PEPTIDES, ISOELECTRIC pH

1. M.Prasad Naidu MSc Medical Biochemistry, Ph.D,. BIOLOGICAL IMPORTANT PEPTIDES

2. 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.

3. 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

4. Glutathione (GSH) • Glutathione has a Pseudopeptide linkage. • GS-H is a Tripeptide – 3 amino acids • It is gamaglutamyl – cysteinyl – glycine • Glutathione is present in RBC and many other tissues. • Reduced glutathione (GS-H) is the active form

5. 2G – SH G - S – S – G Reduced Oxidized Active Inactive

6. 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.

7. 2. Glutathione serves as a coenzyme fro certain enzymes. eg: Prostaglandin PGE2synthetase. 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

8. 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)

9. 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)

10. TRH, a Tripeptide TRH - Thyrotropin-releasing hormone Secreted by hypothalamus, causes anterior pituitary gland to release thyrotropic hormone

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

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

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

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

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

16. Aspartame Aspartame -2AAs It’s 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

17. 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

18. 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.

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

20. 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.

22. 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

23. Isoelectric pH of some proteins

24. 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).

25. 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.

26. 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.

27. Non Protein Amino Acids or Non standard Amino Acids

28. Non standard amino acids

29. Non-α-amino acids β-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.