TVER STATE

MEDICAL UNIVERSITY

BIOCHEMISTRY DEPARTMENT

CHEMISTRY AND FUNCTIONS

OF PROTEINS

ILLUSTRATED BIOCHEMISTRY

Schemes, formulas, terms and algorithm of preparation

The manual for making notes of

lectures and preparation for classes

Tver, 2018

AMINO ACIDS

-amino acids -These are organic acids with at least a minimum of one of its

hydrogen atoms in the carbon chains substituted by an amino group.( Show the

radical, amino and carboxyl groups)

Proteinogenous and Nonproteinogenous Amino acids

- Major proteinogenous (standard) amino acids. (Give the names of each amino

acid)

-

NH2 – (CH2)3 – CH2 –

NH2 – C – NH – (CH2)3 – CH2 –

||

NH

H –

CH3 –

(CH3)2 CH –

(CH3)2 CH – CH2 –

CH3 – CH2 – CH –

|

CH3

HOOC – CH2 –

HOOC – CH2 – CH2 – NH2

|

– C – H

|

COOH

NH2 – CO – CH2 –

NH2 – CO – CH2 – CH2 –

1

2

3

4

5

6

7

8

9

10

11

20

HO – CH2 –

CH3 – CH –

|

OH

HS – CH2 –

CH3 – S – CH2 – CH2 –

12

13

14

15

16

17

18

19

NH2

|

C – H

|

COOH

R

R R

NH СООН

2

-Glycine

Alanine

-Valine

Leucine

Isoleucine

Aspartic acid

Glutamic acid

Asparagine

Glutamine

Lysine

-Arginine

-Serine

-Threonine

-Cysteine

-Methionine

-Phenylalanine

-Tyrosine

-Tryptophan

-Histidine

-Proline

•Rare proteinogenous (standard) amino acids. ( Derivatives of lysine, proline and

tyrosine)

•Nonproteinogenous amino acids. (Name and show them)

NH2

|

HS – (CH2)2 – CH

|

COOH

NH2

|

NH2 – C – HN – (CH2)3 – CH

|| |

O COOH

NH2

|

H2N – (CH2)3 – CH

|

COOH

Ornithine

Homocysteine

Citrulline

NH2

|

H2N – CH2 – CH – (CH2)2 – CH

| |

OH COOH

10

CLASSIFICATION OF PROTEINOGENOUS (STANDARD)

AMINO ACIDS

Amino acids are classified by:

*The structure of the radical (show);

- aliphatic amino acids

-monoaminodicarboxylic amino acids

-amides of amino acids

-diaminomonocarboxylic amino acids

-hydroxy amino acids

-sulfur-containing amino acids

-cyclic (aromatic and heterolytic) amino acids

*the polarity of the radical (show);

-Non-polar (hydrophobic –Ala, Val, Leu, Ile, Trp, Pro.)

-Polar (hydrophilic).

·charged amino acids

-negatively charged (Asp, Glu)

-positively charged (Arg, Lys,His)

-uncharged (Gly,Ser,Thr,Cys,Tyr).

*the level of essentiality (biological classification);

- Essential (VILL MTTPh)Val, Ile,Leu,Lys,Met,Thr, Trp, Phen)

-Semi-essential (Arg,Tyr,His)

- Nonessential (all the rest).

*their acid-base properties;

-acidic (Asp, Glu)

-basic (Lys, Arg, His)

-neutral (all the rest)

PHYSICO-CHEMICAL PROPERTIES OF AMINO ACIDS

•Ionization-(protonization and dissociation) of basic and acidic groups of amino acids

in water. (Explain and open up the biological importance of this phenomenon).

-Show the following:

the acidic groups of the amino acids

the basic groups of the amino acids

the forms in which amino acids can exist( show the neutral, the transition and dipole

states)

11

*The influence of pH on the ionization (charge) of an amino acid

-show how the charges of amino acids in basic and acidic media are changed.

-pH<7, the excess of hydrogen ions{H+}- acidic medium

-pH>7, the excess of hydroxyl ions {OH-}-basic medium

R

H+ | OH

-

(+) +H3N – CH – COO

– (+)

•The Isoelectric state and isoelectric pH point of amino acids.

-Explain the meaning of the isoelectric pH of various amino acids. Prove various

of the isoelectric points of the following amino acids.

-neutral (alanine )

-acidic (aspartic acid)

-basic (lysine )

COO-

|

CH2

|

H3+N – CH – COO

–

NH3+

|

(CH2)4

|

H3+N – СН – COO

–

Asp Ala Lys

7

9,8

IEP

2,8

IEP

CH3

|

H3+N – CH – COO

–

? ?

+ H+ ОH

-

7

рН

12

•The amphoteric and buffer properties of amino acids

-What is amphoterity?

-Why do amino acids exhibit buffer properties?

PROTEINS

They are ;

High –molecules

Nitrogen containing

Organic compounds (substances)

Made up of amino acids

joined in a chain

with the help of peptide bonds

and have a complex structural organization

(Explain all these characteristics)

-Elementary components of proteins (C50-54% , O21-23% , N15-17%, H6,5-7,3% , S0,5% )

-What are oligopeptides, polypeptides, proteins (up to 10 , 10-40 ,>40 amino acids) and

their molecular masses ( the average molecular mass of one (1) amino acid is 110 a.u ) ?

N.B a.u = atomic units

- Biological functions of proteins (Give examples of proteins with different functions and

give the characteristics of the actions of the proteins action in performing these functions)

Function Example of protein Characteristics of the

action

Fermentative (Enzyme)

Hormonal

Receptive

Transport

Structural and Supportive

Contractile

Substrate - energetic

Electro-osmotic

Immunological

Haemostatic

Energo –transformative

-There are about 50,000-100,000 different proteins functioning in the

body of a human being. These different proteins perform the same number of different

functions in the human organism.

13

- How can the different kinds of functions of proteins, their individual and

immune properties be explained?(Sequence and the number of the 20 proteinogenous

amino acids) .Explain your answer.

