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INTRODUCTION
Classification and General Considerations
Proteins are an abundant component in all cells, and almost all except storage proteins are important for
biological functions and cell structure. Food proteins are very complex. Many have been purified and
characterized. Proteins vary in molecular mass, ranging from approximately 5000 to more than a million Dal
tons. They are composed of elements including hydro gen, carbon, nitrogen, oxygen, and sulfur. Twenty α-
amino acids are the building blocks of proteins; the amino acid residues in a protein are linked by peptide
bonds. Nitrogen is the most distinguishing element present in proteins.
Proteins can be classified by their composition, structure, biological function, or solubility properties. For
example, simple proteins contain only amino acids upon hydrolysis, but conjugated proteins also contain non-
amino-acid components.
The analysis of proteins is complicated by the fact that some food components possess similar physico-
chemical properties. Nonprotein nitrogen could come from free amino acids, small peptides, nucleic acids,
phospholipids, amino sugars, porphyrin, and some vitamins, alkaloids, uric acid, urea, and ammonium ions.
Therefore, the total organic nitrogen in foods would represent nitrogen primarily from proteins and to a lesser
extent from all organic nitrogen-containing nonprotein substances. Depending upon methodology, other major
food components, including lipids and carbohydrates, may interfere physically with analysis of food proteins.
Numerous methods have been developed to measure protein content. The basic principles of these methods
include the determinations of nitrogen, pep tide bonds, aromatic amino acids, dye-binding capacity, ultraviolet
absorptivity of proteins, and light scattering properties. In addition to factors such as sensitivity, accuracy,
precision, speed, and cost of analysis, what is actually being measured must be considered in the selection of
an appropriate method for a particular application.
Importance of Analysis
Protein analysis is important for:
1. Nutrition labelling
2. Pricing: The cost of certain commodities is based on the protein content as measured by nitrogen content
(e.g., cereal grains; milk for making certain dairy products, e.g., cheese).
3. Functional property investigation: Proteins in various types of food have unique food functional properties:
for example, gliadin and glutenin in wheat flour for bread making, casein in milk for coagulation into cheese
products, and egg albumen for foaming.
4. Biological activity determination: Some proteins, including enzymes or enzyme inhibitors, are relevant to
food science and nutrition: for instance, the proteolytic enzymes in the tenderization of meats, pectinases in
the ripening of fruits, and trypsin inhibitors in legume seeds are proteins. To compare between samples,
enzymes activity often is expressed in terms of specific activity, meaning units of enzyme activity per mg of
protein.
Protein analysis is required when you want to know:
1. Total protein content
2. Content of a particular protein in a mixture
3. Protein content during isolation and purification of a protein
4. Nonprotein nitrogen
5. Amino acid composition
6. Nutritive value of a protein
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Content in Foods
Protein content in food varies widely. Foods of animal origin and legumes are excellent sources of proteins.

Kjeldahl Method
Principle
In the Kjeldahl procedure, 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. The result of the analysis represents the crude
protein content of the food since nitrogen also comes from nonprotein components (note that the Kjeldahl
method also measures nitrogen in any ammonia and ammonium sulfate).
Historical Background
Original Method In 1883, Johann Kjeldahl developed the basic process of today’s Kjeldahl method to analyze
organic nitrogen. General steps in the original method include the following:
1. Digestion with sulfuric acid, with the addition of powdered potassium permanganate to complete oxidation
and conversion of nitrogen to ammonium sulfate.
2. Neutralization of the diluted digest, followed by distillation into a known volume of standard acid, which
contains potassium iodide and iodate.
3. Titration of the liberated iodine with standard sodium thiosulfate.
Improvements Several important modifications have improved the original Kjeldahl process:
1. Metallic catalysts such as mercury, copper, and selenium are added to sulfuric acid for complete digestion.
2. Potassium sulfate is used to increase the boiling point of the sulfuric acid to accelerate digestion.
3. Sulfide or sodium thiosulfate is added to the diluted digest to help release nitrogen from mercury, which
tends to bind ammonium.
4. The ammonia is distilled directly into a boric acid solution, followed by titration with standard acid.
5. Colorimetry Nesslerization, or ion chromatography to measure ammonia, is used to deter mine nitrogen
content after digestion.
General Procedures and Reactions
 Sample Preparation: Solid foods are ground to pass a 20-mesh screen. Samples for analysis should be
homogeneous. No other special preparations are required.
 Digestion: Place sample (accurately weigh ed) in a Kjeldahl flask. Add acid and catalyst; digest until clear
to get complete breakdown of all organic matter. Non-volatile ammonium sulfate is formed from the
reaction of nitrogen and sulfuric acid.

