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

CHMI E.R. Gauthier, Ph.D.2 Proteins: Building blocks of life!  Silk  Spider web  Horns  Antibiotics  Toxins  Enzymes  Antibodies  Hormones  Membrane transporters  Haemoglobin 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 E.R. Gauthier, Ph.D.3 Amino acids An amino acid is a molecule in which a carbon atom (C  ) is bonded simultaneously to a carboxylic acid (COOH) and an amine group (NH 2 ); Hundreds of amino acids exist in nature; However, only 20 amino acids are used by the cell to make proteins. C H R COOH NH 2 CC 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

CHMI E.R. Gauthier, Ph.D.4 General properties of amino acids. Note that the C  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. 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 E.R. Gauthier, Ph.D.5 General properties of amino acids. Every amino acids has at least two chemical groups that can be ionized:  Carboxylic Acid (COOH)  Amino (NH 2 ) Zwitterion Net charge: +1 Net charge: 0Net charge: -1 Acidic pH Basic pH pKa 1 (pH 2-2.5) pKa 2 (pH )

CHMI E.R. Gauthier, Ph.D.6 Amino acid titration 1. simplest scenario pI = isoelectric point = pH where 100% zwitterion pI = pKa1 + pKa2 2 The fraction of COOH or NH 2 group that is charged at any given pH can be determined with the Henderson-Hasselbach equation: pH = pKa + log A - HA Buffer!

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

CHMI E.R. Gauthier, Ph.D.8 Structure of the 20 amino acids 1. Hydrophobic amino acids All these amino acids are NOT soluble in water. Note: glycine is NOT optically active. Why?

CHMI E.R. Gauthier, Ph.D.9 Structure of the 20 amino acids 1. Hydrophobic amino acids Proline is the only cyclic amino acid;

CHMI E.R. Gauthier, Ph.D.10 Structure of the 20 amino acids 2. Aromatic amino acids

CHMI E.R. Gauthier, Ph.D.11 Structure of the 20 amino acids 2. Aromatic amino acids Tyr, Trp and Phe are very useful:  Their aromatic ring absorb UV light at nm.  Form the basis of protein detection at 280 nm. Absorbance max (nm) Molar Absorbance (M -1 cm -1 ) Phenylalanine Tyrosine Tryptophan

CHMI E.R. Gauthier, Ph.D.12 Structure of the 20 amino acids 3. polar, uncharged amino acids

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

CHMI E.R. Gauthier, Ph.D.14 Structure of the 20 amino acids Tyr, Ser, Thr P=O O-O- O-O- Ser Phospho-Ser Protein kinase Protein phosphatase ATP ADP H2OH2O PO 4 -3

CHMI E.R. Gauthier, Ph.D.15 Structure of the 20 amino acids 4. Negatively charged amino acids

CHMI E.R. Gauthier, Ph.D.16 Structure of the 20 amino acids 4. Negatively charged amino acids pKa1pKa2pKaR What is the pI of glutamate? NaOH pH pKa1 pKaR pKa % A 100% B 100% C 100% D ABCD

CHMI E.R. Gauthier, Ph.D.17 Structure of the 20 amino acids 5. Positively charged amino acids

CHMI E.R. Gauthier, Ph.D.18 Structure of the 20 amino acids 5. Positively charged amino acids: His pKaR pKa2

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

CHMI E.R. Gauthier, Ph.D.20 Analysis of amino acids 1. 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. Elution + + pH >> pI pH << pI

CHMI E.R. Gauthier, Ph.D.21 Analysis of amino acids 2. Detection of amino acids: ninhydrin reagent While Trp, Phe and Tyr can be detected by their A nm, 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 (A 570nm ) the amino acids contained in the fractions of the IEX column. O O OH Ninhydrin Amino acid 2 O O N O O C H R COO - NH 3 + CO 2 Purple!! R-HC=O