Protein Analysis

Introduction

 Proteins are polymers of amino acids

 20 amino acids
 They are a major source of energy, as well as containing
essential amino-acids, such as lysine, tryptophan,
methionine, leucine, isoleucine and valine, which are
essential to human health, but which the body cannot
synthesize
 proteins are used as gelling agents, emulsifiers, foaming
agents and thickeners
 Many food proteins are enzymes which are capable of
enhancing the rate of certain biochemical reactions
 Content of Protein in Foods

Milk (dry) 22-25 Rice 7.5-9

Milk (skim) 3.2 Wheat flour 9.8-13.5
Egg (dry) 35 Walnuts 15-21
Beef chuck roast 18.5 Potato 10-13
 Methods for Protein Determination
Total Spectroscopic Colorimetric
Other Methods
Nitrogen Methods Methods
Kjeldahl Dye Binding Ninhydrin
UV Absorbance
Method Methods Method
Pyrolysis Turbidimetric
Infrared Method Bradford Assay
Methods Method
Fluorescence Anionic Dye
. .
Method Binding Assay
Reaction of
. . .
Copper Salts

. . Biuret Assay .

. . Lowry Assay .

. . Bicinchronic Assay .
Kjeldahl Method

 A food is digested with a strong acid so that it

releases nitrogen
 The amount of protein is then calculated from the
nitrogen concentration of the food
 Need a conversion factor =refers to Malaysian
act /6.25
 based on the conversion of nitrogen to ammonia
 Digestion, Neutralization/Distillation, and
Titration
i) Digestion

 To complete breakdown of all organic

matter
 carbon and hydrogen are oxidized to
carbon dioxide and water in the presence
of sulfuric acid
 The ammonia picks up hydrogen and
ammonium sulfate is formed

Protein + H2SO4 ----------> (NH4)2SO4 + CO2 + H2O + SO4

ii) Neutralization /Distillation

Sodium thiosulfate to neutralize

(NH4)2SO4 + 2NaOH --------> 2NH3(g) + 2 H2O + Na2SO4

NH3(l) + H3BO3 -----------------> NH4 + H2BO3

Ammonia form is distilled into a boric acid solution containing
indicator
iii) Titration

Borate anion ( prportional to the amount of nitrogen) is titrated with

standerdized HCL

H2BO3- + H+ -----------------> H3BO3

Calculation

 Moles of HCL= Moles NH3 = Moles N in the sample

%N = N HCL x (Corrected acid volume/g of sample) x
(14gN/mol)x100
NHCl= Normality of HCl, in moles/1000ml
Corrected acid vol = ( ml std acid for sample – ml std acid for
blank)
14= a.m.u Nitrogen
Calculations

 
 

% protein = %nitrogen x factor (6.25)

Advantages

 Applicable to all foods

 Relatively simple
 Inexpensive
 Accurate – official method
 Modified to be micro method – smaller samples required
Disadvantages

 Measures total N not protein

 Time consuming – at least 2 hrs
 Poor precision than some other methods
 Corrosive (dangerous) method
Dumas Method

 A sample is combusted at high temperature (700-

1000°C) and nitrogen released is measured.
 measured by GC using a thermal conductivity
detector
 Advantages
No hazardous chemicals,Short - 3min,Automatic -
150 sample no attention
 Disadvantages
Expensive equipment
Lowry method

 is a colorimetric method based on:

 the formation of a blue color formed with: tyrosine or
tryptophan in the protein reduces phosphomolybdic-
phosphotungstic reagent
 Violet purplish color produced when cupric ions are
complexed with peptide bonds (Biuret method).
Ultraviolet Absorption (UV) at
280 nm
 Most proteins exhibit strong UV light absorption at
280 nm because they contain chromophoric side
chains such as tyrosine, tryptophan, and
phenylalanine
 Advantages
The estimation of protein can be carried out quickly
and easily on a small quantity of material
not destroyed
 Disadvantages
Other compounds present in foods also exhibit
absorption at 280 nm