Amino Acids, Peptides & Proteins Part 2

Harliansyah, PhD

Dept. of Biochemistry. FKUY(Doctor of Philosophy in Medical Biochemistry)

Amino group of an AA combines with the carboxyl group of another AAA dipeptide will have 2 AA and one peptide (not two) bondPeptides containing more than 10 AA (decapeptide) polypeptide

Characteristics of Peptide BondsRigidPlanarPartial double bond in characterGenerally exists in transconfigurationBoth _C = O and _NH2 groups of peptide bonds are polarInvolved in hydrogen bond formation

Writing of Peptide StructuresThe peptide chains are written with the free Amino end (N terminal residue) at the left, and the free carboxyl end (C terminal residue) at the right.The AA sequence is read from N terminal end to C terminal endIncidentally the protein biosynthesis also starts from the N terminal Acid.

Shorthand to read peptidesThe AA in a peptide or protein are represented by the 3 letter or one letter abbreviation.This is the chemical shorthand to write proteins.

Naming of PeptidesFor naming peptides, the AA suffixes ine (glycine), - an (tryptophan) ate (glutamate) are changed to yl with the exception of C terminal AA.

A tripeptide composed of an N terminal glutamate, a cysteine and a C terminal glycine is called:glutamyl cysteinyl - glycine

Polypeptide Chains- The linking together of many AA by peptide bonds produces polypeptide chains.Residues- AA, when in polypeptide chains, are customarily referred to as residues.Large peptide chains- protein polypeptide chains are typically more than 100 AA residues long. The backbone of the chain is a recurring sequence:alpha - carbon carbonyl carbon - alpha - amino N - alpha - carbon

Biuret Reaction

Peptides of Physiologic ImportanceGlutamine (Glutathione)- a tripeptide composed of 3 AA - gamma glutamyl cysteinyl glycine - wildly distributed in nature - exists in reduced or oxidized states

Functions:a) As a coenzyme for certain enzymes asprostaglandinPGE2 synthaseglycoxylaseb) Prevents the oxidation of sulfhydryl groups of several proteins to disulfide groupsc) In association with glutathione reductase participates in the formation of correct disulfide bonds in several protiensd) In erythrocytes- maintains RBC membrane structure and integrity- protects hemoglobin from getting oxidized by agents such as H2O2

e) Involved in the transport of AA in the intestine and kidney tubules via delta glutamyl cycle or Meister cycle

f) Involved in the detoxification process

g) Toxic amounts of peroxidases and free radicals produced in the cells are scavanged by glutathione peroxidase ( a selenium containing enzyme).

2. Thyrotropin Releasing Hormone (TRH)- a tripeptide secreted by hypothalamusFunction:Stimulate pituitary gland to release thyrotropic hormone

3. Oxytocin- contains 9 AA (nonapeptide)- hormone secreted by posterior pituitary glandFunction:Stimulate contraction of the uterus muscle during delivery

Stimulate contraction of muscle in breasts for milk ejection

4. Vasopressin (ADH antidiuretic hormone)- also a nonapeptide- produced by posterior pituitary glandFunction:Stimulates kidneys to retain water and thus increases the blood pressure

5. Angiotensins- Angiotensin 1 a decapeptide (10AA) which is converted to angiotensin II (8AA)Function: For the release of aldosterone from adrenal gland

6. Methionine Enkephalin- a pentapeptide found in the brain and has opiate like function.Function:It inhibits the sense of a pain.

7. Bradykinin and Kallidin- nona and decapeptides respectively- produced from plasma proteins by snake venom enzymesFunction:Powerful vasodilators

8. Peptide Antibiotics- Antibiotics such as Gramicidin, Bacitracin, tyrocidin and Actinomysin peptide in nature

9. Dipeptide aspartame- Consists of aspartate and phenylalanine- acts as Sweetener ~ used by diabetic patients

10. Gastrointestinal Hormones- Gastrin, Secretin & etc.- gastrointestinal peptides serving as hormones

Identifying the amino acid sequenceEdman Reaction1. DescriptionPhenylisothiocyanate reagent- developed for a technique that allows repetitive sequencing of a peptide2. Process a. Phenylisothiocyanate reacts in alkali with the N terminal group to form aphenylthiocarbamyl derivative of the peptide

In the presence of a strong anhydrous acid, the phenylthiohydantoin derivative of the 1st AA is cleaved from the peptide and can be identified chromatographically.b. The remainder of the peptide is left intact with the next residue in the chain bearing a free amino group. This can in turn react with fresh reagent , and the reaction continues until the impurities produced by incomplete reactions build up and prevent further repetition of the reaction.

c. The procedure has been automated and can be used to determine the AA sequence of peptides 50 to 60 residues in length. Longer peptides must be broken into smaller fragments for sequencing.

B. Sequencing of peptide fragmentsThe direct sequencing of proteins > than 100 residues is difficult- the analysis is considerably aided by first breaking (i.e. cleaving) the protein chain into short fragments- this cleavage is performed by using chemical reagents or enzymes that sever the peptide chain at specific sites- the sequence of these shorter peptide fragments is then determined by the Edman reaction, using an automated sequenator

2. Arranging the peptide fragments in their true order is a problem that also can be overcomed by fragmenting the original protein with several reagents that act at different sites

3. Reagents used to obtain peptide fragments include proteolytic enzymes & chemical reagents

Proteolytic enzymes:i. Trypsin preferentially cleaves peptide bonds on the carboxylic side of basic AA (Arginine, lysine) ii. Chymotrypsin preferentially cleaves on the carboxyl side of aromatic AA (Phenylalanine, tyrosine, tryptophan)iii. Staphylococcal protease- preferentially cleaves on the carboxyl side of the acidic AA (Aspartate, glutamate)

b) Chemical Reagentsi. Cyanogen bromide cleaves on the carboxyl side of methionine residues

ii. Hydroxylamine cleaves at asparagine glycine bonds.