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Group#5 2APH Mia Sheleth V. Manalo, !"c#$%, Jefferson B. Manuel,


Shirmagne Fatima E. Manugas, Jessieca I. Ongsitco

& 


Gluten can be de¿ned as the rubbery mass that remains when wheat dough is washed to remove
starch granules and water-soluble constituents.Gluten was extracted and hydrolyzed by base and
subjected to different qualitative tests. Paper chromatography had been done to determine and
analyze the different amino acid components of the protein. Results were all positive indicating
that gluten contains many amino acids in it.

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Proteins can be considered as polymers of determine tyrosine and tryptophan residues,

amino acids. Amino acids are linked by respectively. The Nitroprusside test is used

covalent peptide bond into linear chain, to find out if sulfur-containing amino acids

which is called peptide or polypeptide chain. are present; test for amides is used to detect

The properties common to all amino acids R-groups of asparagine and glutamine. Test

are due to the relative special arrangements for amides is used to detects R-groups of

of the carboxyl and amino groups. The asparagines and glutamine.

physical and chemical properties unique to


each amino acid are the result of the The objective of the experiment is to
determine the amino acid components of
structure and chemical properties of the R gluten which can be done by partition paper
group. Several amino acids combine to form chromatography which is widely employed
for the separation of amino acids. The
peptide bonds. Proteins contain polypeptide solvent migrates along a strip of paper and
units (several peptide units). When a protein carries amino acid dissolved in it.
is hydrolyzed, it breaks down into smaller
units (tri and dipeptides), eventually forming $  (
amino acids. Specific reactions are used for Alkaline Hydrolysis of Intact Protein
the purpose of identifying amino acids and
Gluten extracted from wheat flour was
proteins in biological media, for qualitative
hydrolyzed by base after it was tested with
and quantitative analysis. The biuret test is different qualitative color tests. The alkaline
used to detect the presence of peptide bonds hydrolysis of protein undergone by adding
10 mL of 4 M NaOH to 0.5 g isolated
while the Ninhydrin reaction is a typical test
protein in a hard glass test tube and then
for an Į-amino acid. Xanthoproteic test autoclaved for 5 hours (15 psi). After the
detects side chains of aromatic amino acids appearance of the mixture was noted, ten
mL of distilled water was added and was Separation and Identification of Amino
transferred into a 250-mL beaker. The Acid by Paper Chromatography
mixture was neutralized with 1 M HCl, and
was tested with red and blue litmus paper to A 1.5 cm margin from one edge of the paper
check if it was already neutral. And the was measure and drawn lightly with a pencil
on the prepared 12 x 15 cm Whatman filter
neutralized mixture was used as a sample for
characterization tests and chromatography. paper # 1. Five equidistant points were
labeled on the line for application of the
* alitative Color Reactions chosen samples. The samples were air-dried
between applications by a capillary tube.
The gluten hydrolysate was tested with The paper was then rolled into a cylinder
different characterization reagents available
without overlapping, and then stapled. It was
in the laboratory namely: Biuret, Ninhydrin, then placed in a pre-equilibrated chamber,
Xanthoproteic, Millon¶s Hopkins ± Cole and covered to allow the solvent to ascend
tests, and Test for Amides. Six test tubes undisturbed. When the solvent reach at least
were prepared for each of the test reactions. 2 cm from the other end, the paper was
Each test tube consists of 1 mL of distilled removed and the solvent front was marked
water added to 0.5 mL of hydrolyzed immediately with a pencil line. The paper
samples.
was air-dried and sprayed lightly with 1%
In Biuret test, 2-3 drops of 0.1 M CuSO4 ninhydrin reagent. Then, it was oven-dried
solution was added to a mixture of the and the presences of blue, purple, or yellow
sample and 20 drops of 2.5 M NaOH. The spots were noted. The spots were encircled
test tube was shaken and the color changes with a pencil and the Rf values were
in the solution were observed. For computed.
Ninhydrin test, 6-10 drops of 0.1%
ninhydrin solution was placed into the
sample and then heated in a boiling water
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bath. The appearance of violet coloration
was taken note of. In the Xanthoproteic test, * alitative Color Tests for Hydrolyzed
10 drops of concentrated nitric acid and Gl ten
concentrated sodium hydroxide was slowly Table shows the comparison of the results of
added to the diluted sample and then mixed. color test reactions done in the hydrolyzed
Color changes after each addition was protein to that of tests done in intact protein.
observed. For the Millon¶s test, 5 drops of
Millon¶s reagent was added to the diluted |    
  

   
sample, and the color changes were noted.  
  

