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20 Disember 2017

Biochemistry : Amino Acid and Protein




SIK1001/SHES1270
BIOLOGY PRACTICAL



EXPERIMENT :  PRACTICAL 5.3 ( AMINO ACID AND PROTEIN )
LECTURER      :  DR NUR AIRINA BT MUHAMAD



MUHAMMAD AMIRUL BIN AMIL ( SIJ17XXXX )
GROUP 1



1.1  Introduction
Proteins are the most versatile macromolecules in living systems and serve crucial functions in essentially all biological processes. They function as catalysts, they transport and store other molecules such as oxygen, they provide mechanical support and immune protection, they generate movement, they transmit nerve impulses, and they control growth and differentiation.
The term structure when utilized in relation to proteins, goes up against a substantially more intricate significance than it improves the situation small molecules. Proteins are macromolecules and have four distinct levels of structure – primary, secondary, tertiary and quaternary.
Because of the idea of the feeble connections controlling the three-dimensional structure, proteins are extremely delicate particles. The term local state is utilized to depict the protein in its most stable normal compliance in situ. This local state can be upset by various outer anxiety factors including temperature, pH, evacuation of water, nearness of hydrophobic surfaces, nearness of metal particles and high shear. The loss of auxiliary, tertiary or quaternary structure because of presentation to an anxiety factor is called denaturation. Denaturation brings about unfurling of the protein into an arbitrary or miscoded shape.
A denatured protein can have a significant distinctive movement profile than the protein in its local shape, as a rule losing natural capacity. Notwithstanding getting to be noticeably denatured, proteins can likewise shape totals under certain anxiety conditions. Totals are frequently delivered amid the assembling procedure and are commonly unwanted, to a great extent because of the likelihood of them causing unfavorable safe reactions when managed. 
 
Notwithstanding these physical types of protein debasement, it is additionally essential to know about the conceivable pathways of protein compound corruption. These incorporate oxidation, deamination, peptide-security hydrolysis, disulfide-security reshuffling and cross-connecting. The strategies utilized as a part of the handling and the detailing of proteins, including any lyophilization step, must be painstakingly inspected to anticipate debasement and to expand the soundness of the protein biopharmaceutical both away and amid sedate conveyance.
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2.1 Objectives
2.1.1 To observe physical and chemical properties of some common proteins
2.1.2 To physical and chemical tests to distinguish between groups of amino acids
2.1.3 To identify unknown proteins

3.1 Materials and Apparatus
Glysine , 0.5% of ninhydrin solution , nitric acid , 0.1% tyrosine solution , 40% of NaOH , Millon’s reagent, NaNO3 solution , 0.1% arginine solution , a-naphtol , bromine , casein , egg albumin , peptone , gelatin

4.1 Method
Ninhydrin Test
1) 1 ml of glysine solution was added to 2 drops of 0.5 % of ninhydrin solution and was boiled slowly for 2 minutes.
2) A blue or purple colour was gave a positive result. The amino acid/protein solution should be neutral to obtain a clearer result.
3) The concentration limit was determined by carrying out a set of serial dilutions until a negative result was obtained.
4) Results were recorded and discussed.

Xanthoproteic Acid Test
1) 1 ml of concentrated nitric acid solution was added to 1 ml of 0.1 % tyrosine solution.
2) The colour change was observed.
3) 40 % NaOH was added until the solution is basic.
4) The change in colour that occurs was recorded.
5) Results were recorded and discussed.

Millon’s Reaction
1) 5 drops of Millon’s reagent was added to 1 ml of tyrosine solution and the solution was heated in a water bath for 10 minutes.
2) The solution was cooled to room temperature and 5 drops of sodium nitrite was added.
3) A red colour will be observed after obtaining positive results
4) Results was recorded and discussed.



Sakaguchi Reaction
1) 1 ml of NaOH (40 %) was added with 3 ml 0.1 % arginine solution and  2 drops of α-naphtol (1 % in ethanol) was added.
2) The solution was mixed well and 4 5 drops of bromine was added
3) The colour formed was observed.
4) Results were recorded and discussed.
Determination of Solution A and B
1) Two protein solutions A and B were given.
2) All the tests above were tested and explained in detail the characteristics of the amino acid present in both solutions.








