72. Quantification of proteins by biuret reaction and Ellmann-method

Question 1

I. Precipitation
1. What is denaturation?

Answer 1

loss of native tertiary structure (3D shape of a polypeptide)

Question 2

I. Precipitation
2. What is precipitation?

Answer 2

loss of protein solubility

Question 3

I. Precipitation
3. What is the relationship between denaturation and precipitation?

Answer 3

• Denaturation exposes the hydrophobic side-chains to the surface, and causes aggregation which leads to precipitation.
• Precipitation can occur without denaturation, and vice versa

Question 4

I. Precipitation
4. Why is the determination of the total protein content of a sample important? Give an example?

Answer 4

• The determination of the total protein content of a sample is very important, because it helps to quantify the efficiency of protein purification.
• For example, urinary protein (proteinuria) can be detected in a person with a kidney disease. In diabetes mellitus, proteinuria can be a sign of nephropathy (kidney disease).

Question 5

I. Precipitation
5A. The role of sulfosalicylic acid (SSA)

Answer 5

The strong organic acid, sulfosalicylic acid (SSA), is a reagent which is able to precipitate proteins of low concentrations in an irreversible manner

Question 6

I. Precipitation
5B. What is the mechanism of sulfosalicylic acid (SSA)?

Answer 6

• With its negative charge, SSA binds to the positively charged moieties of the proteins and due to its aromatic ring, it forms an apolar layer on the surface resulting in decreased water solubility and precipitation of proteins.
• The proteins turn the sample “milky” with low amounts, and form granules which can even sink
  to the bottom of the tube for higher levels of protein.
• The degree of turbidity is graded from a trace amount to a 4+ reaction, and the determination of protein concentration is semiquantitative.

Question 7

I. Precipitation
5C. Give the SSA test results based on this photo

Answer 7

Question 8

I. Precipitation
5D. Give the description of SSA test result “negative”

Answer 8

• No noticeable turbidity/ cloudiness
• Clear from top to bottom of tube

Question 9

I. Precipitation
5E. Give the description of SSA test result “trace”

Answer 9

• Using bright light, turbidity is barely visible

Question 10

I. Precipitation
5D. Give the description of SSA test result “negative”

Answer 10

• No noticeable turbidity/ cloudiness
• Clear from top to bottom of tube

Question 11

I. Precipitation
5F. Give the description of SSA test result “1+”

Answer 11

Turbidity is considerable, but individual granules not visible

Question 12

I. Precipitation
5G. Give the description of SSA test result “2+”

Answer 12

Granulation of particles are visible but no large clumps (flocculation) are visible

Question 13

I. Precipitation
5H. Give the description of SSA test result “3+”

Answer 13

Considerable turbidity with both granulation and flocculation

Question 14

I. Precipitation
5I. Give the description of SSA test result “4+”

Answer 14

Clumps or solid particulate are present; solution may appear clear when all precipitated protein sinks to bottom of tube

Question 15

II. Biuret reaction
1. What is the purpose of biuret reaction?

Answer 15

The biuret reaction can be used for determination of protein concentration

Question 16

II. Biuret reaction
2. What are the features of biuret reaction?

Answer 16

• The biuret reaction is specific for peptide bonds in biological samples.
• All compounds containing at least 2 peptide bonds (carboamide bonds) give a positive biuret probe.
• The reaction is named after the compound biuret, which is the simplest molecule to give a positive reaction, but is not a component of the reagent.
  => Biuret is formed from 2 urea molecules by condensation upon heating

Question 17

II. Biuret reaction
3. What is the principle of Biuret reaction?

Answer 17

The principle of the reaction is that, under alkaline conditions, the deep blue Cu2+-ions form purple complexes with proteins:
- The peptide bond undergoes oxo-enol tautomerism
- The nitrogen-bound hydrogen is transferred to the oxygen (-OH)
- The proton dissociates, so the oxygen becomes negatively charged
- One Cu2+ is complexed with 2 negatively charged oxygens and the lone pairs of the nitrogens -> the complex is purple (which can be measured by a spectrophotometry at 546nm)
- The intensity of the color is proportional to the number of the peptide bonds, so it can be used for the measurement of total protein concentration
=> The method is sensitive enough in the 0,5 - 5 mg/ml protein concentration range

Question 18

II. Biuret reaction
4. Describe the experiment of Biuret reaction

Answer 18

• To each tube, biuret reagent (CuSO4 + NaOH) is added
• Calibration curve is plotted based on known protein concentrations (BSA) and collected absorbance data
• After that, 2 unknown protein solutions are tested for concentration determination
  => Almost a linear relation between the solution concentration to absorbance value
  => Can calculate the concentration by the Lambert-Beer law

Question 19

III. Ellman-method
1. What is the purpose of Ellman-method?

Answer 19

The Ellman reaction is used to determine the presence/concentration of thiol (-SH) groups.

Question 20

III. Ellman-method
2. What are the features of thiol groups?

Answer 20

Thiol groups can be found in cysteine, therefore proteins containing cysteine yield positive reactions.

Question 21

III. Ellman-method
3. What is the Ellman reagent?

Answer 21

• The Ellman reagent is a disulfide, 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB)
• During the reaction, a thiol reacts with the colorless DTNB -> forming a mixed disulfide (TNB adduct), which will liberate the yellow exhibiting acid 5-thio-2-nitrobenzoic acid (TNB).

Question 22

III. Ellman-method
4. How can amount of yellow TN be determined in?

Answer 22

• The amount of yellow TNB can be determined in spectrophotometry at 412nm.
• The absorbance of TNB changes linearly with the concentration, therefore the concentration of thiol can be calculated using the molar extinction coefficient (molar absorptivity).