STRUCTURAL LEVELS OF ORGANIZATION OF PROTEIN

MOLECULES

Primary ( Linear sequence of amino acids joined in a chain with the help of peptide

bonds)

A scheme of the primary structure of a protein

-Show :

-the peptide chain

-the N- and C-terminals of polypeptide chain (ppc)

R1 R2 R3 e.t.c. (n) times Rn

(N С)

R

|

H2N – CH – COOH

R2

|

H2N – CH – CОOH +

R3 Rn

|

H2N – CH – CОOH + ….….… +

Rextreme

|

H2N – CH – C – OH

R1

|

H2N – CH – CОOH +

R1 R2 R3 Rn O

| | | | //

H2N – CH – CО ––– NН – CH – CО ––– NН – CH – CО ………… NН – CH – C – OH

1 2 3 n

– Н2О – Н2О – Н2О

………………….

14

-the radicals of the amino acids

Secondary structure of proteins.

-It is a way of folding of the polypeptide chain (Primary structure ) into a regulate -

spiral or a -structure ( kind of bond –Hydrogen bond)

-Explain the differences between the -spiral and the -spiral

Tertiary structure of proteins

-This is the folding of the -spiral (-helix) or the -structure in space (Globular,

Fibrillar proteins, explain their structure)

– О

||

– C –– N –

|

Н +

– CO — NH–

β-structure

R4

15

-Show how the tertiary structure is stabilized by ionic, hydrogen, covalent (disulfide)

bonds and hydrophobic interactions

Quaternary structure

-It is the joining the group of polypeptide chains with tertiary structures into a unit

functional protein molecule.

What are protomer (subunit), oligomer?

What kinds of bonds stabilize the quaternary structure of a protein ?

( Show them on the above drawn diagram )

16

Which molecules contain information about the primary, secondary , tertiary and

quaternary structures of proteins and where are they located?

Explain the importance of the primary and quaternary structures of proteins in the

performance of their functional activities giving examples of ;

- denaturation of proteins

- sickle –cell anemia

- different structures and functions of myoglobin and hemoglobin

17

PHYSICAL AND CHEMICAL PROPERTIES OF PROTEINS

THE STRUCTURE, HYDRATE AND IONIC LAYERS OF PROTEIN

MOLECULES.

Solubility of Proteins

Show and explain how the number (quantity) of amino acids , the hydrate layer and

the structure (conformation) affect the solubility of proteins

-neutral proteins

-acidic proteins.

Isoelectric Point of proteins

-using the diagram, show the value of the isoelectric point of the following proteins

(pH<7, >7, =7)

-basic proteins

-Explain how changes in the pH medium can affect the charge of a protein molecule

(<7, >7,=7)

Acid medium pH 7 Basic medium

[H+] [OH

–]

(0)

OR

The value of the charge

depends on the amino

acid composition and

pH of the medium.

COO –

NH3+

COO

–

COO –

NH3+

COO

–

NH3+

NH3+

1)

2)

3)

18

- Using given diagrams explain:

-the isoelectric state of a protein molecule

-the isoelectric point (pI) of protein molecules.

Amphoteric Properties of Proteins

- Prove using the diagrams on pages 9 an 10 that, proteins are amphoteric

polyelectrolytes. Which groups of atoms give a protein molecule acidic properties and

which groups give basic properties?

Buffer properties of proteins

-What is a buffer?

-Which groups of atoms exhibit buffer properties?

-Explain the value of the buffer capacity of proteins.

Colloid properties of proteins

- What qualities of a protein molecule enable them to exhibit colloid properties?

(molecular mass, size of the molecules, charge, hydrate layer).Explain your answer.

-Name the colloid properties of protein solutions.(density, rate of diffusion, gel

formation, optical properties, inability to penetrate trough semi-permeable membranes

etc.).

Osmotic properties of proteins (diffusion and osmosis)

-Show on the diagram the osmotic (hydrostatic) pressure.

-Explain what is meant by the acetic pressure of proteins.

-Explain the importance of the oncotic pressure of proteins in the regulation of water

in the organism (Water metabolism).

Semipermeable

membrane

19

Salting-out and the denaturation of proteins

-Using the diagram, explain how the following occur:

-the increase in the solubility of proteins when minute (small) concentrations of

the salts of alkaline or alkaline-earth metals are added to them in solution

-the decrease in the solubility of proteins (salting-out) when high concentrations of

the above mentioned salts are added to them

-Factors that stimulate the above mentioned processes (choose the factors for

denaturation and those for salting-out)

-High temperatures

-Vibration

-Salts of alkaline and alkaline-earth metals

-salts of heavy metals

-Mineral and organic acids

-Organic solvents

-Ionized radiation

-Explain the differences in the structure and function of protein molecules during

denaturation and salting-out.

CLASSIFICATION OF PROTEINS

Proteins are classified by their:

-electrochemical indications (acidic, basic and neutral).Explain and give

examples

-polarity ;polar (hydrophobic), nonpolar (hydrophilic), amphiphilic. Explain these

terms.

-function (transport, enzymes, hormones, antibodies etc)

20

-chemical composition(simple and complex)

Simple proteins(Describe the amino acid composition, the molecular mass, the

charge , the structure and functions of the following:)

-Protamines -Albumins

-Histones -Globulins

COMPLEX PROTEINS

The chemical composition and structure of complex proteins.

-Protein component-Apoprotein

-Nonprotein component-Prosthetic group

Principle of classification (based on the name of the prosthetic group) and classes of

complex proteins.

COMPLEX PROTEIN PROSTHETIC GROUP EXAMPLE OF THE

PROTEIN AND ITS

FUNCTION

1. Chromoproteins

2. Lipid-protein complexes

3. Carbohydro-protein

complexes(Glycoproteins and

Proteoglycans)

4. Phosphoproteins

5. Metalloproteins

6. Neucleoproteins

-The prosthetic groups of complex proteins (heme, lipids, carbohydrates, phosphoric

acid, ions of metals, nucleic acids)

-Examples (hemoglobin and myoglobin, -lipoproteins, prothrombin,

gastromycoprotein, caseinogen (casein), transferring, chromatin.)

CHROMOPROTEINS

CLASSIFICATION OF

CHROMOPROTEINS

PROSTHETIC GROUP FUNCTION OF THE

CHROMOPROTEIN

Hemeproteins

Retinalproteins

Cobamidproteins

Flavoproteins

13

-Prosthetic groups (heme, vitamin A, Vitamin B, FAD).

