Professional Documents
Culture Documents
Title of Experiment
Amino Acids and Protein
B. Objective of Experiment
Before conducting the experiment, students must first understand the
structure of the protein. As long as doing this experiment is expected to:
1. Can prove the existence of a peptide bond.
2. Can understand the xanthoproteat reaction and biuret test against various
contents of protein.
3. Understand the solubility and amphoteric properties of amino acids.
4. Proficient in paper chromatographic separation of amino acids and their
identification.
C. Literature Review
When a protein is hydrolyzed by dilute acids, bases, or hydrolytic
enzymes, the result is a mixture of acids. An amino acid is a carboxylic acid that
also contains an amine group -NH2 an a-amino acid has the amino group on the
carbon atom - the C atom next to the carboxyl group. Thus, proteins are high-
molecular-mass polymers composed of acids. Of the known acids, about 20 have
been identified as the building blocks of plant and animal proteins
(Petrucci.2011:1279).
Fibrous proteins provide structural integrity and strength for many types of
tissue and are the main components of muscle, hair, and cartilage. Other proteins,
usually called globular proteins because of their roughly spherical shape, are the
“worker” molecules of the body. These proteins transport and store oxygen and
nutrients, act as catalysts for the thousands of reactions that make life possible,
fight invasion of the body by foreign objects, participate in the body’s many
regulatory systems, and transport electrons in the complex process of
metabolizing nutrients (Zumdahl.2010:691).
The amino acids contained in protein are α-aminocarboxylic acids. Amino
acids contain amino (-NH2) and carboxylic (-COOH) groups. The structure of the
amino acids can be described as below with variations in the structure of these
monomers occurring in the (R) side chain
Amino acids will bond to form polyamides, the bonds formed are called peptide
bonds
The 20 amino acids can be assembled in any order, which makes possible
an enormous number of different proteins. This variety allows for the many types
of proteins needed for the functions that organisms carry out. The order or
sequence of amino acids in the protein chain is called the primary structure. We
indicate the primary structure by using three letter codes for the amino acids
where it is understood that the terminal carboxyl group is on the right and the
terminal amino group is on the left. For example, one possible sequence for a
tripeptide containing the amino acids lysine, alanine, and leucine
(Zumdahl.2010:693).
Peptides and proteins are polymers that are formed from amino acid units
through a peptide bond between an α-amino group from one amino acid and a
carboxyl group from another amino acid. This peptide bond will form an amide
group. The peptide-forming amino acids are referred to as residues. An example
of a peptide formed from glycine and serine is called glycylserine. For dipeptides
there are two possible combinations as shown below. Tripeptides can form
combined amino acid combinations in six ways. So the more amino acid residues
that make up the peptide, the more possible combinations of these amino acids
are. Peptide names start with the amino acid names from left to right
(Wardiyah.2016:194).
Amino acids are classified on the basis of polarity (distribution of electric
charge) of the R group. Group I: Nonpolar amino acids: These are glycine,
alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and
tryptophan. The R groups of these amino acids have either aliphatic or aromatic
groups and hence become hydrophobic (“water fearing”). Functions of Amino
Acids: Amino acids possess a variety of complex nitrogen-containing molecules.
The most prominent are the nitrogenous base components of nucleotides and the
nucleic acids (DNA and RNA). There are complex amino-acid derived cofactors
such as heme and chlorophyll. Heme is the organic group containing iron,
required for the biological activity such as the oxygen carrying haemoglobin and
the electron-transporting cytochrome. (Azad.2018:14 and 15).
Because many circular proteins are cysteine-rich, the oxidative folding
step in forming the correct connectivity of disulfide bonds is another key step in
their synthesis. However, forming the native disulfide bonds under oxidative
folding conditions remains empirical with unpredictable outcomes. Therefore, the
chemoselective method was introduced for the synthesis of cyclotides in 1997.
With a pair of hydrofluoric acid-stable cysteine-protecting groups such as the
acetamidomethyl group, it greatly reduces the number of disulfide isomers
formed. This method had been used in the synthesis of many cysteine-rich
peptides such as engineered protegrins and defensins (Tam.2012:27023).
