Chemistry of

Amino Acids and Proteins

ภญ.ดร.ภัทรวดี ศรี คุณ

คณะเภสัชศาสตร์ มหาวิทยาลัยบูรพา
E-mail: pattaravadees@buu.ac.th
Objectives

1. มีความรู้ความเข้ าใจเกี่ยวกับโครงสร้ าง หน้ าที่และคุณสมบัติ

ของกรดอะมิโนและโปรตีน
2. สามารถอธิบายกลไกและบทบาทหน้ าที่ของกรดอะมิโนและ
โปรตีนในสิ่งมีชีวิตได้
3. สามารถอธิบายหลักการทดสอบและการวิเคราะห์กรดอะมิ
โนและโปรตีนเบื ้องต้ นได้

2
 o , ni Vs :novJni5' o ! lush , MV

Why learn chemistry of amino acids & proteins?

Wv Macro mutecvlendwwosdrlsinovnon

•( Proteins are the biggest component of

* living organism’s cells and total
body.

_*
monomer w > Protein

Amino acids are

Proteins are essentially the basic structural
important in every biochemical units of proteins.
reactions and pathways.

3
https://www.thoughtco.com/chemical-composition-of-the-human-body-603995
 Chemical Structure of Amino Acids

:
ndrinwwo :1n
'

chz .

peptide bond a. ov.lunb.sn peptidase

(covalent)

Protein Peptide ) Amino acid

• Amino acids are monomers of protein

• Consists of amino group (-NH2), H-atom, R group (or

side chain) bonding with α-C-atom 4
 ioovniolrwgj

Asparagus
1806 Asparagine
o7n :
not amine

Glutamate
Wheat gluten
krrnirv
Cheese (Greek-tyros)
Tyrosine

Glycine Sweet taste (Greek-glycos)

1938 Threonine 5
 Amino Acids
Structure of amino acid

:
( nwntohl

4dm
(general)

nnioln
'

no rid Chiral center

Asymmetric carbon +
9viH
{v
At Structure of amino acid 9 who mw

on
(physiological condition)
www.Y
NHY

od.lu form two :O

,

All molecules with a chiral center are able to rotate plane-

polarized light. 6
Priority of attached group on chiral center indicates number
position ÷
VJMIWWW . VOIIWI ]n
,
> 5Y✓|n7 :

-OCH3 > -OH > -NH2 > -COOH > -CHO > -CH2OH > -CH3 > -H

Chiral carbon
 r
3 Types

Classification of Amino Acids

AA’s are classified according to the location of the amino group.

IO

kHz
c.
y\
@ IIO

8
P L
E 8
2
5 4 3 1
H
{
-
CH -
CH 2
-
CH 2-
-
00 -

2
,

tNHz + NH Ct car box Nu C ,

noontime Nw mw .
ndwniiw

There are 20 genetically encoded-amino acids found

in peptides and proteins

8
Every amino acid has 2 forms of enantiomers

:lWM
formndloohrinsnyw jnsmvlonw ,
9VVr:luW

– L-form (found in physiological pH 7.4) Optically active

– D-form -

mnwitvwmointfovm @ N mhsiwsi
'
configuration
iionnwlonml JVs
llriloiwttilrilwhtm )nI✓1n
'

Stereoisomers

MW

Mirror image rrinow

9ms :on
 form
581 's 17,2
'

Cook ljviovw & 1

1. 101 Coo ,

CH yliiisnh ]

nd .
amino 04 Left 2 Right
2 . 7h
htslnw
Right → D form
Yin
-
°
vn

Left → L form 3
. ,v -

×
w mirror image now

• Lining up C-atoms from 1- to 3-C from top to bottom

• Consider which side of α-amino group is located

• L or left or levorotatory or (-) or Egomania

counterclockwise
• D or right or dextrorotatory or (-) or counterclockwise
now Nwvinnn

10
Two ways to illustrate the stereoisomers

11
 '

1H ni v o n s : w 7 v v o s 11 J ] 5 in o 3 J

L, D system refers only absolute configuration,

but not optical properties

• The binding specificity of a chiral receptor site for a

chiral molecule is usually only favorable in one way.