-Functions (participating in the transport of oxygen, in the processes of vision,

hemopoesis, redox reactions.)

HEMEPROTEINS

Classification of hemeproteins

Hemoglobin

4 (heme + globin)

Four protomers

II. Fermentative

– Cytochromoxidase

-Catalase

– Peroxidase

I. Nonfermentative

- Myoglobin

- Hemoglobin

The structure of nonfermentative hemeproteins

Myoglobin

(heme + globin)

One protomer

14

globin О2

Myoglobin

Globin is the protein component. Describe its molecular mass, the number of

amino acids, structure(secondary and tertiary), and the place of attachment of the

heme (Histidine)

Heme- is the prosthetic group. It is made up of pyrrol rings (tetrapyrrol),

protoporphyrin IX (methane bridges, vinyl groups, propionate, i.e, the remains of

propanoic acid.), an atom of iron (the point of attachment to the protoporphyrin and

globin).What is the valence of iron?

Hemoglobin

-Similarities and differences between hemoglobin and myoglobin.(Compare in

according to the molecular mass, the structure and the affinity for oxygen.)

-Cooperativity during the binding of hemoglobin oxygen and the subsequent

changes in the affinity for oxygen.

Explain the biological importance of the different affinities of hemoglobin and

myoglobin for oxygen.

-Oxygen dissociation curves for hemoglobin and myoglobin in the lungs and other

tissues (in the skeletal muscles).

15

DERIVATIVES OF HEMOGLOBIN

- There are other groups of atoms that can attach to the heme of hemoglobin and

myoglobin, for examples CO, CO2, CN, OH. The complexes formed as a result are

named as follows:

- Oxyhemoglobin - Methhemoglobin

- Carboxyhemoglobin - Cianhemoglobin

16

-Carbhemoglobin.

PHYSIOLOGICAL AND ANOMALOUS TYPES OF HEMOGLOBIN

-Physiological types of hemoglobin

-Primitive (P), fetal(F) and adult hemoglobins.

Periods of change of the components and the physiological types of hemoglobin

PERIOD OF LIFE TYPE OF Hb STUCTURE OF

THE SUBUNITS

AFFINITY FOR

OXYGEN

Fetus

0-2 months

2-4 months

4-9 months

P(primitive)

P(primitive)

F(fetal)

4

2+2

2+2

++

++

+++

Man

0-4 months

4-40 years

40-60 years

F-A

A(adult)

A2(adult)

Change in structure

2+2

2+2

++

+++

60years and older A3 Change in the

structure of -

chains

+

Anomalous hemoglobins. Diseases of hemoglobins (hemoglobinosis)

-Hemoglobinopathy-sickle cell anemia. Reason: alteration of the physico-chemical

and physiological functions of the hemoglobin (Glu and Val).

-Thalassemias-( and ).Homozygous (big) and heterozygous (small)) thalassemias.

Reason: alteration of the synthesis of hemoglobin.

Fermentative hemeproteins

-Cytochromes b, c1, c, a, a2 and others. (Apoproteins, prosthetic groups and metals that

are components of the heme. What are the valences of these metals?)

Catalase and peroxydase (the chemical composition and the reactions these ferments

catalyze).

1. 2 Н2О2

К 2 Н2О + О2

2. R + Н2О2 П RО + Н2О

PHOSPHOPROTEINS

- Using the following as examples, explain the biological importance of the presence

of phosphoric acids in the organism, their chemical composition and places of

attachment.

- Phosphorylase

- Glycogensynthetase - Caseinogen of milk.

-The reciprocal interaction between phosphorylase and glycogensynthetase

CARBOHYDRO-PROTEIN COMPLEXES

Glycoproteins (GP) and Proteoglycans (PG).

Glycoproteins - The relation of the protein component to that of the prosthetic group

- 95:5.

17

- Carbohydrates of glycoproteins;

- Their chemical nature and structure.

- The roles of the carbohydrate components(stability, period of semidecomposition,

functional importance).

Examples of glycoproteins and the functions they perform.

GROUP OF GLYCOPROTEINS FUNCTIONS

1.G.P.’s of blood serum; factors of

coagulation, transport GP’s ,

immunoglobulins and interferons

2.G.P.’s of saliva, gastric juice , urine,

mucin, Castle’s intrinsic factor.

3.G.P.’s of ferments-enterokinase,

cholinesterase, peroxydase.

4.G.P.’s of hormones – gonadotropic and

thyrotropic hormones

5.G.P.’s of membranes

14

CARBOHYDRATE COMPONENTS OF GLYCOPROTEINS

Proteoglycans: The relation of the protein component to the carbohydrate is 5:95

-Carbohydrates of proteoglycans:

-their chemical nature and their structure – these are heteropolysacchyarides

(glycosaminoglycans (GAG’s). Their old name was mucopolysaccharides

-the role of the carbohydrate component (stability, period of semidecomposition

and functional importance)

15

-Sulphur derivatives of glycosaminoglycans.

-Examples of proteoglycans, glycosaminoglycans and their biological functions in the

organism.

*Hyaluronic acid

*Chondrotin sulphate

*Dermatan sulphates

*Keratan sulphates

*Heparin

16

THE STRUCTURAL ORGANISATION OF THE INTERCELLULAR

MATRIX . (FRAGMENTS OF COMPLEXES OF HYALURONIC ACIDS

WITH PROTEOGLYCANS)

METALLOPROTEINS

-Examples of metalloproteins that contain the following:

*Nonheme iron proteins (ferritin, transferrin, hemosiderin)

*Copper (cytochrome oxidases a, a3, thyroxine)

*Sodium, potassium, calcium (ATP-ases)

*Zinc (carbonic anhydrases, alcohol dehydrogenases)

*Molybdenum (xanthine oxidase)

-The functions of metalloproteins:

*Transport

*Depot

*Structural functions

*Fermentative functions

LIPID- PROTEIN COMPLEXES

These complexes are divided into two groups : free-lipid-protein complexes (blood

lipoproteins ) and structural lipoproteins (proteolipids of membranes).

Plasma lipoproteins

blood lipoproteins are chemically composed of the following; triacylglycerols,

cholesterol esters, cholesterol, phospholipids and proteins.