The increase in protein protein content is in line with the increase in yield.
In the salting out process, the desired coagulation of protein is added to a salt
solution so that it increases the solubility (salting in) to its maximum point, then
decreases its solubility (salting out). When this process occurs there is competition
between protein and salt in attracting water molecules for the dissolving process,
then the interaction between protein and protein becomes more important.
The increase in protein content in protein coincides with the increase in yield. In
the salting out process, the desired coagulation of protein is added to a salt
solution so that it increases the solubility (salting in) to its maximum point, then
decreases its solubility (salting out). When this process occurs there is competition
between protein and salt to attract water molecules for the dissolving process,
then the interaction between protein and protein becomes more
important (Nurhayati.2018:29).
D. Apparatus and Chemicals
1. Apparatus
a. Beaker 500 mL (1 piece)
b. Beaker 100 mL (3 pieces)
c. Graduated cylinder 250 mL (1 piece)
d. Test tube rack (2 pieces)
e. Funnel (2 piece)
f. Spray bottle (1 piece)
g. Round flask 500 mL (1 piece)
h. Distillation flask (1 piece)
i. Test tube (10 pieces)
j. Graduated cylinder 25 mL (2 pieces)
k. Stir bar (1 piece)
l. Spatula (2 pieces)
m. Wood clamp (1 piece)
n. Cork plug (1 piece)
o. Bunsen burner (1 piece)
p. Capillary pipe (2 pieces)
q. Drop pipette (5 pieces)
r. Reflux tools (1 piece)
s. Stative and Clamp (1 piece)
t. Rough Cloth (1 piece)
u. Soft Cloth (1 piece)
2. Chemicals
a. Hydrochloride acid 10 % solution (HCl)
b. Hydrochloride acid 20 % solution (HCl)
c. Sodium Nitrite 5% solution (NaNO2)
d. Copper (II) sulphate 2%solution (CuSO4)
e. Aquadest (H2O)
f. Nitrite acid solution 10M (HNO3)
g. Crystal of Glycine powder (C5H5NO2)
h. Crystal of L-Tyrosine (C9H11NO3)
i. Crystal of Urea ((NH2)2CO)
j. Sodium hydroxide 10% solution (NaOH)
k. Crystal of Casein (C10H22O6N)
l. Crystal of Aspartic Acid (C4H7NO4)
m. Ice cube (H2O(s))
n. Litmus Paper
o. Aluminum foil (Al2O3)
p. Filter paper (C22H11O22)
q. Label
r. Matches
s. Tissue
t. Matches
u. Boiling stone
E. Work Procedures
1. Solubility and amphoteric properties
a. Glycine was added to 2 ml of water and tested using litmus.
b. The experiment was repeated using L-aspartic acid and L-tyrosine.
c. 1 ml of 10% NaOH solution was added to the suspension of 0.1
gram of L-tyrosine in 2 ml of water and the results were recorded.
d. A piece of litmus paper is put in the solution and added dropwise
until the solution is acidic.
e. The solution is stirred for 1 minute observe and record the results.
f. The solution is added another 10 drops of acid solution, the result is
recorded.
g. 0.1 gram of casein is put into a test tube. 2 ml of 10% NaOH and 5
ml of distilled water were added to the test tube.
h. The test tube is closed and shaken to form a colloid solution.
i. 2 ml of the solution was stored for further experiments.
2. Reaction with Nitrite Acid
a. 5 ml of 10% HCl solution is added to 0.1 gram of glycine in the test
tube.
b. In another test tube, added 5 ml of 10% HCl solution as a
comparison, the two tubes were cooled to 0 degrees Celsius in ice
water.
c. 1 ml of 5% NaNO2 solution was added in each tube. And the results
are recorded.
d. The casein solution was cooled in ice water then 1 ml of NaNO 2
solution was added, recorded
3. Biuret test
a. 0.5 grams of urea is put into a dry test tube and heated slowly until
the urea melts and forms gas.