  

For the Hopkins-Cole test, 20 drops of
 c) *
Hopkins-Cole reagent was slowly added to

)
the sample, and then mixed. Concentrated
& Violet-blue Green ppt
sulfuric acid of about 20 drops was slowly in color
added along the side of the test tube, shaken, + Blue-violet Violet soln
and the color of the interference was taken in color
note of. Lastly, test for amides was done by ,+-) Orange in Yellow clear
adding 1 mL of 20% NaOH to 10 drops of color soln
the sample and placing the tube in a boiling $. Flesh/peach Flesh/peach
in color soln
water bath. Moistened red litmus paper was
-/ Violet- Violet ring
placed over the mouth of the tube to test the
black ring
evolution of gas during heating. Results   Red ù Blue Red ù Blue
were taken note of. !
Paper Chromatography Analysis of Gl ten
Millon¶s test is a test specific for tyrosine,
Characterization of gluten using paper the only amino acid containing phenol
chromatography yielded different results group. In this test, the phenol group of
compared with color tests of the gluten tyrosine is first nitrated by nitric acid in the
hydrolysate. Table 2 shows the Rf values of test solution. Then the nitrated tyrosine
complexes Mercury (I) and Mercury (II)
standards and the hydrolysate.
ions in the solution to form red precipitate or
|            
  red solution. And since gluten does not
contain tyrosine, both intact and hydrolyzed
 

protein when tested resulted negative in the
color reactions and chromatography.
! )
0+ -
 Standards Hydrolysate
The Hopkins-Cole test is specific for
% 0.51 0.3429 trypthopan, the only amino acid containing
 0.60 0.3571 an indole group. And gluten when tested
) 0.59 0.3714 resulted into an indole violet ring which
 0.67 0.3857 indicates the presence of trypthopan.
 0.73 0.4
Test for amides uses litmus paper to show
whether the protein contains amides.

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Chromatography is a technique that
Subjecting gluten in alkaline hydrolysis separates mixtures into their individual
which is composed of extreme temperature components for example: if we put black
caused the breaking down of peptide bonds. washable ink onto a tissue, the ink will
spread outwards from the place where we
Biuret test is used to look the presence of blotted it however, the various components
peptide bonds. Because biuret solution of the ink can't all move at the same speed
contains Cu ions which react with peptide as it spreads out - so the components will
visibly separate.
bonds, intact protein will yield a positive
result (violet solution) and a completely
hydrolyzed protein will display a negative As we can see from the results, Arginine,
result (green ppt). Histidine, Glycine, Alanine and Proline are
present.
Ninhydrin reacts with ammonia, a primary
amine, or a secondary amine (amino acids The values in the table has its discrepancy
have a primary or alpha amino group, except due to errors done while doing the
for proline which has a secondary amino
experiment.
group). They all turn purple/blue right away
upon heating with ninhydrin. The thing that is measured in
chromatography is the difference between
Some amino acids contain aromatic groups how far a substance (from the mixture)
that are derivatives of benzene. These
travels compared to how far the solvent
aromatic groups can undergo reactions such travels.
as the nitration of benzene ring with nitric
· Rf = (distance traveled by a
acid. This nitration, when used to identify substance) / (distance traveled by the
the presence of an activated benzene ring, is
solvent)
commonly known as the Xanthoproteic test,
because the product is yellow. The intensity
of the yellow color deepens when the The experiment then, successfully
reaction occurs in basic solution. Intact
characterized the different amino acid
protein resulted positive and since histidine
and proline are amino acids, hydrolyzed components of gluten by using the
protein yielded a positive result also.
qualitative color reaction tests and paper
chromatography.


 

The Biochemistry Department (2010).


Ê  
      
 
 Manila: University of Santo
Tomas.

³Gluten´ En.wikipedia.org/wiki/Gluten

http://www.cerlabs.com/experiments/10875
404480.pdf

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