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CHMI 2227E Biochemistry I. Amino acids: Structure General chemical properties. Silk Spider web Horns Antibiotics Toxins. Enzymes Antibodies Hormones Membrane transporters Haemoglobin. Proteins: Building blocks of life!. Proteins are extremely complex molecules;

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CHMI 2227E Biochemistry I


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    1. CHMI 2227EBiochemistry I Amino acids: Structure General chemical properties CHMI 2227 - E.R. Gauthier, Ph.D.

    2. Silk Spider web Horns Antibiotics Toxins Enzymes Antibodies Hormones Membrane transporters Haemoglobin Proteins: Building blocks of life! • Proteins are extremely complex molecules; • Proteins are involved in a vast variety of biological structures and phenomena: • The building block for all proteins are amino acids. CHMI 2227 - E.R. Gauthier, Ph.D.

    3. An amino acid is a molecule in which a carbon atom (Ca) is bonded simultaneously to a carboxylic acid (COOH) and an amine group (NH2); Hundreds of amino acids exist in nature; However, only 20 amino acids are used by the cell to make proteins. Each amino acid differs from all the others by the nature of the R group. Each R group differs from the others in several ways: Size Charge Water solubility Ca COOH H C NH2 R Amino acids CHMI 2227 - E.R. Gauthier, Ph.D.

    4. Note that the Ca is bonded to 4 different groups and is chiral; Two stereoisomers are distinguished: L-amino acids D-amino acids Only the L-amino acids are found in proteins. General properties of amino acids. • L = amino group is on the left in the Fisher representation. • L and D: arbitrary. Has nothing to do with the direction of rotation of polarised light by the amino acid. CHMI 2227 - E.R. Gauthier, Ph.D.

    5. Net charge: 0 Net charge: -1 Net charge: +1 pKa 1 (pH 2-2.5) pKa 2 (pH 9-10.5) Basic pH Acidic pH Zwitterion General properties of amino acids. • Every amino acids has at least two chemical groups that can be ionized: • Carboxylic Acid (COOH) • Amino (NH2) CHMI 2227 - E.R. Gauthier, Ph.D.

    6. pI = isoelectric point = pH where 100% zwitterion pI = pKa1 + pKa2 2 The fraction of COOH or NH2 group that is charged at any given pH can be determined with the Henderson-Hasselbach equation: pH = pKa + log A- HA Buffer! Buffer! Amino acid titration1. simplest scenario CHMI 2227 - E.R. Gauthier, Ph.D.

    7. The twenty amino acids: CHMI 2227 - E.R. Gauthier, Ph.D.

    8. Structure of the 20 amino acids1. Hydrophobic amino acids • All these amino acids are NOT soluble in water. • Note: glycine is NOT optically active. Why? CHMI 2227 - E.R. Gauthier, Ph.D.

    9. Structure of the 20 amino acids1. Hydrophobic amino acids • Proline is the only cyclic amino acid; CHMI 2227 - E.R. Gauthier, Ph.D.

    10. Structure of the 20 amino acids2. Aromatic amino acids CHMI 2227 - E.R. Gauthier, Ph.D.

    11. Structure of the 20 amino acids2. Aromatic amino acids • Tyr, Trp and Phe are very useful: • Their aromatic ring absorb UV light at 260-280 nm. • Form the basis of protein detection at 280 nm. CHMI 2227 - E.R. Gauthier, Ph.D.

    12. Structure of the 20 amino acids3. polar, uncharged amino acids CHMI 2227 - E.R. Gauthier, Ph.D.

    13. In biochem: • oxidation: lose H • Reduction: gain H Disulfide bond Structure of the 20 amino acidsCysteine and cystine CHMI 2227 - E.R. Gauthier, Ph.D.

    14. Ser PO4-3 ATP Protein kinase Protein phosphatase ADP H2O O- P=O O- Phospho-Ser Structure of the 20 amino acids Tyr, Ser, Thr CHMI 2227 - E.R. Gauthier, Ph.D.

    15. Structure of the 20 amino acids4. Negatively charged amino acids CHMI 2227 - E.R. Gauthier, Ph.D.

    16. 100% D pH 9.5 pKa2 100% C pKaR 4.1 pKa1 A B C D 2.1 100% B pKa1 pKaR pKa2 What is the pI of glutamate? 100% A NaOH Structure of the 20 amino acids4. Negatively charged amino acids CHMI 2227 - E.R. Gauthier, Ph.D.

    17. Structure of the 20 amino acids5. Positively charged amino acids CHMI 2227 - E.R. Gauthier, Ph.D.

    18. pKa2 pKaR Structure of the 20 amino acids5. Positively charged amino acids: His CHMI 2227 - E.R. Gauthier, Ph.D.

    19. Structure of the 20 amino acidspKa values of amino acids CHMI 2227 - E.R. Gauthier, Ph.D.

    20. pH << pI pH >> pI + + Elution Analysis of amino acids1. Ion exchange chromatography • Different types of ion exchange resins exist: • Cation exchange: negatively charged/separation of cations • Anion exchange: positively changed/separation of anions. • Obviously, the type of resin to be used will depend on the charge of the amino acid on interest, itself dependent on the pH of the solution. CHMI 2227 - E.R. Gauthier, Ph.D.

    21. O R-HC=O O O CO2 OH COO- OH N H NH3+ C O 2 R O O Amino acid Ninhydrin Purple!! Analysis of amino acids2. Detection of amino acids: ninhydrin reagent • While Trp, Phe and Tyr can be detected by their A260-280nm, the other amino acids cannot; • Ninhydrin reacts with the amine group of amino acids, generating a purple product (yellow in the case of Pro). • The ninhydrin reaction allows one to detect and quantify (A570nm) the amino acids contained in the fractions of the IEX column. CHMI 2227 - E.R. Gauthier, Ph.D.