12
 Note R, S system n7Snw,w
won s :wiv

V 0011101 ri ✓ n )7w

• Consider more
precisely configuration
• Most useful system for
compounds with ≥ 1
chiral center

• Assign priority of each attached group to chiral center by

this following
-OCH3 > -OH > -NH2 > -COOH > -CHO > -CH2OH > -CH3 > -H
/W7|n OY 1ham
• Locate the lowest priority group pointing away
)
4
• Consider decreasing orders of priority groups ( 1 to -1
3 )
• R or rectus or right or clockwise nrnfn nw

• S or sinister or left or counterclockwise § now

13
 mrwdw rinds form W7 R & 5
win 's
-
Vos
,

14
• There are 20 genetically encoded -amino acids found in
peptides and proteins

• Consist of 10 essential amino acids and 10 non essential

amino acids

• 19 are primary amines, 1 (proline) is a secondary amine

• 19 are “chiral”, 1 (glycine) is achiral
Q : OJV 7W Vvooi
Wa -
woo 1J ]

Vdcemi C

no Nw
\ chiral C
R group
a

0011 now Wow

i vn ? amino

a wo rose
,

15
lsimwow amino d rioodw
 Amino acids can be classified by R groups
lirio

Nonpolar:

*
*
67 ¥
A

sdviohrilw ymnriolnvr
'
:O
,

Aromatic R group
Polar but non-ionizable: | As Sd 7)
107 s

'

2h51 n n ; n

16
 [ Jh 0011 now
of in 's No )

Acidic: 5 6 1h51 evw Jntwvm

msn.nntwndwnr.ln.si
,

side
Basic: ljnwlvnoiu aromatic

chain
1

25.2: Stereochemistry of Amino Acids: The natural

configuration of the -carbon is L. D-Amino acids are found in
the cell walls of bacteria. The D-amino acids are not genetically
encoded, but derived from the epimerization of L-isomers (Ch.
25.6). hrwbact Minis
.
Dform,
L
j mafyrenz to .wivvD←8 form , ,
.
L

jsriilr #form lri

.

( wniivlri win )

17
Test!!! Amino acids can be classified by R groups

– Nonpolar
• Aliphatic : 7 aa : ….., Pro**
• Aromatic : 2 aa (3) : Phe, Trp
– Polar, uncharged
• O
6 aa (5) : Ser, Thr, Cys, Asn, Gln, Tyr*
– Electrically charged
• 2 Acidic aa: Asp, Glu
• 3 Basic aa: Lys, Arg, His**

18
*

Fri
ioolvprutndoybody
'

Hydropathy index
“transfer of R group to
: water” amino
daioiw
wilri

,
Gibbs fveeneuevgy
|
• ∆G < 0 favorable Eiiniw
loins

• ∆G > 0 unfavorable
. )
.

Amino
y
a

I
'
.
d) wwj
tilri
 ;
(1 #6) CHIMSV

rionvsmrsmvrio

Margaret Oakley Dayhoff

(1925-1983)
( rinrioaiurioonvrrioirivo
 (1 #6) CHIMSV
• #5) AGLPT
(2

to
Gly(7.2%) vs. Glu(6.3%)

:&
.
Ala(7.8%) vs. Asp(4.3%)
Pro(5.2%) vs. Phe(3.9%)
Leu(9.1%) vs. Lys(5.9%)

Margaret Oakley Dayhoff

(1925-1983)

Thr(5.9%) vs. Tyr(3.2%)

 msinp

(1 #6) CHIMSV
(2 #5) AGLPT
(3 #4) RFYW

q
Fenyl-alanine
tYrosine
tWiptotophan

aRginine
(1 #6) CHIMSV
(2 #5) AGLPT
(3 #4) RFYW
• #4) DNEQ
(4

:
asparagiNe
Q-tamine

asparDic
glutamEke
(1 #6) CHIMSV
(2 #5) AGLPT
(3 #4) RFYW
(4 #4) DNEQ

:
(5 #1) K

sine

… I , J , K , L , M , ….
L M 981 Moi wvvowwiriilniy
,
Uncommon amino acids

• Derived from common amino acids

Lys
• Incorporated into a polypeptide

• Usually have important functions

e.g.

