Download moluceular lab 1

Lab (1)
Molecular Biology Definition :
1-Modern field of science
2-To understand the basic of living organisms’ chemical reactions necessary to
build cell’s nutrients and to perform biological functions.
Structural organic order is as follows:
Organism–System–Organs–Tissues–Cells–Organelles– Molecules-Atoms
The body of Living organisms consists of : two types of Molecules:
Inorganic molecules : (water, salts, acids and bases) they are basic simple
materials that composes more complicated materials of living cell.
Organic molecules: composed primarily of carbon atom (C) .
Cellular organic molecules are four kinds of macromolecules:
Proteins - Carbohydrates - Lipids - Nucleic acids
Amino Acids:
1- Definition : 1-Building of proteins.
2- consists of : Amine group (NH2) with basic properties,
and Carboxyl group (COOH) with acidic properties,
in addition to a side group (R) which determines the
distinctive properties of amino acids.
Amine
Carboxyl
group
group
Chemical formula of Amino acid
2- Kinds of Amino acids :
•Neutral non polar amino acids: e.g. Tryptophan .
•Neutral polar amino acids: e.g. Cysteine.
•Non-neutral amino acids: e.g. Arginine.
3-Levels of Structure in Proteins
I- Primary Structure: The binding of amino acids with peptide bond to form a linear
chain of poly peptide.
H2N
His
Tyr
Ser
)Peptide bond(
Met
Glu
Phe
Glu
Arg
His
Ser
Sequence of amino acids to form linear
chain of polypeptide
COOH
H2N
II- Secondary Structure:
Tyr
Ser
The specific shape of protein results from Hydrogen (H)-bonding of
Met
Glu
Ala
the poly-peptide chain.
Val
Hbond
Cys
Met
Ser
There are 2 forms of secondary structure:
His
Phe
Ser
1- α-helix : e.g. Collagen protein in white fibers, and Elastine in elastic
Glu
Lys
Thr
fibers (both in connective tissues).
2- β- plated sheet: e.g. Keratin protein in hair and horny layer of skin.
Both forms are fibrous proteins that do not dissolve in water
(insoluble)
Glu
Arg
COOH
Secondary structure is formed by
formation of H bonds of Hydrogen atom of
Amine group in one amino acid with
Oxygen atom of carboxyl group of another
amino acid.
III- Tertiary Structure:
There are 3 main types of chemical bonds that contributes to the formation of tertiary
structure:
1-H-bond : ( Binding between parts of near region and far region from poly-peptide )
2-Ionic bond : (Binding between free of Amine group at one side of the poly-peptide
with free of Carboxyl group on the other side of the poly-peptide
3-di-sulfide bond (-S-S-) :(Binding between two atom of sulfide in two amino acide
Distanced from each other by a specific distance , which result in formation of a
Globular protein
that dissolves in water (soluble) . e.g. enzymes.
IV- Quaternary Structure:
The joining of multiple polypeptide molecules(units) into one large complex structure.
and that are in the form of bundle , and have the same chemical bonds that also this
structure are non-covalent.
( chain)
(Iron)
( chain)
(Heme)
Quaternary structure: example is Hemoglobin, it consists of 4 polypeptid coiled chains
to form one complex globular structure.( OC1 – OC2 – B1 – B2 )
Slide
Reagents and dyes Object
Features
(For read)
Intestine with
protein inclusions
Tissue parts
 Bromophenol
stained blue
blue
indicates
 ninhydrin Schiff
Staining technique: presence of
 Chloramine-T
bromophenol blue. Proteins
Schiff
Muscle with protein
inclusion
Tissue parts
stained violet Pseudo-iso-cyanin
Insulin in pancreas indicates
Immuno-peroxidase
presence of
stain
Insulin
Secondary
Structure
Elastic (yellow)
fibers
Elastin
Tissue parts
stained black
indicates
presence of
elasic fibers
Gieson Stain
Proteins
in cells
Insulin
Protein
hormone
Elastine
(Fibrous
helical
protein)
Secondary Structure
Tissue parts stained
Collagen (white)
blue indicates
fibers
presence of collagen
fibers
Tri
chrome
stain
Upper part: fibrous
keratin
Secondary Structure
In horny layer
Keratin in Skin
Keratin
H&E
(Fibrous
plated protein)
Tertiary Structure
Tissue parts staines
Phosphatase in lung
blue indicates
presence of protein
Tertiary Structure
enzyme
Phosphatase in
Collagen
(Fibrous
helical
protein)
Phosphatase
globular (
enzyme )
alimentary duct
Quaternary
Structure
Hemoglobin in red
blood cell
Red blood cells
(RBCs) sained red
naturally due to
presence of
hemoglobin.
Hemoglobin
(blood
pigment that
binds and
transfers
oxygen)
Chemical Experiments
Detection of Amino Acids - Studying Proteins Characteristics
Detection of amino acids in egg white and milk:
•A) Detection of Tryptophan, phenylalanine:
(1) in 2 test tubes Add 3 ml of each sample (egg white, milk)
(2) Add 1 ml of conc. Nitric acid, then heat tube over flame for 1 min.,a yellow color will occur.
(3) Cool tube (in ice ), then add 4 ml of 40% NaOH.
Observation:
Yellow color converts to orange (formation of orange precipitant) indicates presence of both
amino acids.
Results:
Amino acid
Milk
Tryptophan
+ light orange
Phenylalanin
+
Egg white
+ dark
orange
+
2-Detection of amino acids Cyseine and Methionine
1- Put 5 ml of each sample (milk, egg white) in test tubes
2- Add 2 ml NaOH (40%), heat tubes on flame for 5 or more minutes till yellow color develop.
3- Cool tubes, then add 2 ml of lead acetate (5%).
Observation:
dark brown or gray to black ppt. (darken with time)
Result:
Amino acid
Milk
Cysteine
+ dark brown ppt
Methionine
+ dark brown ppt
Egg white
+ black
ppt
+ black
ppt
(1) Affecting Factors on Protein Denaturation:
1) In 5 test tubes, add 5 ml of egg white in each. Mark tubes from 1 to 5.
2) Put tube 1 in boiling water bath (or heat over flame)
3) In tube 2, add 3 ml of NaCl solution (ionic solution)
4) In tube 3, add 3 ml NaHCO (base)
5) In tube 4, add 5 ml HCl (acid)
6) In tube 5, add 5 ml ethanol alcohol (organic compound)
Results:
Tube #
1
2
3
4
5
Added factor
Boiling water bath / flame
NaCl (ionic compound)
NaHCO (base)
HCL (acid)
Ethanol (organic compound)
Observation
(+) Coagulation (like boiled
egg)
(-) no change
(+) bubbles burst that
disappears quickly
(+) coagulation (white
color)
(+) white ring in middle
Conclusion:
-In normal conditions, proteins exists in their natural state.
-Some factors affects the nature of proteins and can change structure of proteins.
-Factors like high temperature, exposure to some chemicals causes denaturation of proteins,
function of protein is lost.