AMINO ACID

STRUCTURE AND FUNCTION OF

BIOMOLECULES

(WEEK 8)
 AMINO ACID
I. DEFINITION
II. GENERAL FORMULA
III. CLASSIFICATION : ● Charge of R ● Type of R
IV. PROPERTTIES OF AMINO ACID: ● Zwitter Ion ● Optic
V. ASAM AMINO BUKAN PENYUSUN PROTEIN
VI. REACTIONS OF AMINO ACID: ● COOH ● NH2 ●R ● Peptide bond
VII. AMINO ACID ANALYSIS: ● Classical chromatography: (paper, thin layer)
● Electrohoresis
● Spectrophotometri
●Advanced chromatography: (ion exchange,
HPLC, GC)
VIII. PROTEIN/PEPTIDE SEQUENCHING*
IX. PROTEIN/PEPTIDE SYNTHESIS*
● Chemical (Organic) synthesis  solid phase site-directed synthesis
● Biochemistry  biotechnology  genetic engineering
X . AMINO ACID SYNTHESIS*
● Chemical (Organic) synthesis  solid phase site-directed synthesis
● Biochemistry  biotechnology  fermentation
I. DEFINITION: What is Amino Acid?

● COOH

● NH2

● Polipeptide/protein monomer
 II. GENERAL FORMULA

H
O
H2N C C OH
R

R functional group differenciate each amino acid to

another → classification → based on the charge or
type of R funtional group
 III. CLASSIFICATION

3.1 Based on the charge of R functional group

Non polar (hidrophobic):val, ala, leu, ile, pro, met, phe, tyr
Polar (uncharged): gly, ser, thr, lys, tyr, asn, gln
Polar (charged): Separation method based on charge:
negative: asp, glu electrophoresis, chromatography, etc.
positive: lys, arg, his

3.2 Based on the type of R functional group

aliphatic: gly, ala, val, leu, ile
aromatic: his, phe, tyr, trp
Acid: asp, glu
Base: arg, his lys
Hyidroxyl: ser, tyr, thr
Sulphur : cys, met
Amide : asn, gln
Carboxylic: asp, glu
Imino: turunan aa
Specific functional-group-based
test/analysis:
identification/qualitative
Specific functional-group-based
staining method:
Spectrophotometri  quantitative
 STRUCTURE OF VARIOUS AMINO ACIDS
Nonpolar, aliphatic R groups Polar, uncharged R groups
O
O O O O O
H2N CH C OH H2N CH C OH
H2N CH C OH H2N CH C OH H2N CH C OH H2N CH C OH
CH CH3 CH OH
H CH3 CH2 CH2
CH3 CH3
OH SH
Glysine (gly) Alanine (ala) Valine (val)
O O
Serine (ser) Threonine (thr) Cysteine (cys)
H2N CH C OH O
H2N CH C OH O O
C
CH2 CH CH3 OH
H2N CH C OH O H2N CH C OH
CH CH3 CH2 HN CH2 H2N CH C OH CH2
CH3 CH3 CH2 CH2 CH2
Leucine (leu) Isoleucine (ile) Proline (pro) S C O C O
CH3 NH2 NH2
Nonpolar, aromatic R groups
O
O
Methionine (met) Asparagine (asn) Glutamine (gln)
O H2N CH C OH
H2N CH C OH CH2
H2N CH C OH
Polar, positively charged R groups
CH2 O
CH2 O
H2N CH C OH H2N CH C OH
CH2 O
HN CH2
OH CH2 H2N CH C OH
CH2
Phenylalanine (phe) Tyrosine (tyr) Tryptophan (trp) CH2 CH2 CH2

CH2 NH
● Val, thr, tyr, phe, met, lys () NH2 C NH
N
NH
 essential aa  synthesized by Lysine (lys)
NH2
Arginine (arg) Histidine (his)
animal or human  must be supplied
from outside (food) Polar, negatively charged R groups
O
● Defficiency  disruption of the total O H2N CH C OH
H2N CH C OH
protein metabolism CH2

● rice, serealia etc.  <<<<  addition

CH2 CH2
C O C O
of essential aa (feed additive)  aa OH OH
industry Aspartat (asp) Glutamat (glu)
 IV. PROPERTIES OF AMINO ACID
4.1 Amphoteric properties of amino acid (aa)  acid/base  +/-  dipolar ion  pH
OH- H+

H+ OH-
not dissociated

aa general formula within water solution

as zwiter ion → aa-dipolar ion
→ zwiter ion
● Isoelectric point → the + & - charge of aa are the same → aa neutral (specific for
each aa) → minimal solubility in water (polar) ↓ → precipitation
● pH at the isoelectric point → isoelectric pH → pI
● Graph and determination of pI:
acid base

 For aa with ionizable pK1 pK2

pH
R function: asp, 13 pK1 = 9,60
glu, his, cys, tyr,lys, arg pH
→ ada 3 pK 7 pI = 1/2 (pK1 + pK2) = 5,97
pK2 = 2,34

0 ekivalen OH-
pI=1/3(pK1+pK2+pK3) 0 0,5 1 1,5 2
4.2 Optical properties of amino acid (aa)
● aa (except glycyne)  asymetric/chyral C atom  rotation of plane polarization
dextrorotatory (+): right → D; levorotatory (-): left → L → protein monomer
COOH COOH
H2N H H NH2 Chyral C atom
Ball and stick model (4 different
CH3 CH3
substituents/
COOH COOH functional
H2N H H NH2 Perspective formula groups are
CH3 CH3 attached)
COOH COOH Projection formula
H 2N H H NH2
CH3 CH3
L-alanine D-alanine
 Number of optical isomer: 2n, n =  chyral C atom.
Example: thr & ile  2 chyral C atom   isomer = 22 = 4  R/S system
COOH COOH COOH COOH
H 2N H H2N H H NH2 H NH2
HO H H OH HO H H OH
CH3 CH3 CH3 CH3
L-threonine L-alothreonine D-threonine D-alothreonine
 V. NON-PROTEIN MONOMER-AMINO ACID

