The purpose of this experiment is to obtain information about structural of a compound with some tests these are biuret ,ninhydrine ,xanthoprotic and heavy metal group by observing colour changes or not.
Casein is a protein that is found in milk and used as a binding agent. Technically, phosphoproteins, collections of proteins bound to something containing phosphoric acid.[1]
Albumin is an umbrella term for a type of protein which is water soluble. Albumin can be found two of the most familiar examples that are in egg whites and in human blood. Albumins are an important class of protein, and they are vitally important to health and well being for many organisms. Many plants and animals contain or secrete albumin. [2]
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Tyrosine is a nonessential amino acid the body makes from another amino acid called phenylalanine. It is a building block for several important neurotransmitters, serotonin, dopamine etc. Neurotransmitters help nerve cells communicate and influence mood. Tyrosine also helps produce melanin (the pigment responsible for hair and skin color) and helps in the function of organs responsible for making and regulating hormones. It is involved in the structure of almost every protein in the body.[3]
Protein Testing:
Biuret Test: The Biuret Test is a general test to detect peptide bond or not. When protein that includes peptide bond reacts with copper(II) sulfate (blue), the positive test is the formation of a violet colored complex.
The Biuret Test works for any compound containing two or more of the following groups.
Ninhydrin Test: The Ninhydrin Test is a test for amino acids and proteins to detect free -NH2 group. When such an -NH2 group reacts with ninhydrin, a purple-blue complex is formed.
Xanthproteic Test: Phenyl group containing an activating group can be nitrated it produces as a yellow product.
The production of a yellow colored product provide by the addition of nitric acid and this test works for the presence of tyrosine or tryptophan in a protein.The addition of strong base will deepen the color to orange. The yellow stains on the skin caused by nitric acid are the result of the xanthoproteic reaction.
Heavy Metal Ions Test: Heavy metal ions precipitate proteins by cross-linking free amino groups and carboxylate groups.
Ions commonly used for testing for the presence of proteins include Zn2+, Fe3+, Cu2+, Sb3+, Ag1+, Cd2+, and Pb2+.
Among the metal ions, Hg2+, Cd2+, and Pb2+ have very high toxicity. They cause serious damage to proteins (especially enzymes) by denaturing them. [4]
APPARATUS
Dropper
Test tubes
Heater
%2Casein
%2Albumin
%2Gelatin
%2Glycine
%2Trypthophan
NaOH
Copper sulfate
Ninhydrine reagent
Nitric acid
Milk
Silver nitrate
Iron nitrate
Sodium nitrate
Water Bath
PROCEDURE
Biuret Test
Firstly,%2 solutions ;glycine,gelatin,albumin,thyrosine and casein were prepared.
These tubes were labelled as 1,2,3,4 and 5
1ml of each solution except casein and put caserin with 1 ml water.
7-10 drops NaOH and 2 drops copper sulfate solution on each of the samples to be tested.
Observations were recorded.
Ninhydrine Test
New test tubes were labelled again.
7-10 drops of ninhydrin reagent were added to each test tube and heated in water bath approximately 5 min.
Observations were recorded.
Xanthoproteic Test
New test tubes were labelled
10-15 drops of nitric acid were added to each test tube and heated in water bath approximately 10min.
Observations were recorded.
Heavy Metal Precipitation
1.2ml milk were put into glycine ,gelatine and albumin labelled as 1,2,3
2.10 drops of %5silver nitrate were added to glycine ,10 drops of iron(III) nitrate to gelatine and 10 drops of sodium nitrate to test tube albumin.
3.These steps were repeated for %2 albumin.
4.Finally,observations were recorded.
OBSERVATIONS
In this experiment,we placed new test tubes into rack by labelled each with number.The reason of that is to apply some tests to each one.These tests are biuret ,ninhydrine ,xanthoproteic and heavy metal tests.Casein,albumin ,gelatin ,glycine and tyrosine were tested with biuret test.Firstly inside blue layer with casein and then light purple at top of tube and blue layer.In albumin,the purple colour diffused in test tube ,and then purple at top and purple layer at bottom. With gelatin ,at top is purple as a first and then purple particles were also observed.For glycine ,blue appeared as homogeneous like dissolved.And then at top is blue ,at bottom like green.Final result for biuret test is for tyrosine.At the beginning, blue layer was remained in suspense and then green ,brown particle was observed at bottom.
Secondly,ninhydrine test results were recorded for each one.With casein,we observed light purple like grey.With gelatin ,glycine and also tyrosine dark purple.With albumin,the color appeared like dark purple due to heater.
Thirdly,the other test is xanthoprotic test to detect easily phenyl group.With casein ,opaque turned into yellow particles.With gelatin and glycine ,the colour is so light yellow like colourless.With tyrosine ,yellow color turned into brown in time.Finally,albumin appeared as cloudy white and then turned into yellow particles.
Final test is heavy metal test that gives information about structure.Milk with silver nitrate turned into pink due to density.With iron and sodium nitrate ,there are no changes.
RESULTS
In this experiment, we learned some test to obtain information that we searched.For example,Biuret test is help to give information about peptide bond or not.If there is a peptide bond ,the color is observed as purple.If the colour is blue,it means no rxn there.Casein ,albumin ,gelatin,glycine and tyrosine were tested.Casein,albumin and gelatin gave purple color and these ones have peptide bond.Because these are proteins that have peptide bonds.However, glycine and tyrosine are not proteins one of them is aminoacid the other is enzyme.Therefore they did not give rxn not observed purple color as we expected.
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With ninhydrine test,except casein,the others gave pozitive rxn appeared as purple color.This test is to help information about free amino group primary or secondary.The other test is xantroprotic test.It gives information about phenyl group.Aromatic group one is yellow.Nitric acid is used to test it.This reaction is also endothermic rxn.Casein and tyrosine gave reaction and observed color changes.The final test is heavy metal test.It helps to give information about structure of a compound.If protein is tested with heavy metal,colour changes are observed also.It is tested with silver ,iron and sodium nitrate also.Silver helps to distrupt the structure that means colour change.With silver,the color turns into pink.
DISCUSSION
The purpose of this experiment is to learn which test why is used.Biuret test is to detect there are peptide bond or not.Normally,biuret test can give pozitive result that means purple color with proteins.In our experiment,casein albumin and gelatin give (+) result however glycine and tyrosine not due to aminoacid or enzyme.Our results are also related to as we expected.Ninhydrine test was applied to same prepared samples.The aim of this test is to detect free aminogroup (-NH2) and to decide primary or secondary group according to the colour.We used heater in this part.The reason of that reaction become a reality due to endothermic.At this part,except casein the others could give (+)result.Glycine that we learned previous experiment can have free aminogroup (primary).With casein,the others colour turned into dark purple.
The other test is xandroprotic test that is used to detect phenly group.It helps to discriminate aromatic structure from others.For casein ,the colour turned into yellow,for tyrosine ,the colour turned yellow to orange like brown.Therefore ,two of them could give pozitive result.As a final test ,heavy metal test was applied to prepared samples.This test shows which sample can distrupt with which one.Silver ,iron and sodium nitrate were used to detect it.Silver can distrupt protein structure.We understood that colour changes.The color turns into pink and the others did not change for milk (nesquick).
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