b. Gas odors were recorded and tested with wet litmus paper at the
mouth of the test tube.
c. Heating is continued until gas formation stops and the rest begins to
solidify.
d. Then the test tube is cooled and the solids are dissolved into hot
distilled water.
e. The solution was filtered and 2 ml of 10% NaOH solution was
added, then 2-3 drops of 2% CuSO4 solution.
f. The solution is stirred and the color is observed. as a comparison, 0.5
gram of urea was dissolved in 3 ml of water, 2 ml of 10% NaOH
solution was added, then 2-3 drops of 2% CuSO 4 were added then
the solution was compared to the results of the observation.
g. 2 ml of distilled water and 2 drops of 2% CuSO4 solution were added
to the 2 ml of casein solution provided.
h. The solution is stirred and observed.
4. Xanthoproteat test
a. 0.1 gram of casein was put into a test tube.
b. 2 ml of nitric acid was added and heated slowly.
c. Observed color.
d. The reaction mixture was cooled and neutralized with 10% NaOH.
e. Then add a little excess base.
f. The color of the solution is noted if a change occurs.
5. Hydrolysis of protein
a. The apparatus for reflux is prepared with a 100 ml flask.
b. 0.5 gram of casein was added to the flask, 2 ml of 20% HCl solution
was added and reflux was carried out for 30-40 minutes.
c. After which the reaction mixture was cooled to room temperature.
d. A portion (5 ml) of the solution was cooled in ice water for
comparison.
e. The results were compared. to the other part, the solution is
neutralized with 10% NaOH, 3 ml 10% NaOH is added and 2 drops
of 2% CuSO4 solution.
f. The solution is heated into a water bath.
g. The results were compared with previous experiments.
6. Separation of amino acids by paper chromatography
a. A sheet of Whatman filter paper measuring 10.6 cm 2 is used, holding
the edges of the paper so as not to leave finger marks.
b. 2 lines are drawn across the paper with a pencil, 2 cm from the top
and bottom edges. from now on the paper should only be handled
outside that part of the line.
c. Crosses are made each 2 cm apart on the transverse line down.
d. Mark with A (for aspartic acid), T (tyrosine), F (phenylalanine) and
C (mixture of the three amino acids). A different capillary pipette
was used for each sample, small, dripped and rounded (1 mm
diameter) 0.1 M aspartate solution on T and D in the same manner
for tyrosine and phenylalanine. At C for mixed blemishes of all
three.
e. The stains are allowed to dry and then stained again in order to get
more material to identify them.
f. Re-dried. filter paper is placed in a 500 ml erlenmeyer around the
mouth, and attached with one piece of tape. Ensure that the paper is
perpendicular to the base of the erlenmeyer. through a long neck
filter funnel, 80% phenol solution is put in fresh water.
g. In such a way that the stain is not submerged (on the surface) of the
solvent.
h. The funnel is removed carefully without touching or dripping the
solvent paper. the mouth of the tube is closed, the solvent figure is
allowed to rise up the paper to the upper limit line (approximately 1-
1.5 hours).
i. The paper is removed with a pair of tongs, the excess phenol is
washed onto the paper with a seton stream from a spray bottle until
dry.
j. Chromatography atomizer is used, the paper is slowly sprayed
evenly, so that the paper becomes moist but not dripping with
ninhydrin solution.
k. Hang the paper in the air to dry for about 15 minutes. colored amino
acid stains will appear.
l. The stains were circled with a pencil, then the distance from the
starting line to the center of each spot was measured, and e marks
indicated the final surface of the solvent.
m. Calculate the value of Rf (Rf is the distance between the spots
divided by the distance of the solvent) for each amino acid, note
whether the amino acids in the mixture are well separated, and put
accordingly (compare the Rf).
F. Observation Result
1. The solubility and amphoteric properties
No Activity Result
.
a. Glycine crystal + 2 mL H2O shake
→
Soluble in water colorless
solution.
3. Buret test
No Activity Result
.
