Issuu on Google+

‫بسم الله الرحمن الرحيم‬


In nature, mostly L-AAs form proteins

L and D isomers are mirror images, they are chiral


Nutritional classification: 1- Essential amino acids: valine methionine tryptophan

isoleucine lysine threonine phenyl alanine

leucin

2- Semiessential amino acids: arginine histidine 3- Non essential amino acids: They are the remaining 10 amino acids.


Metabolic classification: according to metabolic or degradation products of amino acids they may be: 1- Ketogenic amino acids: which give ketone bodies . Lysine and Leucine are the only pure ketogenic amino acids. 2- Mixed ketogenic and glucogenic amino acids: which give both ketonbodies and glucose.These are: isoleucine, phenyl alanine, tyrosine and tryptophan. 3- Glucogenic amino acids: Which give glucose. They include the rest of amino acids. These amino acids by catabolism yields products that enter in glycogen and glucose formation.


Edman Degradation • Phenylisothiocyanate adds to the N terminal amino group of a peptide. • When the medium is made anhydrous and acid, a derivative of the N terminal residue is liberated and can be identified by mass spectrometry or gas chromatography. • Then the next N terminal residue may be identified by another such cycle. • Problem is that the yield drops off dramatically • Most powerful sequencing method


Edman degradation with Phenyl isothiocyanate, PITC


Separation and Analysis of AA’s • Acid hydrolysis: If a peptide (protein) is placed into 6 N HCl and heated overnight at 110 C, peptides will be hydrolyzed to a mixture of free amino acids.

• Drawback: Tryptophan is destroyed.


Analysis of Amino Acid Composition • Ion-exchange chromatography: Will allow identification of the kinds and abundances of amino acids in a mixture.


1.Reaction with Ninhydrin

•

Used to visualize spots or bands of amino acids separated by chromatography or electrophoresis.

•

Deep purple color formed with traces of any amino acid.

2.Fluorescamine reagent


N terminal identification • Some reagents (e.g., dinitrophenol, dansyl chloride) combine with the free amino group at the N terminus of a polypeptide chain • The peptide can then be acid-hydrolyzed and the derivative identified by chromatographic, or other, methods


C terminal identification • Carboxypeptidase is an enzyme that removes C terminal amino acids. Brief digestion of a polypeptide with carboxypeptidase will liberate the amino acid at the C terminus.


Fragmentation • Trypsin – Highly specific endopeptidase that splits chains on the C (right) side of Arg and Lys – A digestive enzyme of our small intestines, made in the pancreas – Trypsin like enzymes are ubiquitous


Fragmentation • Chymotrypsin – Splits polypeptide chains on the C side of Phe, Trp, Tyr – Also a proteins-digesting enzyme of our intestines made in the pancreas – Not as specific and reliable as trypsin


Fragmentation • Cyanogen bromide, CNBr – A chemical (non-enzymic) agent that cuts on the carboxyl side of Met – Met is converted to O-homoseryllactone in the process – Highly specific and reliable


Protein structure - bonding • 5 bonds or forces determine structure – Peptide bond – Hydrogen bond – Disulfide bond – Ionic bond – Hydrophobic force


Peptide bond • Peptide bond joins amino acids • Bond at both ends – Increases range of possible proteins • 2 peptides can result from bonding of 2 amino acids • 1.0 x 1026 peptides can be formed from 20 amino acids

From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular Biology. Oxford: Oxford University Press. p23


Primary protein structure

Primary structure of insulin

• Linear sequence of amino acids forms primary structure • Sequence essential for proper physiological function

Bettelheim & March (1990) Introduction to Organic & Biochemistry (International Edition) Philadelphia: Saunders College Publishing, p299


Sickle cell anemia • Replacement of single glutamine with valine in one polypeptide chain of hemoglobin alters structure and function

Bettelheim & March (1990) Introduction to Organic & Biochemistry (International Edition) Philadelphia: Saunders College Publishing, p301


Secondary protein structure • Peptide chains fold into secondary structures: α - helix β - pleated sheet – Random coil


α - helix

• Shape maintained by hydrogen bonds between C=O and N-H groups in backbone • R groups directed outward from coil From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular Biology. Oxford: Oxford University Press. p28


Properties of the alpha helix ∀ φ ≈ ψ ≈ −60° • Hydrogen bonds between C=O of residue n, and NH of residue n+4 • 3.6 residues/turn • 1.5 Å/residue rise • 100°/residue turn


β - pleated sheet

• Structure maintained by hydrogen bonds between C=O and N-H groups in backbone • R groups directed above and below backbone

From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular Biology. Oxford: Oxford University Press. p29


Random coil • Not really random structure, just nonrepeating

From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular Biology. Oxford: Oxford University Press. p27

– ‘Random’ coil has fixed structure within a given protein – Commonly called ‘connecting loop region’ – Structure determined by bonding of side chains (i.e. not necessarily hydrogen bonds)


Tertiary protein structure • Secondary structures fold and pack together to form tertiary structure – Usually globular shape

• Tertiary structure stabilised by bonds between R groups (i.e. sidechains)


Tertiary structure - H bond

Hydrogen bond

• H bonds weak allowing to be broken and reformed easily – Allows structural change • produces ‘functional’ molecules


Tertiary structure - disulfide bond – Covalent bond between sulfur atoms on two cysteine amino acids

From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular Biology. Oxford: Oxford University Press. p32


Tertiary structure - ionic bond

• Ions on R groups form salt bridges through ionic bonds From: Summerlin, LR. (1981) Chemistry for the Life Sciences. New York: Random House, p459


Tertiary structure - hydrophobic forces • Close attraction of nonpolar R groups through dispersion forces • Very weak but collective interactions over large area stabilise structure • Repel polar and charged molecules/particles

Bettelheim & March (1990) Introduction to Organic & Biochemistry (International Edition) Philadelphia: Saunders College Publishing, p302


Quaternary protein structure • Arrangement of multiple tertiary structures into single functional complex • Allows for changes in structure/function in response to chemical stimuli

From: Elliott, WH. Elliott, DC. (1997) Biochemistry and Molecular Biology. Oxford: Oxford University Press. p27


From primary to quaternary structure


Examples on Peptides: 1- Dipeptide ( tow amino acids joined by one peptide bond): Example: Aspartame which acts as sweetening agent being used in replacement of cane sugar. It is composed of aspartic acid and phenyl alanine. 2- Tripeptides ( 3 amino acids linked by two peptide bonds). Example: GSH which is formed from 3 amino acids: glutamic acid, cysteine and glycine. It helps in absorption of amino acids, protects against hemolysis of RBC by breaking H2O2 which causes cell damage. 3- octapeptides: (8 amino acids) Examples: Two hormones; oxytocine and vasopressin (ADH). 4- polypeptides: 10- 50 amino acids: e.g. Insulin hormone


Classification of proteins I- Simple proteins: i.e. on hydrolysis gives only amino acids Examples: 1- Albumin and globulins: present in egg, milk and blood They are proteins of high biological value i.e. contain all essential amino acids and easily digested. Types of globulins: α1 globulin: e.g. antitrypsin: see later α2 globulin: e.g. haptoglobin: protein that binds hemoglobin to prevent its excretion by the kidney β-globulin: e.g. transferrin: protein that transport iron γ-globulins = Immunoglobulins (antibodies) : responsible for immunity.


proteins