 Not  amino acid  

L-Alanin ()    -Alanin

 Energy storage process in vertebrate
 Part of Phantotenic acid & coenzyme A
 Not L amino acid
D-fenil alanin  antibiotik gramisidin S
D-valin  aktinomisin D (inhibitor sintesis RNA)
D-alanin  peptidoglikan dinding sel bakteri gram positif
D-glutamat  --
 Additional subsituent/functional group
 Colagen (12 %)
 animal structural protein
HO

HO

L-Proline L-Hydroxyproline L-Lysine L-Hidroxylysine

 VI. REACTIONS WITH AMINO ACID
H O
- Reactive functional groups :
H2N C - OH
- Reactions: - with NH2
- with COOH R
- with R
- peptide bond formation (polymeration → polypeptide → protein)

 6.1 Reactions with NH2 functional group

HNO2 + + H + + H 2O + N 2

• N2  manometric determimination   -amine  aa, peptide, protein (Kjiedahl

method )
• Lyisine + HN2O  slow reaction
• Proline or hydroxyproline + HN2O 
 H H2O
O +
R'
H2O

HCHO +

 COOH can be tritated using phenolphthalein or timolphtalein indicator

  Nynhydrine Reduced nynhydrine
O O
OH CO2 OH H
+ NH3 + + C O
OH H
O O R
Oxydized nynhydrine
-3H2O O O

Exception: - pro & hypro  >NH  2o  yellow -N=

- asn  brown
O O
Vissible spectrophotometri analysis (quantitative) aa-Nynhydrine (blue)

 Fluoroscamine

O +
HO
O O
O Fluorospectophotometri
aa-fluoroscamine derivative
analysis (quantitative)
(fluorescens )
  Edman method
S S
-H+
-N=C + -N=C

Phenylisothiocyanate
cyclysation H+
HOH

Phenylthiohydantoin

 Sanger method

NO2 HF NO2
O2 N -F + O2N

FDNB (fluorodinitrobenzene)
  Dansylcloride
HCl

H3C H3C
N N
H3C O H3C O
S Cl + S Cl
O O

 Dabsylchloride
O
H3C
N NN S Cl +
H3C
O
HCl

O
H3C
N NN S Cl
H3C
O
 6.2 Reactions with –COOH functional group (carboxylic end)
HOH
+ HOR’
esterification

decarboxylation
+ CO2

decarboxylation
+ CO2

Allergic reaction,
induces secretion
histidine histamine of gastrium fluid
(intestine)
 6.3 Reactions with –R functional group

O HOH

+ HO P OH
Phosphorylation
HO
Serine Phosphatidic acid Phosphatidic serine (phosphorylated aa)

H2
2
Formation of disulfide bond
Cysteine Cystine

+ Ag (Heavy metal)
Cysteine Denatured/precipitated protein
 6.4 Peptide bond formation Peptide bond
HOH

+
Dipeptide
aa1 aa2

HOH
aa3

Protein-general formula
Tripeptide
Numenclature: glycylalanylserine

n
HOH aan
Protein
Polypeptide
 VII.AMINO ACID ANALYSIS

7.1 Paper/Thin Layer Chromatography

S= aa standards
1, 2, & 3 = samples
S 1 2 3 S 1 2 3
separation based on the solubility of the samples within mobile phase
qualytative  migration/RF (dibandingkan dg standar)
quantitative  spot area; or spot  cut  dissolved  spectrophotometri analysis

7.2 Electrophoresis
- - + -
pH=7
pH=7 pH=7

+ +
S 1 2 3
paper, gel gel, liqid liquid
separation based on the difference of the charges of the samples (aa)
qualytative  migration/RF (dibandingkan dg standar)
quantitative  spot area; or spot  cut  dissolved  spectrophotometri analysis
 6
7.3 Spectrophotometri trp

Absorbance
Nonvisible spectrophotometri  UV 4
7.3.1
Qualitative (tyr & trp)
Without 2 tyr
Quantitative (all aa)
Coloring Determination of protein phe
concentration based on the tyr or trp 0
reagent 230 250 270 290 310
content Wavelength (nm)

7.3.2.1 Nynhydrine
lys + glacial acetic
Visible spectrophotometri
acid + nynhydrine
7.3.2 Quantitative  red
With 7.3.2.1 Fluoroscamine   400 nm
Coloring Florespectrophotometri
met + NaOH +
reagent Quantitative Na-nitroprucide +
7.3.2.1 Specific coloring reagents HCl  orange
Visible spectrophotometri   580 nm
Quantitative/qualitative
 7.4 Ion-Exchange Chromatography

Reservoir of buffer allows

sample to percolate slowly
through column
+

Solutions of amino acids at

pH 3.0 is poured onto a
cation-exchange column

Amino acids with greatest

positive charge (red) bind the
coloumn most tightly and
therefore move most slowly.
Those with the least amount
of positive charge (blue)
move fastest and elute first

Chromatogram
Absorbance

Fractions are collected

Detector from the bottom of the
column and analyzed
quantitatively
Time (minute)
 7.5 OTHER CHROMATOGRAPHY METHOD

Other chromatography method will be discussed in

protein session, as well as protein/peptide
sequenching (VIII), protein/peptide synthesis (IX),
and oligonucleotide synthesis (X)