1. 0,5gram urea heat
→
White (melt)
Litmus paper has
Testing with litmus paper heat
→
cool
changed to blue
+ hot aquades filter
→ White solution
+ 2 mL NaOH 10% White solution
Blue solution
0,5 gram urea + 3 mL H2O + 2 mL NaOH
10%
+ 2-3 drops CuSO4
2. 2 mL casein + 2 mL H2O + 2 drops CuSO4 Purple solution
shake
4. Xanthoproteate test
No Activity Result
.
1. 0,1 gr casein + 2 mL concentrated HNO 3 Orange yellow
heat let it
→
Orange solution
Cool + 2 mL NaOH 10%
5. Protein hydrolysis
No Activity Result
.
1. 0,5 gram casein + 2 mL HCl 20% Colorless solution
Reflux for 30 minutes. Make the solution Brown solution
into two test tube
a. Brown solution
5 mL solution chill with
→
ice
b. Purple solution
In room temperature, solution + 3 mL NaOH
10% solution + 2 drops CuSO4
c. white precipitation +
Heated
purple solution
G. Discussion
Asam amino mengandung suatu gugus amino yang berada dalam bentuk
kation amonium dan gugus karboksil yang berada dalam bentuk anion
karboksilat. Sehingga asam amino mengandung gugus yang bersifat basa dan
gugus bersifat asam dalam molekul yang sama. Suatu asam amino mengalami
reaksi asam-basa internal yang menghasilkan suatu ion dipolar, yang juga disebut
zwitterion. Karena strukturnya ini maka asam amino tidak selalu bersifat seperti
senyawa-senyawa organik. Asam amino mempunyai titik leleh yang tinggi (di
atas 200 0C), tidak larut dalam pelarut organik tetapi larut dalam pelarut polar
(wardiyah.2016:190).
1. Kelarutan dan sifat amfoterik
Percobaan ini bertujuan untuk mengetahui kelarutan dan sifat amfoter dari
tiga jenis asam amino yaitu glisin, L-aspartat, L-tirosin. Ketiga bahan uji
ditambahkan dengan akuades. Penambahan akuades ini bertujuan untuk menguji
tingkat kepolaran jenis asam amino yang diujikan. Apabila suatu zat bersifat
polar, maka zat tersebut dapat larut dalam air. Begitupun sebaliknya. Hasil yang
diperoleh pada glisin yaitu larutan bening dan sangat mudah larut dalam air. Hal
ini dikarenakan
glisin merupakan
asam amino yang
paling sederhana
sehingga glisin
mudah
menyesuaikan dengan berbagai situasi. Pada glisin juga kebebasan gugus
aminanya lebih besar dibandingkan dengan karboksil. maka kedua gugus gugus
amin dan karboksil di dalam asam amino akan saling bereaksi menghasilkan ion
zwitter. oleh karena itu struktur dipolar ini maka glisin mudah larut di dalam air.
Setelah mengamati perubahan yang terjadi pada glisin, selanjutnya menguji
pH-nya, menggunakan kertas lakmus merah hasil yang diperoleh yaitu kertas
lakmus merah tetap berwarna merah yang berarti bersifat asam. Hal ini
menunjukkan bahwa glisin bersifat amfoter, artinya dapat bersifat asam jika dalam
larutan asam dan basa dalam larutan basa. Adapun persamaan reaksinya yaitu :
(Natzir,2016: 15)
Percobaan kedua yaitu menguji sifat amfoterik menggunakan L-tirosin dan
kasein. L-tirosin dari percobaan pertama ditambahkan dengan NaOH
menghasilkan larutan bening dan terdapat endapan putih. Penambahan NaOH
berfungsi untuk sebagai akseptor proton sehingga menyebabkan larutan bersifat
basa. Larutan kemudian diuji pH-nya dengan kertas lakmus merah, dan hasilnya
yaitu kertas lakmus berubah warna menjadi biru, yang berarti larutan bersifat
basa. Selanjutnya, larutan ditambahkan dengan HCl yang berfungsi untuk