– 6-N-Methylysine (Lys) : myosin (contractile protein)

– γ-Carboxyglutamate (Glu) : prothrombin

25
Biological Functions of Proteins
1. Enzymes
2. Peptide hormones
3. Toxins
4. Structural proteins
5. Storage proteins
6. Protective protein
7. Transport proteins
8. Contractile proteins (movement)
9. Roles in gene functions
10. Roles in blood clotting
26
1. Being enzyme
e.g.. amylase, protease

2. Being peptide hormone

e.g. insulin

27
 3. Being toxin

4. Being structural protein

e.g. collagen, keratin

28
 5. Being storage protein

6. Being protective protein

e.g. immunoglobulin

29
 7. Being transport protein
e.g. hemoglobin

8. Being contractile protein

e.g. actinomycin,
myocin filament

30
 9. Playing roles in gene
functions
e.g. histone

10. Playing roles in blood

clotting
e.g. fibrinogen,
thrombin
31
 General properties of amino acids

nnwnhwnrnhrmswv Buffer

• in Biology acid-base buffer

'

rlonw 5 total
/ charge in 0
list Vriglrinist
-

no :
show
to :) VJ to
I vnisnrn
nhwnrn zwittev
his :
rivers “amphoteric compound” ion

Isoelectric point; pI
32
 n' ovylriw
NaOH
*
Gly

Gly

:
oisltto
mow
Willowy
• Isoelectric point (pI);

• is where molecule has net

charge=0 or being as
1noTw
“zwitteriron”
33
 NaOH
*
Gly
amino
- n' SH oononn
group

Na-OH +NH -CH -COOH

3 2

3 H-OH +NH -CH -COO-Na+

3 2

2 H-OH NH2-CH2-COO-Na+

1
nntsonniv INUNAOH a ,N
liuriwonrnowlwlnonwlwnmvihlunrn
Nautto :N5sH ooh

34
 NaOH
*
Gly
pKa1

Na-OH +NH -CH -COOH

3 2

3 H-OH +NH -CH -COO-Na+

3 2

2 H-OH NH2-CH2-COO-Na+
fovmvosnrn
} form voo INTO
\ pKa2
wwfovmlevili ↳
1
“Buffer”

ftp.ptt.ph#
-
niimwnrnosl ✓ form nrnoiivlviiw
if!!
"

35
 base acid

Henderson-Hasselbalch Equation

36
• Only weak base or weak acid can be buffer.
• Each aa can be buffer at different pH depending on its
pKa >> จานวนครัง้ /ตาแหน่งในการแตกตัว, บัฟเฟอร์ กรด-บัฟเฟอร์ เบส
37
Cys
pKa1 = 1.96
OH-
H-+

pI = 5.66
“Zwitterion”

pKaR = 8.18 H-+ OH-

H-+
OH-

pKa2 = 10.28
• in Physics Optical activity
– Polarization (หมุนระนาบแสงเดี่ยว)

39
– absorb light at 280 nm of aromatic R groups
irlvnvinmvoww { Vimv rslsi
(e.g. Tyr, Trp, His) rioozzmout

%
Main
:

atsnnnnwaiwnblwo
"
prints
"
"
"

40
• in Chemistry 9V9vmmn
Joviwismovwwwwrriw N' no

– Amino group
amino
• Ninhydrin reaction nine ivnv

firms product Swigs

side
• Except Pro
* yellow color win :

nivm
chain
5
'
rios

amino an

41
Peptides & Proteins

trans form
- Planar structure
- Electron moving
- Double bond-like effect
>> strong (covalently)
-bonding

42
• Terminology of peptide tripeptide
– Oligopeptide
(contains few aa)

tetrapeptide

pentrapeptide

– Polypeptide
usually ≥10 aa are joined together (interchangeably with the
term “protein”)
43
What about the properties of amino acids in peptide?

• Number of the ionized R

groups contribute to the
overall acid-base properties
of peptide

Arm
( pnvipttrvqo

• refer to isoelectric pH (pI) -

• Used to separate peptides

and proteins

44
Biological active peptides and polypeptides

“Simple proteins” 45
Some proteins contain chemical groups other than amino acids

“Conjugated proteins”
Prosthetic group

Carbo

Glycoprotein 46
Conjugated proteins

47
 Once amino acids form polymer, it is for a reason…

• Form structure to function, and to be stabilized

wwno7unsnw1si9risonwlubodysiiw@lIO.vm
Primary Secondary Tertiary Quaternary
}

%
structure structure structure structure
( structure no
'T
simple )
ftnhwpolypcptide
*
%
nioinmuiw vnitnninwsiniu
Hiway won ,n ;
function
lsiformiws .VN '
,iw%n3°)
( jovnn 48
.
.
(a) Primary structure
5) unions ) J7V0o en 2 ,
510in n' 727W

