other by hydrogen bridges

between nitrogenous bases.

◦ However, this connection must be appropriate on mutual basis.

◦ It always resists adenine-thymine (A-T), guanine-cytosine (G-C).

• Tertiary structure◦ The two polynucleotides are coiled, in other words a double spiral is

formed; like twisted ladder.

◦ The same nitrogenous bases come together and are joined by two

chain H bridges.

Deoxyribonucleic Acid (DNA)

137

Molecular Structure of DNA

Primary structure

Secondary structure

Tertiary structure

138

DNA has two main functions in the organism.1. To carry genetic code.

2. Having the ability of replication.

In the cell division, genetic code are transferred

completely and unchanged to the offspring.

In order for a new DNA molecule to come into being

(DNA replication), the double helix, the tertiary

structure, has to break down and make up the

secondary structure.

Deoxyribonucleic Acid (DNA)

139

The separated DNA strands bring about a mold for the

newly formed DNA strands.

Against this pattern, a strand of DNA similar to the

structure of the peer-strand from which the strand itself

is separated may form.

Because the nitrogenous bases in the DNA strand we call

mold are only able to match their respective

nitrogenous bases.

Deoxyribonucleic Acid (DNA)

140

The strands here are called semiconservative

replicates. Semiconservative replication would

produce two copies that each contained one of the

original strands and one new strand.

In the DNA molecule, three nitrogenous bases on top of

one another encode an amino acid called codon.

Deoxyribonucleic Acid (DNA)

141

142

In order for RNAs to be formed, primary DNAs are required.

Is the RNA structure different from the DNA structure?• Instead of deoxyribose, it carries ribose.

• Uracil is found instead of thymine.

• It is not a double-helix; one strand.

There are three main types of RNA; mRNA, rRNA, tRNA

Ribonucleic Acid (RNA)

143

144

145

Transcription

DNA Transcription

146

DNAs determine the sequence of amino acids in the

structure of proteins.

All three nitrogenous bases in the polynucleotide chain

of a DNA encode an amino acid (codon).• Here, the order of the nitrogenous bases in the nucleus of the

DNA is genetic code.

The first step in protein biosynthesis is that the

genetic information in DNA is transported to

ribosomes where protein synthesis occurs. This is

done by mRNAs; Transcription.

mRNA (Messenger RNA)

147

An RNA template is formed that represents the genetic information and DNA portion necessary for protein synthesis. It passes into the cytoplasm and binds to the ribosomes.The amino acid code is valid for all forms of life,

including viruses, that have been researched up to now.The genetic code of DNA is carried by the base sequence

of RNA.The base sequence of the mRNA determines the

amino acid sequence of the protein.

mRNA (Messenger/ RNA)

148

149

The second type of RNA is found on the endoplasmic

reticulum. It is rRNA that is located in the organelle

called the ribosome. RNA forms a large part of the

ribosomes.

rRNA (Ribosomal RNA)

150

They are RNA molecules capable of

combining with specific amino acids

and transporting them to ribosomes.

There are 3 important features.• They can covalently bind a particular amino

acid.

• In their own structure, they have a

anticodon which completes the amino acid

in the mRNA code.

tRNA (Transfer RNA)

anticodon

151

152

Chromoproteins A chromoprotein is a conjugated protein that contains a

pigmented prosthetic group (or cofactor); like porphyrines. Hemoglobin and respiratory enzymes are chromoproteins

whose prosthetic group is heme. Again, the prosthetic group is riboflavin in flavoproteids,

the prosthetic group is melanin in proteins found in hair and wool. The catalase enzyme is also a chromoprotein whose

prosthetic group is heme. The chromoproteins that carry the heme structure as a

prosthetic group are called hemoproteids.• Hemoglobin, myoglobin, cytochromes and peroxidases are

hemoproteids.

153

The pyrrole ring is the first and simplest substance of porphyrins. When the side chains such as propionic acid, acetic acid or methyl, ethyl, hydroxyethyl, vinyl are added to the pyyrol ring, the structure called porphobilinogen comes to the fore. Porphobilinogen is called monopyrrol and is considered to be the precursor of porphyrins.• If the four porphobilinogen rings are connected to each other

by methine (=CH-) bridges, porphyries will form.

• Porphyrins are a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges.

Porphyrins

154

Pyrrole Ring Porphobilinogen (PBG)

155

If 4 acetic acid and 4 propionic

acid are attached to the

porphyrin ring, uroporphyrins

are formed.• Uroporphyrins I

• Uroporphyrins III

• Coproporphyrin I

• Coproporphyrin III

Porphyrins

Porphin Ring

156

Porphyrins

Hemoglobin• They are chromoproteins carrying HEM as a prosthetic

group.