During digestion, protein nitrogen is liberated to form ammonium ions; sulfuric acid oxidizes organic matter
and combines with ammonium formed; carbon and hydrogen elements are converted to carbon dioxide and
water.
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 Neutralization and Distillation: The digest is diluted with water. Alkali-containing sodium thiosulfate is
added to neutralize the sulfuric acid. The ammonia formed is distilled into a boric acid solution containing
the indicators methylene blue and methyl red.
(NH4)2SO4 +2NaOH→2NH3+Na2SO4+2H2O
NH3+H3BO3(boric acid) → NH4 +H2BO3− (borate ion)
 Titration: Borate anion (proportional to the amount of nitrogen) is titrated with standardized HCl.
H2BO3− +H+ →H3BO3
Calculations
Moles of HCl = moles of NH3
=moles of Ninthe sample
A reagent blank should be run to subtract reagent nitrogen from the sample nitrogen.

where:
NHCl = normality of HCl, in mol/1000ml
Corrected acid vol. = (ml std. acid for sample) - (ml std. acid for blank)
14 = atomic weight of nitrogen

A factor is used to convert percent N to percent crude protein. Most proteins contain 16% N, so the conversion
factor is 6.25 (100/16 = 6.25).
%N/0.16 = %protein OR %N×6.25 =%protein
Applications

Advantages: Disadvantages:

1. Applicable to all types of foods 1. Measures total organic nitrogen, not just
protein nitrogen
2. Inexpensive (if not using an automated 2. Time consuming (at least 2h to complete)
system)
3. Accurate; an official method for crude protein 3. Poorer precision than the biuret method
content
4. Has been modified (micro Kjeldahl method) 4. Corrosive reagent
to measure microgram quantities of proteins

Biuret Method
Principle
A violet-purplish color is produced when cupric ions are complexed with peptide bonds (substances
containing at least two peptide bonds, i.e., biuret, large peptides, and all proteins) under alkaline conditions.
The absorbance of the color produced is read at 540nm. The color intensity (absorbance) is proportional to the
protein content of the sample.
Procedure
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1. A 5-ml biuret reagent is mixed with a 1-ml portion of protein solution (1–10mg protein/ml). The reagent
includes copper sulfate, NaOH, and potassium sodium tartrate, which is used to stabilize the cupric ion in the
alkaline solution.
2. After the reaction, mix is allowed to stand at room temperature for 15
or 30min, the absorbance is read at 540nm against a reagent blank.
3. Filtration or centrifugation before reading absorbance is required if
the reaction mixture is not clear.
4. A standard curve of concentration versus absorbance is constructed
using bovine serum albumin (BSA).

Applications The biuret method has been used to determine proteins in

cereal, meat, soybean proteins, and as a qualitative test for animal feed.
The biuret method also can be used to measure the protein content of
isolated proteins.
Advantages:
1. Less expensive than the Kjeldahl method; rapid (can be completed in
less than 30min); simplest method for analysis of proteins.
2. Color deviations are encountered less frequently than with Lowry, ultraviolet (UV) absorption, or
turbidimetric methods.
3. Very few substances other than proteins in foods interfere with the biuret reaction.
4. Does not detect nitrogen from nonpeptide or nonprotein sources.
Disadvantages:
1. Not very sensitive as compared to the Lowry method; requires at least 2–4mg protein for assay.
2. Absorbance could be contributed from bile pigments if present.
3. High concentration of ammonium salts inter fere with the reaction.
4. Color varies with different proteins; gelatin gives a pinkish-purple color.
5. Opalescence could occur in the final solution if high levels of lipid or carbohydrate are present.
6. Not an absolute method: color must be standardized against known protein (e.g., BSA) or against the
Kjeldahl nitrogen method.
Lowry Method
Principle
The Lowry method combines the biuret reaction with the reduction of the Folin–Ciocalteau phenol reagent
(phosphomolybdic-phosphotungstic acid) by tyrosine and tryptophan residues in the proteins. The bluish color
developed is read at 750nm (high sensitivity for low protein concentration) or 500nm (low sensitivity for high
protein concentration). The original procedure has been modified by Miller and Hartree to improve the
linearity of the color response to protein concentration.
Procedure
The following procedure is based on the modified procedure of Hartree:
1. Proteins to be analyzed are diluted to an appropriate range (20–100μg).
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2. K Na Tartrate-Na2CO3 solution is added after cooling and incubated at room temperature for 10min.
3. CuSO4-K Na Tartrate-NaOH solution is added after cooling and incubated at room temperature for 10min.
4. Freshly prepared Folin reagent is added and then the reaction mixture is mixed and incubated at 50◦C for
10min.
5. Absorbance is read at 650nm.
6. A standard curve of BSA is carefully constructed for estimating protein concentration of the unknown.
Applications
Because of its simplicity and sensitivity, the Lowry method has been widely used in protein biochemistry.
However, it has not been widely used to determine proteins in food systems without first extracting the
proteins from the food mixture.
Advantages:
1. Very sensitive
(a) 50–100 times more sensitive than biuret method
(b) 10–20 times more sensitive than 280-nm UV absorption method
(c) Similar sensitivity as Nesslerization; however, more convenient than Nesslerization
2. Less affected by turbidity of the sample.
3. More specific than most other methods.
4. Relatively simple; can be done in 1–1.5h.
Disadvantages:
For the following reasons, the Lowry procedure requires careful standardization for particular applications:
1. Color varies with different proteins to a greater extent than the biuret method.
2. Color is not strictly proportional to protein con centration.
3. The reaction is interfered with to varying degrees by sucrose, lipids, phosphate buffers, monosaccharides,
and hexoamines.
4. High concentrations of reducing sugars, ammonium sulfate, and sulfhydryl compounds interfere with the
reaction.