memberikan suasana asam pada larutan, dan menguji pH-nya, dan hasilnya yaitu
kertas lakmus biru tetap berwarna merah yang berarti larutan bersifat asam. Jadi
dapat disimpulkan bahwa L-tirosin bersifat amfoter karena dapat bereaksi dengan
larutan asam dan larutan basa. Hal ini sesuai dengan teori yang menyatakan
bahwa asam amino tertentu bersifat amfoterik yang dikarenakan oleh keberadaan
gugus –NH2 atau –COOH yang bebas, dan akan bereaksi pada penambahan asam
atau
basa Reaksi yang terjadi yaitu :
(Larutan
berwarna bening
(Lakmus
biru→biru)
Dan terdapat endapan putih)
(Natzir,2016: 15)
Percobaan ketiga yaitu menggunakan kasein yang dilarutkan dalam air
diperoleh larutan berwarna putih kemudian menambahkan larutan NaOH yang
berfungsi untuk memberikan suasana basa. Hasil yang diperoleh setelah
penambahan air yaitu larutan keruh yang menandakan kasein sukar larut Hal ini
sesuai dengan teori yang menyatakan gugus R yang terdiri dari banyak atom
karbon atau bersifat aromatik, maka asam amino sukar larut dalam air
(Natzir,2016: 15)
2. Reaksi dengan Asam Nitrit
Tujuan percobaan ini adalah untuk mengetahui adanya gugus amin bebas
pada asam amino dan protein yang ditandai dengan terbentuknya gas N2. Pada
Percobaan ini menggunakan glisin dan kasein sebagai bahan uji. Percobaan
pertama yaitu mereaksikan glisin dengan HCl. Penambahan HCl bertujuan
memberikan suasana asam yang akan bereaksi dengan NaNO2 membentuk HNO2.
Kemudian menambahkan larutan NaNO2 yang menghasilkan larutan bening dan
terdapat gelembung gas N2 yang banyak. NaNO2 dalam hal ini berfungsi sebagai
penyedia ion nitrit. Adanya gelembung gas N2 yang terbentuk disebabkan karena
gugus amin yang ada pada glisin bereaksi dengan asam nitrit, dengan mekanisme
reaksi:
(Natzir,2016: 18)
Sebagai pembanding, HCl direaksikan dengan NaNO2 tanpa menggunakan kristal
glisin dan diperoleh larutan bening dan terdapat gelembung gas. Hal ini tidak
sesuai dengan teori. Dimana teori mengatakan tidak terdapat gelembung
dikarenakan tidak adanya gugus amin pada HCl, karena HCl tidak termasuk asam
amino. Adapun reaksinya yaitu
HCl + NaNO2 HNO2 + NaCl
(asam (Natrium (asam (garam)
klorida) nitrat) nitrat)
(Natzir,2016: 18)
Hal ini membuktikan bahwa kasein tidak bereaksi dengan asam nitrit
karena gugus amin yang bebas hanya satu sedangkan rantainya panjang, sehingga
gas N2 yang dilepaskan sangat sedikit sehingga tidak terlihat. Adapun reaksinya
yaitu:
3. Uji Biuret
Pengujian ini bertujuan untuk mengetahui adanya ikatan peptida pada
protein yang ditandai dengan terbentuknya larutan berwarna merah ungu.
Percobaan pertama menggunakan urea yang dipanaskan hingga berubah fasa
menjadi larutan bening kekuningan. Larutan ini bersifat basa,
yang ditandai dengan perubahan warna pada kertas lakmus
merah menjadi warna biru.
Larutan kemudian dilarutkan dengan air panas untuk melarutkan padatan
urea dan menghasilkan larutan bening. Selanjutnya larutan disaring sehingga
diperoleh larutan bening yang kemudian ditambahkan NaOH yang berfungsi
memberikan suasana basa pada larutan agar dapat terjadi perubahan warna dan
mencegah adanya endapan Cu(OH)2 yang akan memecah ikatan peptida pada saat
ditambahkan CuSO4. Peubahan yang terjadi yaitu larutan berwana biru tua.