• It is formed by four groups, each in parallel with the globin,

perpendicular to the peptide chain.

• Hemoglobin is mainly synthesized by the liver in fetus. It is

also synthesized in bone marrow in adults.

• Hemoglobin is rich in histidine, and is a good buffer.

• The most important task is to create oxyhemoglobin, which

is combined with atmospheric oxygen in the lungs to

transport oxygen to the tissues.

157

Heme Hemoglobin

158

Porphyrins• Each of the 4 groups in the hemoglobin can bind an oxygen

molecule.

• Carbaminohemoglobin is a compound of hemoglobin and carbon

dioxide, and is one of the forms in which CO2 exists in the blood.

• When combined with CO, carboxyhemoglobin is formed.

• Methemoglobin is a form of the oxygen-carrying metalloprotein

hemoglobin, in which the iron in the heme group is in the Fe3+

(ferric) state, not the Fe2+ (ferrous) of normal hemoglobin.

Methemoglobin cannot bind oxygen.

• Fe++ can be replaced with some anions. Combine with cyanide to

form cyanomethemoglobin.

• When oxyhemoglobin is treated with H2S, sulfhemoglobin forms.

159

PorphyrinsMyoglobin• Keeps the oxygen brought by

hemoglobin.

• In the structure;◦ There is a single polypeptide chain

containing 153 amino acids.

◦ Globular structure.

◦ 1 molecule of Heme as a prostatic

group.

• It is found in the muscle tissue.

160

Erythrocytes are destroyed at the end of their life for about 125 days, and as a result, the porphyrin ring is opened. It comes in an open chain containing 4 pyrrole rings, which are called bile pigments. It is assumed that all of the substances included in the group

of bile pigments are theoretically derived from a (proto) Bilin substance. In the disintegration of hemoglobin, Biliverdin results from

the breakdown of the heme moiety of hemoglobin in erythrocytes. Bilirubin is formed by the reduction of one of the methine

groups linking the porphobilinogen rings of Biliverdin to the methylene group.

Bile Pigments

161

Mesobilirubin occurs when the bilirubin vinyl groups are converted into ethyl groups. In the other two methine (=CH) groups that link the

porphobilinogen rings, mesobilirubinogen is formed by reduction to the methylene (CH2) group. In this, two side porphobilinogen rings form stercobilinogen by entering two H atoms. In clinical biochemistry, mesobilirubinogen and

stercobilinogen are called bilinogenes or urobilinogens. • Urobilinogen can be reduced to form mesobilirubinogen, and

mesobilirubinogen can be further reduced to form stercobilinogen.• Urobilin was isolated in urine and stercobilin was isolated in

stool for the first time. They are actually found in many tissues and other body fluids.

Bile Pigments

162

Bile Pigments Features• Mesobilirubinogen and stercobilirubinogen are colorless,

urobiline and stercobilin are yellow, mesobilirubin and bilirubin are orange blossom and biliverdin is bluish green color.

• The Na and K salts of biliverdin and bilirubin are dissolved in water, but salts of Ba and Ca are insoluble.

• If concentrated nitric acid and bile-colored substances are layered, green, blue, violet, red colors come into contact on the contact face. This is called the Gmelin’s test. With this test, it is possible to diagnose the jaundice.

• When the bilirubin is reacted with freshly prepared diazo reagent, azo-bilirubin in red forms. It is used to quantify bilirubin in blood.The reaction name is Van den Bergh.◦ The diazo substituent is a mixture of sulfanilic acid, HCl and sodium nitrite.

163

Bilirubin is the bile-colored substance in the plasma collection to bilirubinemi, removing the bilirubinuria urine, skin and mucosa into painting these tissues collected icterus (jaundice) is called. According to their causes icterus divided into three.1. Mechanics/retention Icterus (Post-hepatic): It is also called

obstructive jaundice and caused by an interruption to the drainage of bile containing conjugated bilirubin in the biliary system. The most common causes are gallstones in the common bile duct. Beside this, inflammation, parasites, tumors can also be reasons. both direct and indirect bilirubin levels are increased in serum. In the urine, bilirubin (+), urobilinogen is normal. Stool is colorless.

Icterus (Jaundice)

164

Icterus (Jaundice)2. Hepatocellular Icterus (Hepatic): It can be caused by acute or

chronic hepatitis, hepatotoxicity, cirrhosis, drug-induced hepatitis. Cell necrosis reduces the liver's ability to metabolize and excrete bilirubin In the serum, indirect bilirubin increased.

In the urine, urobilinogen is (+).

Stool is light color.

3. Hemolytic Jaundice (Pre-hepatic): Pre-hepaticular jaundice is caused by anything which causes an increased rate of hemolysis (breakdown of red blood cells). In the serum, indirect bilirubin increased .