. : 7 :
we ah 5 'd n
,

The most important element of 1st structure is the sequence

of amino acid residues. 49
 line
imvnhv or
pro
9

vdivlvoi

ทาให้ α-helix ไม่เสถียร

?
nninhhlriisv

in non Mr J In
H
.

bond 9w IN who studies

-

air R group ขนาดใหญ่/มีประจุ สามารถรบกวน

α-helix ได้ { ) |
|nw '
onj in

nsrworio
his

L -
] 57

helix
] m
a i

yvv
: M W

laminorielnw

Left-handed α-helix
Right-handed α -helix
50
 • The β conformation organizes polypeptide chains into sheets.
carboxylic group
amino
N C
-
-

group
anti-parallel

C N
parallel
N C

hydrogen bonded

N C
These structures are rigid and stable. 51
 myoglobin
pdb code: 1WLA

Bacteriorhodopsin
pdb code: 1AP9

Anti-parallel
-sheets
of lectin
Parallel -sheets pdb code: 2LAL
carbonic anhydrase
pdb code: 1QRM 52
 • general structure in physiological
condition
• can be active (ready to work)
• called “protein folding”. '

fnrwrnsiyzo imrvsrio 95 onflvrilnvvi function

a, protein folding onnivuuioo :

nbworiomrniow
Wviowvqnvyw Add pattern ,wwow )

Fibrous Polypeptides strands that “bundle” to form elongated

fibrous assemblies; insoluble.
form Nviowimow

Globular Proteins that fold into a “spherical” conformation.

Proteins will fold so that hydrophobic amino acids are on the

inside (shielded from water) and hydrophilic amino acids are on
the outside (exposed to water) 53
• consists of subunits of polypeptides bounded by weak
bonding
• advantage for not to synthesize long protein

54
 * *
*
Summary

3D structure of a protein is determined by its amino

acid sequence.
Functions of a protein depends on its structure.
Isolated protein usually exists in a stable structure
forms
The most important forces stabilizing the 3D
structures maintained by noncovalent interaction
They share some common structure patterns that
help to recognize the understanding of protein
architecture.

55
Techniques in Amino acid and Protein Analysis

• Protein separation and purification

• Quantitative analysis

• Qualitative analysis

• Amino acid sequencing

• Protein structure determination

56
 Protein separation and purification M W
n on w J 7 W V n 9 w n n s F ,

Salting out

is 5 o v r o V

w r ri w 1 no no vir
Increasing ammonium
nn r 2 o , w
'

sulfate concentration
= .

1 i i Tv
Saturated or
subsaturated
ammonium sulfate

Atarting volume of
sample + pH-
adjusting buffer 57
Electrophoresis Noun

58
 Protein identification
Blotting

Immunodetection

59
Chemiluminescent detection of protein bands

60
 V { ( ✓ rniw
msilnst.nl wiwvoo

Quantitative analysis : Protein concentration

Methods using UV-visible spectroscopy

1. Direct measurement at 280nm

2. Biuret Method

3. Bradford Method

4. BCA method

5. Lowry Method

6. Turbimetric method

61
 Iinoinlrtonln
' '

nnoivn 9v1ab
260hm
a.

npnfwnd 28°
nm

Jg
9vpNAr Historic
protein [ DNAI i
'

rw{ 4ns )

Since determination of protein concentration at A280nm

is not sensitive but convenience, DNA/protein ratio is
used for checking purification of DNA extraction.
62
 { ws .

jan
.

63
Assay Description Advantages Disadvantages
Bradford Coomasieblue dye Assays are generally • Interference from
binds to protein and simple/quick to SDS or other
undergoes a shift in perform detergents at high
absorbance concentration
• Small linear range
BCA Cu2+ ions and forms a • Available in format
colored product that is compatible with
measured reducing agents
spectrophotometrically • Less protein-to-
(Biuret reaction) protein variation
than Bradford
Lowry Similar to BCA (Biuret Very well cited in • Assay time may be
reaction) liturature longer than others
• May not be
practical for large
sample groups
• Precipitates may
form 64
Recommended reading

65