17

Pre-requisite for the formation of these complexes are NB; lipids are

hydrophobic

The structure of the lipoprotein complexes consists of hydrophobic and

hydrophilic components.

CLASSIFICATION OF PLASMA LIPOPROTEINS

18

1 – according to the classification based on density .

2 - according to the classification based on electrophoretic mobility.

Classification Cholymicrons

VLDL LDL

HDL

2 -

lipoproteins

Pre--

lipoproteins

-lipoproteins -lipoproteins

Chemical

composition %

Proteins= 2

Phospholipids

=3

Cholestrol=2

Cholesterol

ester =3

Triacylglycero

l (TAG)=90

Proteins =10

Phospholipid

s =18

Cholesterol

=7

Cholesterol

ester =10

TAG =55

Proteins =22

Phospholipids

=21

Cholesterol =18

Cholesteryl

ester =42

TAG =7

Proteins =50

Phospholipids

=27

Cholesterol =4

Cholesterol

ester =16

TAG =3

Apoprotein B, C B,C B A

Place of synthesis The

epithelium of

the small

intestines

In the cells of

the liver and

also in the

epithelium of

the small

intestines

In the plasma

of blood

In the cells of

the liver

Function The transport

of exogenic

lipids from the

small

intestines

The transport

of endogenic

lipids from

the liver and

the mucous

The transport

of cholesterol to

the tissues of

the body

The transport

of cholesterol

from the body

tissues to the

liver

19

membrane of

the small

intestine

Density (g/ml) 0.92-0.98 0.96-1.00 1.00-1.06 1.06-1.21

Diameter of the

units (nm)

More than 120 30-100 21-25 7-15

VLDL- Very low density lipoproteins

LDL –Low density lipoproteins

HDL- High density lipoproteins

-Arterogenic and antiarterogenic plasma lipoproteins. (Show how these properties

of lipoproteins depend on their chemical composition, size and function)

THE RELATIVE COMPOSITION OF THE DIFFERENT CLASSES OF

PROTEINS AND LIPIDS OF PLASMA LIPOPROTEINS

10%

18%

7%

10%

55%

50%

27%

4%

16%

3%

proteins phospholipids Cholesterol Cholesterol ester Triacylglycerols

2% 3% 2% 3%

90%

22%

21%

8%

42%

7%

Chilo-

microns

VLDL

LDL HDL

20

-Structural Lipoproteins (Proteolipids of cell membranes and their subunit

structures) NB ;These are membrane –bound lipoproteins.

-The protein –to-lipid relation in different membranes (the cell membrane, the

mitochondrial membrane, myelin-50:50, 80:20, 20:80.)

-The lipids of cell membranes (phospholipids, cholesterol, plasmalogen,

sphingolipids)

-The biological importance of proteins found in cell membranes (structural, antigens,

receptors, ferments, transport, etc)

INTERRELATIONSHIP BETWEEN THE PROTEINS AND LIPIDS OF CELL

MEMBRANES

-The unitary model of the cell membrane , ie, the “sandwich” model by Danielli and

Dawson (1931).The presence of a phospholipid bilayer , with pores covered by

different proteins.

-The liquid-mosaic model of the cell membrane by Singer and Nicholson (1972).The

phospholipid bilayer is of liquid consistency , and it has proteins both on the surface

and those that permeate (penetrate) the whole width of the bilayer.

Cholesterol decreases the fluidity of the membrane and helps in the structural

organization of the bilayer.

THE LIQUID-MOSAIC MODEL OF CELL MEMBRANES

21

-Lipid and protein molecules of membranes are always in regular defined motions

(lateral displacement or turning)

-The asymmetry of membranes is due to the different structures, chemical

compositions and functions on the different sides of the membrane.

MECHANISMS USED FOR THE TRANSPORT OF LIPOPHILIC AND

HYDROPHILIC SUBSTANCES THROUGH THE CELL MEMBRANE

-Transport mechanisms according to the laws of diffusion.(Normal (I) and facilitated

(II)diffusion)

-The active transport of substances through their concentration gradient (protein-

carriers, ATP, symport (IIIa) and antiport (IIIb) .

The vesicular transport of macromolecules through the cell membrane (endo- (IV)

and exocytosis (V).)

22

23

THE BIOLOGICAL FUNCTIONS OF MEMBRANES (EXPLAIN).

-Divisional: membranes divide the intra- and extracellular spaces.

-Integrative (uniting): membranes unit many different uncoordinated reactions to form

a structural unit.

-Transportal: membranes take part in the transport of different substances between the

cell and the extracellular environment.

-Osmotic: this function is performed due to the different concentrations of substances

(for example, cations of sodium and potassium) on opposite sides of the cell

membrane .

-Electric: membranes enable the conditions for differences of electric potentials.

-Energo-transformational: membranes provide with the transformation of electric and

osmotic energy into the chemical energy of ATP.

-Receptional: membranes are able to receive signals from the external environment

due to the presence of special protein-receptors on their surfaces.

The received signal is transmitted into the cell.

-Regulatory function: membranes take part in the formation of intracellular regulators

of substances exchange -3,5’-cyclic ADP and 3,5’-cyclic GMP.

-Metabolic: ferments of membranes take part in the transformation of substances

characteristics of the cell (natural) and substances that are “foreign” to the cell.

-Antigenal: glycoproteins of cell membranes define the ability of the membranes to

cause the formation of specific antibodies.

-Adhesive: adhesion or contact with other cells depends on the zones of familiarity

(recognition) containing carbohydrates.

