chmi 2227 - e.r. gauthier, ph.d. 1 chmi 2227e biochemistry i amino acids: - structure - general...

CHMI 2227 - E.R. Gauthier, Ph.D. 1

CHMI 2227EBiochemistry I

Amino acids:-Structure-General chemical properties


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.


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 (NH2);

Hundreds of amino acids exist in nature;

However, only 20 amino acids are used by the cell to make proteins.

CH

R

COOH

NH2

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


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.


General properties of amino acids.

Every amino acids has at least two chemical groups that can be ionized:Carboxylic Acid (COOH)Amino (NH2)

Zwitterion

Net charge: +1 Net charge: 0 Net charge: -1

Acidic pH Basic pH

pKa 1(pH 2-2.5)

pKa 2(pH 9-10.5)


Amino acid titration1. simplest scenario

pI = isoelectric point = pH where 100% zwitterion

pI = pKa1 + pKa22

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!


The twenty amino acids:


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?


Structure of the 20 amino acids1. Hydrophobic amino acids

Proline is the only cyclic amino acid;


Structure of the 20 amino acids2. Aromatic amino acids


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.

Absorbance

max

(nm)

Molar

Absorbance

(M-1 cm-1)

Phenylalanine 257.4 197

Tyrosine 274.6 1420

Tryptophan 279.8 5690


Structure of the 20 amino acids3. polar, uncharged amino acids


Structure of the 20 amino acidsCysteine and cystine

Disulfide bond

In biochem:-oxidation: lose H-Reduction: gain H


Structure of the 20 amino acids Tyr, Ser, Thr

P=O

O-

O-

Ser

Phospho-Ser

Proteinkinase

Proteinphosphatase

ATP

ADP H2O

PO4-3


Structure of the 20 amino acids4. Negatively charged amino acids


Structure of the 20 amino acids4. Negatively charged amino acids

pKa1 pKa2pKaR

What is the pI of glutamate?

NaOH

pH

pKa1

pKaR

pKa2

9.5

4.1

2.1

100% A

100% B

100% C

100% D

A B C D


Structure of the 20 amino acids5. Positively charged amino acids


Structure of the 20 amino acids5. Positively charged amino acids: His

pKaR

pKa2


Structure of the 20 amino acidspKa values of amino acids


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.

Elution

+ +

pH >> pI pH << pI


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.

O

O

OHOH

Ninhydrin Amino acid

2

O

O

N

O

O

CH

R

COO-

NH3+

CO2

Purple!!

R-HC=O

chmi 2227 - e.r. gauthier, ph.d. 1 chmi 2227e biochemistry i amino acids: - structure - general...

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