The document describes 4 experiments related to analyzing proteins: 1) The ninhydrin reaction identifies amino acids by producing a purple color. 2) The xanthoproteic reaction detects aromatic amino acids in proteins by producing a yellow then orange color with nitric acid and sodium hydroxide. 3) The biuret reaction identifies proteins using copper sulfate which complexes with peptide bonds to form a blue-violet color in alkaline conditions. 4) The biuret method is used to quantitatively determine protein content by measuring absorbance of biuret complexes at 540nm and comparing to a standard curve.

1. 1
Lesson 1. Amino acid and Protein
1.1. Experiment 1 : Ninhydrin reaction
• Principle
- α-amino acids can be qualitatively and quantitatively estimated by their
reaction with ninhydrin.
- The reaction produces hydrindantin, ammonia, aldehyde, and carbon
dioxide
- Then hydrindantin react with ammonia and ninhydrin to make
Ruhemann's Purple.

2. Ninhydrin reaction
2

3. 1.1. Experiment 1 : Ninhydrin
reaction
Reagants:
- Amino acid solution
- Ninhydrin solution, 0.1%
- CuSO4 (copper II sulfate) solution, 5%
Equipment:
- Filter papers
- Oven
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4. 4
Procedures
- Take a filter paper
- Add one drop of amino acid solution to make a small circle in the
middle of the paper
- Dry the paper in oven
- Add one drop of ninhydrin solution in the circle
- Dry the paper in oven
→ Description and note down the color immediately
- Add one drop of 5% copper sulfate solution
- Dry the paper in oven
→ Description and note down the color immediately

5. 1.2. Experiment 2: Xanthoproteic
reaction
Principle:
- This reaction tests for the presence of aromatic amino acid
(benzene rings - Phe, Tyr, Trp) in proteins.
- Aromatic amino acids react with the concentrated nitric acid leads
to the nitration of the aromatic ring and formation of yellow nitro-
products (nitro derivatives) (This reaction need heating time)
When the strong alkali solution (NaOH) is added the colour of
obtained products turns from yellow to orange.
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6. 6

7. 1.2. Experiment 2: Xanthoproteic reaction
Reagants:
- Albumin solution
- HNO3 (nitric acid) concentrated solution
- NaOH (sodium hydroxide) solution, 10%
Equipment:
- Test tubes
- Pipets
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8. 1.2. Experiment 2: Xanthoproteic reaction
Procedures:
- Take 5 drops albumin solution in a test tube
- Add 5 drops of concentrated nitric acid
- Mix, put thermostat, see the change color, take out
- Observe the change of color (yellow)
- Add slowly 10% sodium hydroxide drop by drop and mixing. The
color will change in the alakine medium
- Note down the changes
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9. 1.3. Experiment 3: Biuret reaction
Principle
• The reaction takes place with compounds containing two or more
peptide bonds,
• It can be used for both qualitative and quantitative analysis of
peptides and protein.
• The blue-violet color of the Biuret reaction is due to the complexion
of copper (Cu2+) in alkaline solution with the peptide bonds in the
protein.
NaOH
Protein (Peptide bond) ------------------->
CuSO4
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10. 10

11. 1.3. Experiment 3: Biuret reaction
Reagants:
- Albumin solution, 1%
- CuSO4 solution, 1%
- NaOH solution, 10%
Equipment:
- Test tubes
- Pipets
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12. 1.3. Experiment 3: Biuret reaction
Procedures:
- Take 5 drops of a protein solution (albumin) in a test tube
- Add 5 drops of NaOH 10%
- Add 2 drops of CuSO4 1%
- Mix and observe the change of color
12

13. Experiment 4: Quantitative determination of
protein content by Biuret method
Principle: The Biuret complex can be quantitatively measured by
spectrophotometer (maximum absorbance at 540 nm)
Reagants:
Albumin solution 0.1%
Albumin solution (unknown concentration):
Distilled water
Biure reagant solution:
Equipment:
Test tubes
Spectrophotometer 13

14. Experiment 4: Quantitative determination
of protein content by Biuret method
Procedures:
Step 1: Standard curve plotting (Albumin 0,1%)
Label 6 test tubes as from 1 to 6 and place them in a test tube rack
Add to each tube the solutions in the following table:
Calculate the protein concentration in each tube of standard
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No Protein 0.1% (ml) Distilled water (ml) Biuret reagant (ml)
1 0 1.0 4
2 0.2 0.8 4
3 0.4 0.6 4
4 0.6 0.4 4
5 0.8 0.2 4
6 1.0 0 4

15. Experiment 4: Quantitative determination of
protein content by Biuret method
- Mix well and allow standing at room temperature for 30 min
- Read the absorbance for each tube at 540 nm
- Record your result
- Plot the standard curve using concentration of protein in standard tubes
at 540 nm.
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16. Experiment 4: Quantitative determination of
protein content by Biuret method
Step 2: Determination of protein content in a given sample
- Take 1 test tube and label it as 7
- Add 1 mL of a protein solution (albumin ?) with unknown protein
content
- Add 4 mL of Biuret reagant
- Allow standing at room temperature for 30 min
- Read the absorbance for the tube at 540 nm.
- Record your result
- Determine protein content based on the standard curve
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17. Experiment 4: Quantitative determination of
protein content by Biuret method
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