5.1 Result
Ninhydrin Test :
For a serial dilution of Glycine :
Solution
Observation ( colour intensity )
Indication
Stock
Dark purple

Diluted X10
Slight purple

Diluted X100
Pale purple

Diluted X1000
Very pale purple

Diluted X10000
Colourless



For other solutions :
Types of solution
Observation
Indication
Glysine
Purple colour
Positive
Peptone
Colourless
Negative
Gelatin
Colourless
Negative
Arginine
Colourless
Negative
Casein
Colourless
Negative
Albumin
Colourless
Negative
Solution A
Colourless
Negative
Solution B
Colourless
Negative

Xanthoproteic Acid Test
Solution
Before adding nitric acid
After adding nitric acid
Adding NaOH (40%)
Indication
Glysine
Colourless
Colourless
Colourless
Negative
Peptone
Colourless
Yellow
Orange
Positive
Gelatin
Colourless
Colourless
Colourless
Negative
Casein
Colourless
Colourless
Colourless
Negative
Tyrosine
Colourless
Very light yellow
Orange
Positive
Solution A
Slightly cloudy
Cloudy and light yellow
Orange solution with orange precipitate
positive
Solution B
Colourless
Colourless
Colourless with very minute amount of orange precipitate
Negative









Millon’s Reaction
Solution
Observation
Indication
Glysine
Colourless
Negative
Peptone
Red colour formed
Positive
Gelatin
Colourless
Negative
Arginine
Colourless
Negative
Casein
Colourless
Negative
Tyrosine
Red colour formed
Positive
Albumin
Red colour formed
Positive
Solution A
Red colour formed
Positive
Solution B
Colourless
Negative


Sakaguchi Reaction
Solution
Observation
Indication
Glysine
Red colour formed
positive
Peptone
Pale yellow ( unchanged )
Negative
Gelatin
Pale yellow ( unchanged )
Negative
Arginine
Pale yellow ( unchanged )
Negative
Casein
Pale yellow ( unchanged )
Negative
Albumin
Red colour formed
Positive
Tyrosine
Yellow  ( unchanged )
Negative
Solution A
Red colour formed
Positive
Solution B
Pale yellow ( unchanged )
Negative

Determination of Solution A and B
Solution A : Albumin
Solution B : Caesin






6.1 Discussion
Based on the experiment, Amines (including α-amino acids) react with ninhydrin to give a dark purple product. The α-amino acids typically give a blue-purple product. However, a secondary amine, gives a yellow-orange product. It’s ruined to detect α-L-amino acids and also be used also to detect free amino and carboxylic acid groups on proteins and peptides. All amino acids that have a free amino group will give positive result (purple color) , while not free amino group (proline)will give a (yellow color). Ninhydrin then condenses with ammonia and hydrindantin to produce an intensely blue or purple pigment, sometimes called ruhemann's purple.
The Xanthoproteic Acid Test is used to differentiate between aromatic amino acids
which give positive results and other amino acids. Amino acids containing an
aromatic nucleus form yellow nitro derivatives on heating with concentrated HNO3.
The salts of these derivatives are orange in color. Concentration HNO3 reacts with the aromatic rings that are derivatives of benzene giving the characteristic nitration
reaction. Amino acids tyr. and typ. contain activated benzene rings which are easily nitrated to yellow colored compounds. The aromatic ring of phenyl alanine dose not
react with nitric acid despite it contains a benzene ring, but it is not activated, therefore it will not react.
Millon’s reaction is specific for tyrosine, the only amino acid containing aphenol group, a hydroxyl group attached to benzene ring. All phenols (compound having benzene ring and OH attached to it) give positive results in Millon’s test. The phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then the nitrated tyrosine complexes mercury ions in the solution to form a brick-red solution or precipitate of nitrated tyrosine, in all cases, appearance of red color is positive test.
Sakaguchi test is a specific test for detection of amino acid containing gauanidium group
[R-NH-C= (NH2)2+-NH2]. In other words it’s a test for guanidines, for instance, arginine. In alkaline solution, arginine react with α-naphthol and sodium hypobromite /chlorite as an oxidize agent, to form red complexes as a positive result. We managed to found out several factors that caused the failure of our test which are :
          The reagents that have been used are already expired
          The apparatus that have been used was contaminated with the old solutions that may interfere the result of the experiments
          Using the wrong solution for the tests as all of us know that almost all the solutions are colourless



7.1 Conclusion
In conclusion, Ninhydrin (triketohydrindene hydrate) is an oxidating agent which leads to the oxidative deamination of alpha-amino groups. It is very vital for the detection and the quantitative analysis of amino acids. Next, xanthoproteic  test is used to determine the presence of tyrosine, trypthopane, and phenylalanine to the protein. Amino Acid Tyrosine (Hydroxyphenylalanine) and other phenolic compounds give this reaction. Millon’s test is used for the detection of amino acid tyrosine. The Sakaguchi test is a chemical test used for distinguishing the presence of arginine in proteins. Sakaguchi reagent consists of 1-Naphthol and a drop of sodium hypobromite .

8.1 References
·         http://www.particlesciences.com/news/technical-briefs/2009/protein-structure.html
·         https://www.ncbi.nlm.nih.gov/books/NBK21177/
·         Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J (2004). Molecular Cell Biology (5th ed.). New York, New York: WH Freeman and Company