In the process, urobilinogen is very high.

Stool is dark color.

165

Serum and Plasma• After the blood collected in a tube containing a suitable

anticoagulant agent that can inhibit coagulation, if to be centrifuged, it is separated into two layers; a liquid supernatant which has a yellow-pink color and a shaped elements which are cells.

• The liquid part of the blood that separates in this way is called plasma. Plasma contains 8% plasma proteins, 1% inorganic salts, and the rest contain substances such as lipids, carbohydrates and amino acids. The fibrinogen fraction of the plasma proteins that make up the 8% portion is contained in the plasma.

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166

If the fibrinogen present in the plasma passes into the

fibrin and the blood coagulates, the remaining light

yellow portion is taken as the serum..

Plasma(Water, proteins, lipids,

carbohydrates, hormones, etc.)

.)

Erythrocytes

Leukocytes and thrombocytes

Source: WikiMedia

Normal

Hematocrit

(HCT)

Anemia

(HCT )

Polycythemia

(HCT )

Major Proteins of Fluids and Tissues in Organism

167

Coagulation of blood• It occurs when the fibrinogen, a soluble protein found in

circulating blood, changes. When necessary, this fibrinogen

transforms into a fibrin by a series of reactions. This is what is

called coagulation (blood clotting/clotting).

• The conversion of fibrinogen to fibrin is provided by an enzyme

called thrombin. This enzyme is not active in the circulation.

• Many factors play a role in blood coagulation, mainly Ca.

• Coagulation of blood is not a simple event, but a complex and

controlled mechanism process; it is called Coagulation

Cascade.

Major Proteins of Fluids and Tissues in Organism

168

Thrombocytes are one of the blood cells. • When blood is taken from the circulating

blood, these platelets stick to the wall of the tube, and if it is a hemorrhage, it sticks to the edges of the wound and splits.

• This fragmentation results in the release of a substance called thromboplastin.

• Thromboplastins are found in circulating blood. Prothrombin is an inactive form of thrombin and thromboplastins convert prothrombin to thrombin. Thrombin also provides blood clotting by allowing the fibrinogen to pass into the fibrin.

Canine

Feline

Source: eClinPath

Major Proteins of Fluids and Tissues in Organism

169

Coagulation Cascade Animation

170

Plasma and serum proteins

FibrinogenIt is glycoprotein. It has ellipsoid-fibrillar structure. High

viscosity in solutions. It plays the most important role in blood

coagulation.

Serum

Albumin

It is mostly a lipoprotein structure. It's an ellipsoid shape. It is

important in preserving the osmotic pressure. It binds and

dissolves nonsoluble matters and transports them in circulating

blood.

Serum

Globulin

α-globulins: It's in glycoprotein structure. Ceruloplasmin and

prothrombin can be the examples.

β-globulins

γ-globulins: These are called antibodies or immunoglobulins.

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171

Muscle Proteins

Sarkoplasmic Proteins Myogen, Myoglobin, Myoalbumin, Globin-X

Fibrillar ProteinsMyosin, Actin (Globular actin/G-actin,

Fibrillary actin/F-actin)

Milk and Egg Proteins

Milk Proteins Casein, Lactobombin, Lactoglobulin, Immunoglobulins

Egg Proteins

Egg white: Ovalbumin, Konalbumin, Ovomucoid,

Ovoglobulins, Mucin, Avidin

Egg yolk: Vitellin, Livetin, Phosphitin (all are

phosphoprotein).

Major Proteins of Fluids and Tissues in Organism

Ası. T. 1999. Tablolarla Biyokimya, Cilt 1

Engelking LR. 2014. Textbook of Veterinary Physiological Chemistry. 3rd

edn. Academic Press.

Smith JG (2010). Organic Chemistry, 3rd Edition, McGraw-Hill.

Smith JG (2012). General, Organic, & Biological Chemistry 2nd Edition,

McGraw-Hill.

Sözbilir Bayşu N, Bayşu N. 2008. Biyokimya. Güneş Tıp Kitapevleri, Ankara

References

173

Question 1Cevap: B

Which of the following is the simplest known amino

acid?

a) Alanin

b) Glycine

c) Treonin

d) Triptofan

e) Glumatic acid

174

Question 2Cevap: C

It is called decomposition of proteins because of various

exposures.

a) Deamidation

b) Renaturation

c) Denaturation

d) Decarboxylation

e) Deamination

175

Question 3Cevap: A

Which molecule forms the first and simplest basic

substance of porphyrins?

a) Pyyrol ring

b) Porphobilinogen

c) Fibrinogen

d) Stercobilin

e) Biliverdin

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