24

NUCLEOPROTEINS

Deoxyribonucleoproteins (DNP) and Ribonucleoproteins (RNP).These are complex

proteins made up of simple proteins (protamines and histone)+DNA or RNA

Historical information

1868 - Meischer isolated and separated nucleic acids from the nucleus,

1943 - Avery, Macleod, MacCarthy found out that, the DNA of a virulent bacteria

converts a non-virulent culture of bacteria into virulent ones. DNA carries information

on heredity

1949 - Chargaff and his colleagues discovered the regularity (conformity) of the

formation of the DNA structure

1953-Watson and Crick created the DNA model

1967 - Kornberg synthesized the DNA of a virus

1970 - Corana synthesized an artificial(synthetic)gene

1978 - Arber, Smith, Nathanson discovered the phenomenon of DNA restriction

2000 - Scientists all over the world came close to totally decoding the human

DNA

2002 - It was allowed in England to synthesize different organs and tissues by

means of cloning

-Chemical compositions of nucleoproteins

-The following substances are formed as a result of the total hydrolysis of

nucleoproteins (DNP and RNP)

-amino acids

-pentose

-phosphoric acid

N-containing molecules of (A,G,T,C,U)

-2,4-dioxypyrimidine (Uracil)

-5-methyluracil (Thymine )

-2-oxo-4-aminopyrimidine

-6-aminopurine (Adenine)

25

-2-amino-6-oxopurine (Guanine)

During partial hydrolysis the following substances are produced:

-oligopeptides and proteins (protamines, histones)

-nucleosides,ribose-5-phosphate,mononucleotides,oligonucleotides,nucleic acids

NUCLEOSIDES (NITROGEN BASE+RIBOSE OR DEOXYRIBOSE)

- Principle of formation.

Ribonucleosides Deoxyribonucleosides

-Adenosine - deoxyadenosine

-Gyanosine - deoxyguanosine

-Cytidine - deoxycytidine

-Uridine - thymidine

Ribose-5-phosphate

-Principle of formation

MONONUCLEOTIDES (NITROGEN BASE + RIBOSE + PHOSPHORIC

ACID)

-Principle of formation (as on the previous page).

-Names of ribonucleotides

-Adenylic acid (adenosine monophosphate AMP)

-Gyanylic acid (gyanosine monophosphate GMP)

-Cytidylic acid (cytidine monophosphate CMP)

-Uridylic acid (uridine monophosphate UMP)

26

-Thymidylic acid (thymine monophosphateTMP)

- Names of deoxyribonucleotides:

- Deoxyadenosinemonophosphate ( d-AMP etc)

- Unusual mononucleotides and their nitrogen bases (5-methyladenine, 1-

methyladenine).

The biological importance of these mononucleotides.

(Defense, regulatory)

NUCLEIC ACIDS (These are polymers made up of different mononucleotides joined together by

phosphodiesteric bonds)

These acids may either contain ribose or deoxyribose and also they may either contain

Uracil or Thymine. Based on these nucleic acids may either be called RNA or DNA.

-Principle of formation –complex- esteric covalent bonds(3’,5’)between the ribose of

the previous mononucleotide (C3-OH) and the phosphoric acid of the next

mononucleotide.(C5-OH)

DNA and RNA have primary, secondary and tertiary structures

The primary structure of nucleic acids:

27

-linear sequence of mononucleotides in a polynucleotide chain.

-3’and 5’ terminals of nucleic acids

-Show and explain the given positions of the diagram on page 26

Differences between RNP (ribonucleic proteins) and DNP (deoxyribonucleic proteins)

Difference RNP DNP

Content Ribose

Adenine

Gyanine

Cytosine

Uracil

Deoxyribose

Adenine

Gyanine

Cytosine

Thymine

Proteins Little Many

mm 4.10*4-0,6.10*6 1.10*11-10.10*11

Location Cytolasm nucleus

Structure Irregular Regular

2 –spirals

Biological function Transfer of information Storage of information

DNA and DNP

Secondary structure of the DNA.

- Key to the solution of the secondary structure of DNA

Chargaff’s equivalence rules

(I) A=T ;G=C A = 1; G =1

T C

(II) A+G = C+T A+G =1

C+T

(III) A+C=T+G A+C =1

G+T

(IV) Coefficient of specificity

G+C 1 (in animals)

A+T

(V) The DNA does not change during the lifetime of an organism and does not

depend on nutrition or the external environment.

(Contemporary (modern) view at the above – mentioned (V) problem - AIDS,

radiation)

Results of the x-ray diffraction photograph and its analysis (the work by Maurice

Wilkins and Rosalind Franklin)

-Periodicity of the polymeric chain of the DNA along the 0,34 and 3,4nm axis

-Watson and Crick model

28

-Two polynucleidotide chain (double helical structure)

-Chains are antiparellel (5’,3’ and 3’,5’)

-External side of the spiral – phosphoric acid connected with a deoxyribose.

-Internal components of the spiral – nitrogen bases, which are located

complementary by each other (A::::::::::::T, G C)

-Principle of complementarity. (Explain it using diagrams)

-Tertiary structure of DNA-superspiral-chromosome

-46 chromosomes (23pairs)-These are 46 molecules of DNA containing genetic

information

-The biochemical protein of a gene –This is a fragment of the DNA that codes one or

more proteins.

-Genetic text –triplet of mononucleotides (codogene – show it)

-Genome-the total sum of all the genes in the nucleus.

-Quantity of genes in the nucleus (this quantity is equal to the different number of

proteins in an organism).

-Different types of genes (structural, regulatory, etc).

THE STUCTURAL ORGANISATION OF THE DNA IN THE CROMATIN

29

The principle of formation:

-Histones + a fragment of the DNA (~200 pairs of mononuleotides ) =nucleosome

-During cell division, the genetic material is grouped in the form of chromosomes.

-During the active synthesis of proteins ,the chromosomes untwist and form active

chromatin

Deoxyribonucleic protein (DNP) =DNA + proteins

RNA AND RNP

-Principle of classification of RNA (according to their molecular mass, location and

function).

-m- RNA (i-RNA)

-t-RNA

-r-RNA

m-RNA (messenger-RNA)

-they are formed after the copying of information from the DNA.

-they serve as the template on which the specific amino acid sequence of a protein

molecule is built.

-they code one or more polypeptide chains

-the genetic code for m-RNA; triplet (codon)

-m-RNA + protein = informosome

r-RNA (ribosomal-RNA)

-functions of r-RNA; they serve as the frames for future ribosomes

-the biological role of ribosomes (they take part in the formation /synthesis of

proteins).

t-RNA (transfer-RNA)

30

-chemical composition

-quantity of mononucleotides

-the secondary structure of t-RNA (the formation of the “clover leaf” and the

functionally important parts)

1-the acceptor arm- CCA

2-the anticodon

3-pseudouridine arm (loop); this is the part used to attach to the ribosomes

(TC-loop)

4-dihydrouridine loop (arm) or the D-arm ,this is the point where the ferment

aa-t-RNA-synthetase attaches.This ferment is responsible for the joining of specific

amino acids to the given t-RNA. (D-loop).

