Protein

Question 1

What is protein?

Answer 1

• Polymer of 20 α- amino acids

• N is the most distinguished element: among the
composing elements of C,H, N, O, S

• Most abundant components in cells: 50% of dry cells by weight

• Linkage between amino acids in protein: peptide bonds

Question 2

Analysis of protein can be complicated due to:

Answer 2

1) Some food components possess similar physicochemical properties as protein but considered as non protein

2) Non protein N could be from:
• free amino acid
• small peptides
• nucleic acid
• phospholipids
• amino sugar

Question 3

Method of protein analysis

Answer 3

1) Nitrogen in the sample (Kjeldahl Method)
2) Peptide Bonds (Biuret Method)
3) Aromatic acid ( Lowry Method)
4) UV absorptivity of proteins (Ultraviolet 280nm
absorption method)
5) Free amino group (Dye binding method)

Question 4

Principle of Kjedahl Method

Answer 4

• The percent Total Kjeldahl Nitrogen (TKN) measured is converted to the equivalent protein content by using numerical factors depending on the food being analyzed

• In the Kjeldahl method, proteins and other organic food components in a sample are digested with sulfuric acid in the presence of catalysts.

• The total organic nitrogen is converted to ammonium sulfate.

• The digest is neutralized with alkali and distilled into a boric acid solution.

• The borate anions formed are titrated with standardized acid, which is converted
to nitrogen in the sample

Question 5

Procedure

Answer 5

1) Digestion

2) Distillation & Neutralization

3) Titration

Question 6

DIgestion

Answer 6

• Catalyst (CuSO4) and K2SO4 to accelerate digestion/boiling point increasing agent
• Nonvolatile ammonium sulfate is formed from the reaction of nitrogen and sulphuric acid
• Protein nitrogen is liberated to form ammonium ions

Question 7

2) Distillation & Neutralization

Answer 7

• The digest is diluted with water. NaOH is added to neutralize the sulphuric acid. The ammonia formed is distilled into a boric acid

• solution containing the indicators methylene blue and methyl red

Question 8

3) Titration

Answer 8

• Borate anions (proportional to the amount of nitrogen) are titrated with acid.
• Titrate the distillate with standardized hydrochloric acid 0.1 N until the color of the mixture turns to pink/red

Question 9

Advantages of Kjedahl Method

Answer 9

• Applicable to all types of foods.
• Relatively simple.
• Inexpensive.
• Accurate and official method for crude protein content.

Question 10

DIsadvantages of Kjedahl Method

Answer 10

• Measures total organic content, not just protein nitrogen.
• Time consuming (at least 2 hrs).
• Poorer precision than the biuret method.
• Corrosive reagent

Question 11

Biuret Method

Answer 11

1) Can be used to detect the amount of protein in a given solution by colorimetric estimation

2) The amount of protein is estimated by estimating the presence of peptide bonds present in the proteins

3) KOH = Provides the alkaline condition
Hydrated Copper sulphate = provides Cu2+ ions
Potassium sodium tartrate = Stabilizes the complex

4) More protein in the solution -> More peptide bonds -> More chetale complex formed -> More color developed

Question 12

Advantages Buiret Method

Answer 12

• Less expensive compared to kjeldahl
• Very fast can be completed in 30 mins
• Does not detect nitrogen from non peptide or non protein sources
• Very few substances other than proteins in foods interfere with the biuret reaction

Question 13

Disadvantages of Biuret Method

Answer 13

• Not very sensitive compared to Lowry method
• Color varies with different proteins
• Color must be standardized against a known protein, e.g, BSA

Question 14

Advantages of Lowry Method

Answer 14

1) Very sensitive compared to most method
2) More specific than most methods
3) Relatively fast and simple ( done btw 1-1.5hrs)

Question 15

Disadvantages of Lowry Method

Answer 15

1) Color change varies with proteins
2) Color intensity is not proportional to protein conc