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Protein

Makhluk hidup terbuat dari


apa ?
Karbohidrat :

rantai dari
molekul glukosa dan gula lainnya
yang berulang

Lemak (Lipid) :

Memiliki
struktur yang terbentuk dari satu
molekul gliserol dan tiga molekul
asam lemak

Protein :

Rantai dari asamasam amino yang berbeda-beda

Struktur Kimia PROTEIN


PROTEIN : POLIMER ASAM-ASAM AMINO YANG
BER-IKATAN PEPTIDA
PENYUSUN UTAMA Protein : ASAM AMINO

Protein
Terdapat

20 jenis asam amino yang

dibutuhkan oleh tubuh


Sel

tubuh kita hanya dapat mensintesis 11

jenis asam amino non esensial (endogen)


9

asam amino lainnya adalah asam amino

esensial (eksogen) yang kita peroleh dari


bahan pangan

Amino Acids - 1

Amino Acids - 2

Secara umum ASAM AMINO


berstruktur:
C alpha, ASIMETRIK (Kec. GLISIN)
dengan simbol (L) /LEVO

Only L-amino acids are constituents of


proteins.
L and D isomeric nomenclature is similar

Sifat Asam Amino dan Protein


1.

ION ZWITTER

2.

pH TINGGI :
GUGUS KARBOKSIL TERDISOSIASI

3.

pH RENDAH :
GUGUS AMINO TERDISOSIASI

4.

pH ISOLEKTRIK Protein

Biochemistry 2/e - Garrett & Grisham

Copyright 1999 by Harcourt Brace &

STRUKTUR PROTEIN
1. STRUKTUR PRIMER
struktur linear yang tersusun dari ikatan
polipeptida
2. STRUKTUR SEKUNDER
Pelipatan struktur primer dari suatu polipeptida
3. STRUKTUR TERSIER
Pelipatan dari struktur-struktur sekunder
membentuk suatu bentuk yang khas
4. STRUKTUR KUARTENER
Struktur kompleks gabungan dari beberapa
struktur tersier suatu polipeptida

Primary

Assembly

Secondary

Folding

Tertiary

Packing

Quaternary

Interaction

PROCESS

STRUCTURE

Biology/Chemistry of Protein
Structure

Struktur primer

Struktur sekund
(alpha helix)

Struktur sekunderStruktur tersie

Biochemistry 2/e - Garrett & Grisham

Copyright 1999 by Harcourt Brace &

Biochemistry 2/e - Garrett & Grisham

Copyright 1999 by Harcourt Brace &

Mengapa Telur terlihat


berbeda setelah dimasak ?
Telur merupakan bahan pangan
kaya protein.
Ketika telur dimasak,
menyebabkan perubahan struktur
protein, peristiwa ini disebut
Denaturasi.

Perubahan bentuk protein akan


mengakibatkan protein menjadi
lebih mudah untuk dicerna
Daging juga disusun atas asamasam amino..

SIFAT KIMIA PROTEIN

SIFAT KIMIA PROTEIN


3a. SALTING IN
MENINGKATNYA Kelarutan Protein
Akibat penambahan SEDIKIT GARAM

3b. SALTING OUT


BERKURANGNYA Kelarutan Protein
Akibat penambahan GARAM yang
BERLEBIH

Klasifikasi Protein
1. BERDASARKAN SUSUNAN
MOLEKUL

1a. Protein Fibriler (Serabut)


Tersusun dari rantai molekul yang panjang
sejajar dengan rantai utama, elastis, tidak
larut pada larutan encer
Contoh : kolagen, miosin, gluten
1b. Protein Globular (Bola)
rantai molekul memadat membentuk
struktur bola, lebih mudah larut, mudah
terdenaturasi
Contoh : laktalbumin, albumin

Protein Berdasarkan Fungsinya


1. Enzim : papain
2. Pengangkut : hemoglobin,mioglobin,transferin
3. Pergerakan : pergeseran 2 protein
Aktin dan miosin pada otot (pada suhu
ruang berkurang sehingga daging liat)
4. Antibodi contoh : lizozim
5. Hormon contoh : insulin

Uji Kualitatif asam amino Protein

Uji Ninhidrin. 0.5 mL larutan ninhidrin 0.1% + 3 mL larutan protein.