-there are 20 proteinogenic (standard) amino acids and there are over 60 t-RNA.

(Explain the reason of the t-RNA excess).

*one amino acid is transported by 2-3 tRNA, but one t-RNA can only transport

a definite amino acid.

*aminoacyl-t-RNA (aa-t-RNA) is the active form in which amino acids are

transported.

STRUCTURE OF THE t-RNA

31

PHYSICO-CHEMICAL PROPERTIES OF NUCLEIC ACIDS AND

NUCLEOPROTEINS

-Molecular mass

-Charges of the molecules

-Colloid properties

-Viscosity

-Ability to denaturate and renaturate

-The molecular hybridization of nucleic acids as a method of defining the level of

homologousness (identity) of organisms. (Defining the degree of relationship)

-The DNA of man and monkey are highly homologous

GENETIC ENGINEERING

This is a direction of molecular genetics aimed at working out methods for the

receiving and transplanting of needed genes into the DNA of a host, thereby changing

the genetic properties of the host cell.

-Methods of genetic engineering (basic stages)

-receiving the gene of interesting DNA fragment

-the joining of this DNA to a vector molecule (a plasmid, etc)

-introducing the needed gene into the host-cells.

-selection of cells in which the reproduction of the introduced needed gene occurs.

Defense mechanisms of the DNA against foreign nucleic acids

-Restriction enzymes (restrictases)

-Minor nitrogen bases (2-methyladenine, 5-methylcytosine)

- the way of DNA defense against hydrolases

- the participation in the regulation of transcription.

FREE MONONUCLEOTIDES ,DINUCLEOTIDES AND

COFERMENTS(COENZYMES)

-free mononucleotides (ATP, ADP, AMP, GTP, GDP, GMP, etc)

-macro ergs -their biological role (ATP is a universal macro erg)

denaturation

renaturation

I II

32

-Cyclic mononucleotides(c-AMP, c-CMP) and their biological role (secondary

messengers)

-Dinucleotides (NAP,NADP, FAD) and their biological roles (coenzymes).

33

TEMPLATE SYNTHESES (BIOSYNTHESIS OF DNA, RNA AND

PROTEINS)

Ways of transfer of genetic information:

Biosynthesis of Nucleic Acids

Biosynthesis of DNA (Replication)

-What serves as the template? (mother DNA)

-How many daughter DNA molecules are formed?

Removal of RNA

primers and filling the

gap with DNA

polymerase

34

-What are the constituents of the newly formed DNA molecules?( the mother chain

and the newly formed daughter chain)

-Stages of replication

-Identifying the origin of replication and the unwinding of the DNA strands

(DNA-unwinding and DNA-binding proteins)

-Initiating the synthesis of DNA (primase, RNA-primer)

-Elongation (DNA-polymerase III, Okazaki fragments, DNA-polymerase I,

ligase)

-Winding (reconstitution) of the strands into a double helix structure (spiral).

-Termination (Reconstitution of chromatin)

*Predecessors of the daughter chains (RNA-primers, macroergs, Okazaki fragments)

*Leading (forward) and lagging (retrograde) strands. Explain the differences

between these two chains.

*DNA replication system (DNA polymerases, fragments, enzymes, etc)

*What is the speed of daughter chains formation?

*The frequency of mistakes during replication (10-10

).

DAMAGE AND REPARATION (CORRECTION OF MISTAKES ) OF DNA -Reasons of damage (UV-rays ,radiation, drugs, xenobiotics etc)

-Types of DNA damage (Show it using diagrams on page 26)

-The tearing off (removal ) of a group of atoms from the purines and pyrimidines of

the nucleus

-Removal of nitrogen bases

-The sticking together of purine and pyrimidine bases (with the formation of dimers )

-Deletion of nucleotides (mononucleotides)

-Substitution of some nucleotides (mononucleotides)

DNA REPARATION

35

1-Identifying the damaged part

2-Deletion of damaged part

3-Alignment of “needed” nucleotides

4-Rejoining (sewing together) of the repaired strand

-Repair system-Endonucleases (E),DNA-repair-polymerase(E):DNA-ligase ;Explain

their roles using the diagram

-Genic Mutations

-Biological results of mutation :

Neutral

Negative (hereditary pathologies like sickle -cell anemia)

positive (factor of evolution)

BIOSYNTHESIS OF RNA (TRANSCRIPTION, RECOPYING)

-Differences between the processes of transcription and replication

Replication Transcription

Biosynthesis of DNA Biosynthesis of RNA

Thymine, deoxyribose Uracil, Ribose

The whole chromosome is copied Recopying of information from different

fragments of the chromosome(from the

genes)

36

A new daughter DNA copy is formed t-RNA, m-RNAand r-RNA are formed

Double strand molecule of daughter DNA

identical to the mother DNA

Single –strand molecule of RNA

Stages of transcription

-Identifying the origin of transcription (promoter, DNA template ,-subunit of the

RNA polymerase)

-Unwinding of the DNA fragment (-subunits of the RNA polymerase )

-Initiation (-subunit of the RNA polymerase )

-Elongation (-subunit of the RNA polymerase ,ATP,GTP,UTP,CTP)

-Termination (terminative part of the gene i.e. termination of the protein)

Proceessing-This is the post transcriptive modification of RNA’s (the formation of a

suitably “mature” m-RNA molecule for translation)

Splicing –Removal of the intervening sequences of introns from the transcript and

the splicing together of exons

Caping-The chemical modification of the 5’end of the mRNA transcript (defense

against enzymes disintegration )

Poly(A)tail-(polyadenylation) –The chemical modification of the 3’end of the m-

RNA transcript and the formation of parts used for binding with ribosome and for

defense against enzymes disintegration

- Enzymes (ferments)of transcription (DNA-dependent, RNA- polymerase I,II ,III)

- 80-100 RNA-polymerase molecules take part in transcription at once

37

-RNA polymerase

-RNA

-Reverse transcription (RNA-dependent, DNA-polymerase )

-Viruses contain reverse transcriptases. The biological results of the functions of these

viral transcriptases are as follows: viral hepatitis, HIV, etc.

