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

peptides, isoelectric pH, Non AAs

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Page 1: peptides, isoelectric pH, Non AAs

M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.

Page 2: peptides, isoelectric pH, Non AAs

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.

Page 3: peptides, isoelectric pH, Non AAs

1. Glutathione 3 2. TRH 3 3. Enkephalins 54. Angiotensin- II 85. Oxytocin 96. Vasopressin 97. Bradykinin 98. Aspartame 2

Page 4: peptides, isoelectric pH, Non AAs

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

Page 5: peptides, isoelectric pH, Non AAs

2G – SH G - S – S – G

Reduced Oxidized Active Inactive

Page 6: peptides, isoelectric pH, Non AAs

Functions of Glutathione1. 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.

Page 7: peptides, isoelectric pH, Non AAs

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)

Peroxidase2GSH + H2O2 G – S – S – G - + 2H2O

Page 8: peptides, isoelectric pH, Non AAs

5. Glutathione is involved in the transport of amino acids in the intestine and kidney tubules via γ-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)

Page 9: peptides, isoelectric pH, Non AAs

Conjugation for detoxification:Glutathione helps to detoxify several compounds by transfering the cysteinyl group e.g:

a. Organo phosphorus compoundsb. Halogenated compoundsc. Nitrogenous substances ( chloro

dinitro benzene)d. Heavy metalse. Drugs.

The reaction is catalyzed by glutathione- s – transferase (GST)

Page 10: peptides, isoelectric pH, Non AAs

TRH - Thyrotropin-releasing hormoneSecreted by hypothalamus,

causes anterior pituitary gland to release thyrotropic hormone

Page 11: peptides, isoelectric pH, Non AAs

Methionine enkephalin - Enkephalins (5 Aas)

Opiate-like peptide.Found in brain. Inhibits sense of pain.

Page 12: peptides, isoelectric pH, Non AAs

Angiotensin- II (8 AAs)

Pressor or hypertensive peptideStimulates release of aldosterone from adrenal cortex.

Page 13: peptides, isoelectric pH, Non AAs

Oxytocin – (9 AAs)

Oxytocin secreted by posterior pituitary gland

Contains 9 amino acids(nonapeptide)Oxytocin causes contraction of uterus.

Page 14: peptides, isoelectric pH, Non AAs

Vasopressin (antidiuretic hormone) (9 Aas)

Secreted by posterior pituitary glandcauses kidney to retain water from urine

Page 15: peptides, isoelectric pH, Non AAs

Plasma bradykinin Bradykinin – 9 AAs

Vasodilator peptideProduced from plasma proteins by snake venom enzymes.

Page 16: peptides, isoelectric pH, Non AAs

Aspartame -2AAs

It’s a dipeptide produced commercially by combination of aspartic acid and phenylalanine.

It is above 200 times sweeter than sucroseIt is used as a low calorie artificial sweetner in soft drink industry

Page 17: peptides, isoelectric pH, Non AAs

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

Isoelectric PH

Page 18: peptides, isoelectric pH, Non AAs

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.

Page 19: peptides, isoelectric pH, Non AAs

Zwitterion or Dipolar ion

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

Page 20: peptides, isoelectric pH, Non AAs

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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Page 22: peptides, isoelectric pH, Non AAs

From the graph it is evident that the

buffering action is maximum in and

around pK1 or at pK2 and minimum at plAmino 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

Page 23: peptides, isoelectric pH, Non AAs

ProteinsLysozymePancreatic ribonucleaseMyoglobinHemoglobinHuman serum immunoglobulinsCarboxypeptidaseCaseinFibrinogenHuman serum albuminEgg albuminPepsin

PI119.67.06.86.4 – 7.26.04.65.54.84.61.0

Isoelectric pH of some proteins

Page 24: peptides, isoelectric pH, Non AAs

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).

Page 25: peptides, isoelectric pH, Non AAs

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.

Page 26: peptides, isoelectric pH, Non AAs

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.

Page 27: peptides, isoelectric pH, Non AAs

Non Protein Amino Acidsor

Non standard Amino Acids

Page 28: peptides, isoelectric pH, Non AAs

Amino acids Functionsα – Amino acidsOrnithineCitrulline Intermediates in the biosynthesis of ureaArginosuccinic acid

ThyroxineTriiodothyronine 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.

Non standard amino acids

Page 29: peptides, isoelectric pH, Non AAs

Non-α-amino acidsβ-Alanine Component of vitamin

pantothenic acid and coenzyme A

γ-Aminobutyric acid A neurotransmitter produced (GABA) from glutamic acid

δ- Aminolevulinic acid Intermediate in the synthesis (ALA) of porphyrin (finally heme)

Taurine Found in association with bile acids.