Dipanaskan selama 10 menit
Uji Millon. 5 tetes pereaksi Millon Hg2(NO3)2 + 3 mL larutan protein,
dipanaskan. Oksidasi asam nitrat pada asam amino yang mempunyai gugus
OH seperti tirosin.
Uji Hopkins-Cole. 2 mL lar. protein + pereaksi Hopkins-Cole. + 3 mL H2SO4
pekat melalui dinding tabung, apabila positif mengandung triptofan.
Uji belerang. 2 mL larutan protein + 5 mL NaOH 10%, dipanaskan selama 5
menit. + 2 tetes larutan Pb-asetat 5%, pemanasan dilanjutkan, diamati
warna yang terjadi.
Uji Xanthoproteat. 2 mL larutan protein +1 mL HNO3 pekat, kemudian
dipanaskan. Hasil positif terhadap asam amino yang mengandung cincin
benzena: seperti fenilalanin, tirosin, dan triptofan
Uji Biuret. 3 mL larutan protein + 1 mL NaOH 10% + 1-3 tetes larutan
CuSO4 0.1%

Uji Kualitatif asam amino Protein

Endapan
Hitam

PEMURNIAN PROTEIN :
1. BERDASARKAN UKURAN MOLEKUL
Menggunakan Membran
SEMIPERMIABLE atau KROMATOGRAFI
KOLOM
2. BERDASARKAN MUATAN PROTEIN
Menggunakan KROMATOGRAFI
PENUKAR ION (ION EXCHANGE
Chromatography)

Translation of mRNA into Protein

Biochemistry 2/e - Garrett & Grisham

Amino Acids Can Join Via Peptide Bonds

Copyright 1999 by Harcourt Brace &

Cross links between peptide chains:


Disulfide linkages between individual
cysteines are called cystines:

Insulin is the smallest protein, with 51


amino acids First protein to be fully sequenced (by

Fred Sanger in 1953). For this, he won his first


Nobel Prize

Preserve the structure during


purification
Consider that the structure may be lost
Activity assay a good test

Methods in Protein Biochemistry

Ammonium Sulfate
Precipitation
Very high ionic
strength Proteins
precipitate Salting Out
Modest
Purification but
Also Useful to
Concentrate
the Sample

Ammonium sulfate precipitation salting out proteins

At high concentrations of this strong salt, water is highly


ordered
High concentration of strong chaotropic salts strips water
away from protein
Lower availability of solvent (water)
This favors protein interactions rather than protein - solvent
interactions causes aggregation of proteins (they become
Each
protein has a
insoluble)
different solubility so
this is a method to
isolate groups of
protein
Precipitation is
reversible and usually
non damaging to
structure of the
enzyme
Ammonium Sulfate is
most commonly used.
Urea is also used but

Column Chromatography

Flow-through

Eluate

Ion-Exchange Chromatography

Ion exchange chromatography

Ion exchange resins contain charged groups.


If these groups are acidic in nature they interact with positively charged proteins and are called cation exchangers.

Positively
CH2-COO
+
charged (basic)
If these groups are basic in nature, they interact with
negatively
charged
molecules and are called anion
+
protein
or enzyme
exchangers.
CH2-COO
+
CM cellulose
cation exchanger
-

CH2-CH2 -NH+(CH2CH2) - CH2-CH2 -NH+(CH2CH2)


DEAE cellulose
anion exchanger

Negatively
charged (acidic)
protein or enzyme

Ion exchange chromatography


For protein binding, the pH is fixed (usually near neutral) under low salt
conditions. Example cation exchange column
+

CH2-COO-

+
+
CH2-COO- +

CM cellulose
cation exchanger

Positively charged protein


or enzyme bind to the
column

Negatively
charged proteins
pass through the
column

Ion exchange chromatography


To elute our protein of interest, add increasingly higher amount of salt
(increase the ionic strength). Na+ will interact with the cation resin and Clwill interact with our positively charged protein to elute off the column.
-+ +
CH2-COO
- +

CH2-COO +

+ Increasing
[NaCl] of the
elution buffer

CM cellulose
cation exchanger

CH2-COO
CM cellulose
cation exchanger

+
- Na Na+2

- Na+

CH2-COO
Na+2

ClCl- +
+
Cl
+
- +
Cl

Affinity Chromatography

Gel Filtration Chromatography

Characteristic

Procedure

Solubility:

1.
2.

Salting in
Salting out

Ionic charge:

1.
2.
3.

Ion exchange chromatography


Electrophoresis
Isoelectric focusing

Polarity:

1.
2.

Adsorption chromatography
Paper chromatography
Reverse-phase chromatography
Hydrophobic interaction
chromatography

Molecular size:

1.
2.

Dialysis
Gel electrophoresis
Gel filtration chromatography
Ultracentrifugation

Binding specificity:

1.

Affinity chromatography

Using SDS-PAGE to Assess Purity