Human Immunodeficiency Virus HIV-infection( AIDS)

-Source of HIV infection –Blood, spinal fluid ,breast milk, sperm, secretions of the

reproductive organs

-The most dangerous means of transmitting the infection (hemotransfusion and sexual

contacts )

-Hypotheses of the origin of the HIV infections of man

(Mutation of the genes of viruses found in monkeys)

Structure of HIV

-Membrane of the virus, proteins-antigens(gp41,gp120)

38

-Matrix of virus (p17,p18)

-Nucleocapsid (p24,p25)

-Contents of the nucleocapsid (RNA, reverse transcriptase, endonuclease)

-Genom of HIV-I (3structural and 5 regulatory genes)

Stages of HIV infection

-HIV binds with the glycoproteins on the external surface of the membrane of host -

cells

-Fusion of HIV membrane with the host cell .

The nucleocapsid of the virus then penetrates into the cytoplasm of the host cell.

-With the help of proteases, the viral RNA is released from the nucleocapsid

-The biosynthesis of the pro-viral DNA on the template of the viral RNA with the

help of reverse transcriptases

-Integration of the pro-viral DNA genom into the( DNA) genom of the host cell

-Latent period (there is no transcription of the pro-viral DNA). Diagnosis of HIV is

doubtful .

-Provocation (increase in body temperature, alcohol intoxication ,changes in

hormonal status) and the activation of transcription of the pro-viral DNA

A large number of viral RNA is formed .

-Production of all viral components with the help of the viral RNA and the

formation of daughter viruses

-Cytolysis of cell membranes which leads to the release of the viruses and the

infection of other cells of the host

-There is a rise of viral antigens in the blood and a corresponding rise in the level of

antibodies

-Clinical manifestations of the disease.

-Laboratory diagnosis of HIV infections

-Establishment of the fact of infection

-Defining the stages of pathology development.

-Establishing the prognosis of the disease and its effective treatment .

Infection is proved by the presence of the following substances in the blood serum:

-HIV antigens

-HIV antibodies

-Pro-viral DNA

- Methods of analysis

-Immunofermentative analysis

-Method of hybridization of nucleic acids with specific DNA probes

-Method of polymeric chain reactions

-Doubtful results of diagnosis and actions of the doctor (within3-6 months repeated

medical checks)

-Final diagnosis of HIV infection (clinics, epidemiological laboratory diagnosis )

BIOSYNTHESIS OF PROTEINS(TRANSLATION) AND ITS REGULATION

Stages of translation

I Activation of amino acids

39

II Initiation of translation

III Elongation

IV Termination

V Post translatory modification

- Participants of translation (ferments, amino acids ,tRNA, rRNA, mRNA, factors of

initiation, factors of elongation, factors of termination, macroergs, etc)

Activation of amino acids

- amino acid

- ATP

- aminoacyl(aa)-t-RNA-synthetase, tRNA

40

Components:

41

Ribosomes

m-RNA

f-met-t-RNA (N-formylmethionine)

Initiating factors (IF-1,IF-2,IF-3)

-GTP

-Initiating codons (AUG,GUG)

-Peptidyl part(P) of the ribosome

-Amino acyl (acceptor part ,A-part ) of the ribosome

-Chemical components of the initiating complex

III Elongation

Components:

-aa, tRNA, aa- tRNA………….

-Peptidyl transferase

-mRNA

-Ribosome

-GTP-translocase

-Elongation factors (EF-1,EF-1,,EF-2)

IV TERMINATION

-Terminating codons of the mRNA (UAA,UAG,UGA)

-Termination factors (RF-1, RF-2,RF-3)

*RF-releasing factor

STAGES

-the dissociation of the ribosome into subunits

42

-disintergration of the m-RNA

-the speed of the biosynthesis of proteins (polysomes-80 ribosomes)

V POSTRANSLATORY MODIFICATIONS (PROCESSING)

*The formation of the spatial structures of the molecules.

- a molecule containing information about the structure of proteins (DNA)

*Modifying the protein molecule

-the attachment of prosthetic groups-the formation of phosphoproteins

,nucleoproteins,

lipoproteins, chromoproteins, glycoproteins ,metalloproteins.

-the chemical modification of amino acids (hydroxyproline, hydroxylysine)

-the limited proteolysis and activation of proteins(pepsinogen-pepsin, proinsulin-

insulin)

-the movement mechanisms of proteins to their respective points of action or

function.

("Postal index", receptors)

Regulating the speed of protein biosynthesis at the level of transcription.

(The hypothesis of Jacob and Monod - the Operon hypothesis)

COMPONENTS:

-structural genes

-regulatory genes

-promoter

-operator

-DNA-dependent RNA-polymerase

-m-RNA

-protein-repressor

43

-ferments

-substrates

-hormones

-end products

-The Jacob and Monod operon hypothesis is confirmed by incubating bacteria in a

medium containing either glucose() or amino acids() :

-the protein-repressor joins the substrate in the incubation medium, therefore:

-it changes confirmation and abandons the operator

-the RNA-polymerase then moves to the structural gene and enhances the copying of

information from these structural genes. (formation of m-RNA)

-on the basis of the m-RNA, the enzymes that are needed for the metabolism of the

substrate in the incubation medium are synthesized.

-as the substrate becomes exhausted , the protein repressor again joins the operator ,

thereby blocking the work of the RNA-polymerase and hence the copying of

information from the structural genes is stopped.

-the biological importance of the speed regulation of protein synthesis at the level of

transcription-adapting to the changing conditions of the environment.

INDUCTORS OF PROTEIN SYNTHESIS (ANABOLIC MEANS)

-Hormonal means:

*anabolic steroids (derivatives of male sex hormones)

*insulin

-Nonhormonal means :

*Predecessors of nucleotides (Ionosine, potassium orotate)

Regulating the speed of protein biosynthesis at the level of Translation.

Antibiotics specifically inhibit the stages of protein synthesis (antibacterial

preparations)

-Actinomycin acts at the level of transcription, it binds with the DNA-coding

chain

- Tetracycline inhibits protein synthesis by blocking the A-site, thereby

inhibiting amino acids and aminoacyl-t-RNA.

-Erythromycin inhibits the activity of translocase in the 70S ribosomes

.

