peptides, isoelectric pH, Non AAs

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BIOLOGICAL IMPORTANT PEPTIDES

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Biologically important Peptides

• Peptides are short sequences of amino acids held
  by peptide bonds.
• Each peptide chain will have 2 ends an amino
  terminal N and a carboxy terminal C.
• Peptide bonds are digested by peptidases and
  proteases.

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Biologically important peptides

• 1. Glutathione 3
• 2. TRH 3
• 3. Enkephalins 5
• 4. Angiotensin- II 8
• 5. Oxytocin 9
• 6. Vasopressin 9
• 7. Bradykinin 9
• 8. Aspartame 2

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Glutathione (GSH)

• Glutathione has a Pseudopeptide linkage.
• GS-H is a Tripeptide 3 amino acids
• It is gama glutamyl cysteinyl glycine
• Glutathione is present in RBC and many other
  tissues.

• Reduced glutathione (GS-H) is the active form

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• 2G SH G - S S G
• Reduced Oxidized
• Active Inactive

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Functions of Glutathione

• Reduced glutathione is essential for maintaining
  the normal structure of red blood cells.
• Glutathione (reduced) performs specialized
  functions in erythrocytes
• It maintains RBC membrane structure and
  intergrity.
• It protects hemoglobin from getting oxidized by
  agents such as H2O2.
• glutathione keeps iron in ferrous state in
  haemoglobin there by preventing formation of
  methaemoglobin.
• RBCs with lowered level of reduced glutathione
  are more susceptible to haemolysis.

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• 2. Glutathione serves as a coenzyme fro certain
  enzymes.
• eg Prostaglandin PGE2 synthetase.
• 3. It is essential for the formation of correct
  disulfide bonds in several proteins.
• 4. Toxic amounts of peroxides and free radicals
  produced in the cells are scavanged by
  glutathione peroxidase (selenium containing
  enzyme)
• Peroxidase
• 2GSH H2O2 G S S G - 2H2O

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• 5. Glutathione is involved in the transport of
  amino acids in the intestine and kidney tubules
  via ?-glutamyl cycle or meister cycle .
• 6. It keeps the enzymes in an active state by
  preventing the oxidation of sulfhydryl (-SH-)
  group of enzyme to disulfide (-s-s-) group.
• 7. As a conjugating agent in detoxification
• (liver)

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• Conjugation for detoxification
• Glutathione helps to detoxify several compounds
  by transfering the cysteinyl group
• e.g
• a. Organo phosphorus compounds
• b. Halogenated compounds
• c. Nitrogenous substances ( chloro dinitro
  benzene)
• d. Heavy metals
• e. Drugs.
• The reaction is catalyzed by glutathione- s
  transferase (GST)

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TRH, a Tripeptide

• TRH - Thyrotropin-releasing hormone
• Secreted by hypothalamus,
• causes anterior pituitary gland to release
  thyrotropic hormone

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Enkephalins

• Methionine enkephalin - Enkephalins (5 Aas)
• Opiate-like peptide.
• Found in brain.
• Inhibits sense of pain.

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Angiotensin- II

• Angiotensin- II (8 AAs)
• Pressor or hypertensive peptide
• Stimulates release of aldosterone from adrenal
  cortex.

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Oxytocin

• Oxytocin (9 AAs)
• Oxytocin secreted by posterior pituitary gland
• Contains 9 amino acids(nonapeptide)
• Oxytocin causes contraction of uterus.

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Vasopressin

• Vasopressin (antidiuretic hormone) (9 Aas)
• Secreted by posterior pituitary gland
• causes kidney to retain water from urine

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Bradykinin

• Plasma bradykinin Bradykinin 9 AAs
• Vasodilator peptide
• Produced from plasma proteins by snake venom
  enzymes.

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Aspartame

• Aspartame -2AAs
• Its a dipeptide produced commercially by
  combination of aspartic acid and phenylalanine.
• It is above 200 times sweeter than sucrose
• It is used as a low calorie artificial sweetner
  in soft drink industry

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Isoelectric PH
Iso electric PH (PI) of an amino acid / protein
is the PH at which it has both Ve and Ve
charges in equal quantities and as a whole it is
electrically neutral. Hence it does not move in
an electric field .protein precipitated.
Iso electric PH (PI) PK
1 PK 2 (for Monoamino, Monocarboxylic AA)
2
Ex for glycine 2.4 9.8 6.1
2
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• Amino acids / Proteins are ampholytes - contain
  both acidic (-COOH) and basic group(-NH2)
• They can donate proton and accept a proton.
  Hence they are ampholytes.

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• Zwitterion or Dipolar ion
• Amino acids / Proteins act as zwitter ion
  (dipolar ions) containing positive and negative
  ionic groups.

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• All the ionisable groups present in the protein
  will influence the pI of the protein.
• At isoelectric pH proteins will not migrate in
  electric field .
• Proteins have minimum solubility.
• Hence easily precipitated.
• Proteins have minimum buffering capacity .
• Proteins have low viscosity.

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• From the graph it is evident that the
• buffering action is maximum in and
• around pK1 or at pK2 and minimum at pl

• Amino acids can release H and act as
• weak acids.
• It can be quantitatively described by

Henderson Hasselbalch equation pH pKa log
A- (Conjugate base)
HA (Acid)
It predicts maximum buffering occurs 1 pH around
pKa
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Isoelectric pH of some proteins
Proteins Lysozyme Pancreatic ribonuclease Myoglobin Hemoglobin Human serum immunoglobulins Carboxypeptidase Casein Fibrinogen Human serum albumin Egg albumin Pepsin PI 11 9.6 7.0 6.8 6.4 7.2 6.0 4.6 5.5 4.8 4.6 1.0
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The pI value is characteristic for each protein.
In a solution at a pH value above its pI, a
protein will have a net negative charge(Anion)
below its pI, it has a positive charge(cation).
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Iso electric Focusing(IEF)

• By this technique proteins are seperated
  (immobilized) at Isoelectric pH during
  electrophoresis.
• As the electrophoresis occurs proteins migrate
  to positions corresponding to isoelectric pH.
• Serum proteins can be seperated by to 40
  different bands.

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• Curdling of milk lactic acid formed during
  curdling of milk brings the pH of milk to 4.6(Iso
  electric pH of casein) where casein is
  precipitated.
• Heat and Acetic acid Test
• Acetic acid is added to urine to bring the pH to
  around 4.8 (Iso electric pH of albumin) and then
  heated to detect albumin in urine.

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• Non Protein Amino Acids
• or
• Non standard Amino Acids

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Non standard amino acids
Amino acids Functions
a Amino acids Ornithine Citrulline Intermediates in the biosynthesis of urea Arginosuccinic acid Thyroxine Triiodothyronine Thyroid hormones derived from tyrosine. S-Adenosylmethionine Methyldonor in biological system. Homocysteine Intermediate in methioninen metabolism. A risk factor for coronary heart diseases. Homoserine Intermediate in threonine, aspartate and methionine metabolism. 3,4-Dihhydroxy phenylanine (DOPA) A neurotransmitter, serves as a precursor of melanin pigment.

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• Non-a-amino acids
• ß-Alanine Component of vitamin
  pantothenic acid and coenzyme A
• ?-Aminobutyric acid A neurotransmitter produced
• (GABA) from glutamic acid
• d- Aminolevulinic acid Intermediate in the
  synthesis
• (ALA) of porphyrin (finally heme)
• Taurine Found in
  association with bile acids.