(Natzir,2016: 20)
Fungsi penyaringan adalah untuk memisahkan endapan dari filtratnya. Hal
ini tidak sesuai dengan teori dimana menurut teori larutan berubah warna menjadi
merah ungu yang menandakan adanya ikatan peptida. Menurut teori bahwa tujuan
dari pengujian biuret yaitu untuk menunjukkan adanya
ikatan peptida pada protein dengan cara menambahkan
CuSO4 yang akan bereaksi dengn biuret atau senyawa lain
dengan struktur yang sejenis, menghasilkan larutan berwarna
ungu atau merah muda Reaksi pada saat urea dipanaskan
yaitu:
Sebagai pembanding, urea dilarutkan dengan air kemudian ditambahkan
dengan NaOH menghasilkan larutan bening dan menambahkan CuSO4 yang
menghasilkan larutan biru yang menandakan bahwa tidak terdapat ikatan peptida.
Hal ini disebabkan karena pada percobaan ini urea tidak dipanaskan sehingga
tidak terbentuk biuret yang dapat menunjukkan uji positif berupa warna merah
ungu.
Percobaan selanjutnya, larutan kasein direaksikan dengan CuSO4
menghasilkan larutan berwarna biru keunguan. Hal ini menandakan terdapat
ikatan peptida, karena larutan kasein yang digunakan adalah larutan yang telah
dicampurkan dengan NaOH pada percobaan sebelumnya dan kasein juga
merupakan protein yang tersusun dari beberapa asam amino yang membentuk
ikatan peptida sehinga meskipun tidak dipanaskan akan tetap berwarna ungu.
Reaksinya yaitu:
(Natzir,2016: 21)
4. Uji Xanthoproteat
Pengujian ini digunakan untuk mengetahui adanya gugus benzena pada
protein. Hasil positif pengujian ini ditandai dengan larutan berwarna jingga yang
menandakan cincin aromatik telah mengalami nitrasi menjadi benzena. Pada
percobaan ini menggunakan kasein yang ditambahkan HNO3,
kemudian dipanaskan menghasilkan larutan berwarna kuning. Larutan
kemudian ditambahkan NaOH yang berfungsi untuk menetralkan dan selanjutnya
menambahkan NaOH berlebih sampai bersifat basa, karena dalam bersifat basa
reaksi xanthoproteat akan semakin jelas. Pada saat ditambahkan NaOH berlebih
larutan berwarna kuning keruh.
Hal ini menandakan terjadi nitrasi pada cincin benzena yang terdapat pada
kasein. Hal ini sesuai dengan teori bahwa bila protein ditambahkan asam nitrat
kemudian dipanaskan maka akan terbentuk larutan berwarna juning. Adapun
reaksinya yaitu
(Natzir,2016: 22)
5. Hidrolisis protein
Hidrolisis protein adalah penguraian protein menjadi monomer –
menomernya. Percobaan ini menggunakan kasein yang ditambahkan dengan HCl
kemudian direfluks yang bertujuan untuk memutuskan ikatan peptida sehingga
terurai menjadi asam – asam aminonya. Hasil setelah dipanaskan diperoleh
berwarna coklat.
Azad , Sabina . 2018. "Amino acids: Its types and uses." International Journal of
Clinical and Diagnostic Pathology (ISSN: 2617-7226 ).
Nurhayati, Mappiratu, and Musafira. 2018. "Pembuatan Ponsentrat Protein dari
Biji Kelor (Moringa oleifera l.) dan Analisis Profil Asam Amino."
Kovalen (ISSN: 2477-5398).
Petrucci, Ralph H. , F. Geoffrey Herring, Jeffry D. Madura, and Carey
Bissonnette. 2011. General Chemistry Principles and Modern
Applications. Canada: Pearson Canada Inc.
Tam, James P., and Clarence T. T. Wong. 2020. "Chemical Synthesis of
Circular." The Journal of Biological Chemistry Vol. 287, NO. 32.
Wardiyah. 2016. Kimia Organik. Jakarta Selatan: KEMENKES.
Zumdahl, Steven S. , and Donald J. DeCoste. 2010. Introductory Chemistry a
Fundation. USA: Brooks/Cole cengage Learning.