-Puromycin due to its structural analogy with the aminoacyl-adenosine terminal

of the t-RNA, acts as the acceptor of the polypeptide chain-peptidyl-t-RNA (binds

with the p-part),the termination of protein synthesis is therefore inhibited.

44

45

BIOCHEMISTRY OF THE IMMUNE SYSTEM

Immunity from the biochemical point of view;

*Identification

*Neutralization

*Destruction of genetically foreign structures

The most important elements of the immune system;

-T-lymphocytes ,B-lymphocytes and the antibodies they synthesize, macrophages.

-Hormonal factors (the complementary system and properdines)

-Macrophages

The structure of Antibodies (immunoglobulins)

Chemical composition and structure

-4 polypeptide chains

-2 heavy chains (H-chains)

-2 light chains (L-chains)

-the variable fragment (VL and VH)

-the constant fragment (CL and CH)

-the hypervariable part (antigen-binging sites)

46

THE CLASSIFICATION OF IMMUNOGLOBULIS BASED ON THE TYPE OF

HEAVY CHAINS

IgA, IgG, IgD, IgM, IgE.

General structure Light and heavy chain composition of the

antibodies

Types of heavy

chains

H

H

HM

Classes of

antibodies

(immunoglobulin,Ig)

IgA

IgG

IgM

Quaternary structure (L2H

2)n

n=2 or 4

L2H2 (L2H2

M)5

Fraction in serum 10-20% 70-80% 5-10%

The specificity of antibodies (this is defined by the structure of the variable portions)

VARIABILITY OF ANTIBODIES

Quaternary

structure

2 light chains 2 heavy chains

Structure of the

chains

VL I L CL VH IH CH1CH2

There are 2 domains in the There are 4 domains in the H-chain

L-chain

V-the variable part( they are various for different antibodies)C-the constant part. I- the

intermediate portion

Representation

of the antibody

Variability of the

antibodies VL IL CL

~350 ~10 1type for

each

In all,=350*10*1=3500.There are

3500 different kinds of L-chains

VH IH CH1CH2CH3

~200 ~10 one for each

200*10*1=2000.There are 2000 different

kinds of H chains

SUM=3500*2000=7*106 (~10 million ) different kinds of antibodies ,ie,10m are

formed as a result of the combination of of ~600 different peptide

chains(350+200+10+10+1+3)

47

Schematic representation of immunoglobins origin

3 Processing:the movement of the 4 Interaction with T-helpers

-In the human organism there are 107 different clones of B-lymphocytes. Each of

these cells has its own surface Ig.

-antigens that enter the body are bound by surface Ig’s of specific clones of B-

lymphocytes.

-the antigen together with the surface Ig and the MHC (Major Histocompatibility

Complex) protein move into the cell.(Formation of the endosome).

-The MHC of the B-lymphocyte changes conformation. (Processing)(3)

5 The proliferation and secretion of antibodies by T-lymphocytes

1 Binding the antigen 2 Formation of the endosome

Antibody

MHC protein

Surface Ig

B-lymphocyte B-lymphocyte

3 Processing. Changed MHC proteins

come to the surface of the B-lymphocyte 4 Interaction with T-helpers

T-helper

B-lymphocyte B-lymphocyte

B-lympho-cyte

B-lympho-cyte

48

-The changed MHC protein moves to the surface of the B-lymphocyte and it is

recognized by T-Helpers. (4)

-T-Helpers secrete factors of proliferation.(4)

-These factors activate the proliferation specific clones of B-lymphocytes and

strengthens the synthesis of specific antibodies.

The variability and specificity of antibodies -In the process of evolution, the human organism came into contact with a large

number of foreign agents (antigens).This enhanced the formation and accumulation of

a great number of new genes-antibodies.

-Somatic mutations in the genes and the formation of genes from mini-genes also

caused a multiple increase (~600) in the variability of antibodies.(107)

Interaction of antibodies with antigens

Pentameric structure of the IgM molecule The antigen- antibody

complex

-Binding sites. (variable portions)

-Types of bonds. (Hydrogen bonds, hydrophobic interactions)

-The formation of antigen-antibody associator and their neutralization with the help

of humoral factors and macrophages.

49

HUMORAL FACTORS OF THE IMMUNE SYSTEM

Complement- This is a family of specific blood-plasma proteins

-In normal physiological conditions ,the complement proteins are in the inactive state

in the blood plasma.

-There are two ways of activating them:

-the classical path; this form of activation arises as a result of the

binding of an antigen to an antibody

-the alternate path; this form of activation occurs in response to

polysaccharides or antigens.

-Both paths activate the C3 component of the complement system

-The cascade mechanism of activating the complement system is realized as a result

of the activities of proteases and the release of histamines

-The Functions of the Complement System

*Interaction with components of bacterial membranes

*Enhances the attachment of leucocytes and the phagocytosis of bacteria

*Lysis of bacteria-Components of the complement system penetrate into cells

bacterial membranes and make holes in it. The free movement of Na+ and Ca

2+ ions

50

into, and K+ out of the cell ,causes the hydration and an increase in volume of the cell,

which leads to the destruction of the cell membrane.

The Properdine System - a family of specific blood plasma proteins (Properdine,

-glycoproteins, D-proteases)

-This system is activated by the lipopolysaccharides of bacteria and insulin.

-Function of the properdine system; they activate the complement system (the

formation of C5-C9 ) even in the absence of an antigen and also, they enhance the

proteolysis of antigens .

-Interferons- This is a heterogenic group of proteins. They form a group of basic

defense proteins against viral infections and the formation of tumors.(They suppress

the growth of malignant tumors)

*They are formed in the cell as the result of the penetration of viral nucleic acids into

the cell

*They limit viral infections (Inhibit the biosynthesis of viral proteins at the level of

translation)

*They activate the destruction of both viral and host m-RNA and r-RNA

*This leads to the destruction of host cells. Together with the host cell, the virion also

dies .The sacrifice of a small number(quantity) of host cells may lead to saving the

whole organism from sickness(death).

*They synthesize macrophages and T-cells

*They increase the cytolytic activity of macrophages ,T-cells and killer cells

An alteration in the functioning of the Immune System leads to the following:

-Hypersensitivity

-Immunotolerance(the absence of selection)

-Immunodeficiency (the total or partial loss of some components of the immune

system as a result of damages).