protein analysis

1. General Method of Analysis of
Protein
MADE BY: Lalit Singh
Student of M-pharm (Pharmaceutical
Analysis)
ISF College of Pharmacy,Moga

2. Protein Definition

3. kjeldahl apparatus

4. Kjeldahl Method
•1.Digestion
The purpose of this step is to break down the bonds that hold the polypeptides
together, and convert them to simpler chemicals such as water, carbon dioxide
andammonia.
•The food sample to be analyzed is weighed into a digestion flask and then
digested by heating it in the presence of sulfuric acid (an oxidizing agent which
digests the food), potassium sulfate (K2SO4) (to speed up the reaction by raising
the boiling point of the digesting acid) and a catalyst, such as copper, selenium,
titanium, or mercury (to speed up the reaction).
Ammonia gas is not liberated in an acid solution because the ammonia is in the
form of the ammonium ion (NH4
+) which binds to the sulfate ion (SO4
2-) and thus
remains in solution:
The general equation for the digestion of an organic sample is shown below:
Protein + H2SO4 → (NH4)SO4 + H2O + CO2

5. •
2.Distillation:
• The purpose of the distillation step, is to separate the
ammonia (that is, the nitrogen) from the digestion mixture.
• This is done by, raising the pH of the mixture using (NaOH ).
This has the effect of changing the ammonium(NH4
+) ions
(which are dissolved in the liquid) to ammonia (NH3), which is
a gas as indicated in the following equation.
(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O
• The ammonia gas is led into a trapping solution (an acid )
where it dissolves and become an ammonium ion once again
NH3 + HCl (0.1 N) NH4 +Cl- + HCl (left back)
(in excess)
3. Titration :
• Left back HCl (0.1 N ) is titrated with standard NaOH (0.1 N).

6. Titration :

7. Calculation
• Let the weight of the organic substance be x gm and V ml of (N) HCl is
required for complete neutralization of ammonia evolved.
• V ml (N) HCl = V ml of (N) NH3
• 1000 ml of a N NH3 contain 17 gm of NH3 or 14 gm of Nitrogen
• Amount of Nitrogen present in V ml of (N) NH3 = 14 x V x N = y gm
1000
• Percentage of nitrogen = Weight of Nitrogen (y gm) x 100
• Weight of Substance (x gm)
• Where ,
• N = Normality of Acid Used ; V = Volume of Acid used up
• %N× 6.25(Correction Factor) = %protein

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

9. Dumas (Nitrogen Combustion)
Method
• Principle
• Samples are combusted at high temperatures (700–1000◦C)
with a flow of pure oxygen. All carbon in the sample is
converted to carbon dioxide during the flash combustion.
• Nitrogen-containing components produced include N2 and
nitrogen oxides. The nitrogen oxides are reduced to
nitrogen in a copper reduction column.
• Procedure
• Samples (approximately 100–500 mg) are weighed into a
tin capsule and introduced to a combustion reactor in
automated equipment. The nitrogen released is measured
by a built-in gas chromatograph at a high temperature
(600◦C).

10. General components of a Dumas nitrogen analyzer.
A, the incinerator; B, copper reduction unit for converting nitrogen oxides to
nitrogen; and GC, gas chromatography column.

11. • Applications
• The combustion method is an alternative to the
Kjeldahl method and is suitable for all types of
foods.
• Advantages:
1. Requires no hazardous chemicals.
2. Can be accomplished in 3 min.
• Disadvantages:
1. Expensive equipment is required.
2. Measures total organic nitrogen, not just protein
nitrogen

12. Protein Assay
Biuret Assay Lowry Assay
Bicinchoninic
Acid(BCA) Assay

13. 1.Biuret Assay
• Principle :-
• A violet-purplish color is produced when
cupric ions are complexed with peptide bonds
under alkaline conditions
• The absorbance of the color produced is read
at 540 nm. The color intensity (absorbance) is
proportional to the protein content of the
sample.

14. The Biuret
Reagent
NaOH
Potassium
Tartrate
CuSO4.5H2O
Chelate Cu+2 -> Stabilise it

15. Procedure For Biuret Assay
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 30 min, 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).

16. Advantages:-
1.Less expensive than the Kjeldahl method; rapid (can be
completed in less than 30 min); simplest method for
analysis of proteins.
2. Very few substances other than proteins in foods interfere
with the biuret reaction.
3. Does not detect nitrogen from non peptide or non protein
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 interfere with the
reaction.

17. 2. Lowry Assay
Step 1
Step 2
NaOH Potassium
Tartrate
CuSO4.5H2O
Folin–Ciocalteau
reagent

18. Folin–Ciocalteau
reagent
Phosphotungstic acid Phosphomolybdic acid
Oxidizing Reagent

19. 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).

20. Procedure Lowry Assay
1. Proteins to be analyzed are diluted to an appropriate range
(20–100 μg).
2. 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.
3. After the reaction mix is allowed to stand at room
temperature for 10 min
4. Freshly prepared Folin reagent is added and then the
reaction mixture is mixed and incubated at 50◦C for 10 min.
5. Absorbance is read at 650 nm.
6. A standard curve of BSA is carefully constructed for
estimating protein concentration of the unknown.

21. • 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.
2. Less affected by turbidity of the sample.
3. More specific than most other methods.
4. Relatively simple; can be done in 1–1.5 h.
• 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 concentration.

22. Bicinchoninic Acid(BCA) Assay
• Proteins and peptides (as short as dipeptides) reduce
cupric ions to cuprous ions under alkaline conditions ,
which is similar in principle to that of the Biuret reaction.
• The cuprous ion then reacts with the apple-greenish
bicinchoninic acid (BCA) reagent to form a purplish
complex (one cuprous ion is chelated by two BCA
molecules).
• The color measured at 562nm is near linearly proportional
to protein concentration over a wide range of
concentration from micrograms up to 2mg/ml. Peptide
bonds and four amino acids (cysteine, cystine, tryptophan,
and tyrosine) contribute to the color formation with BCA.

23. Procedure
1. Mix (one step) the protein solution with the BCA
reagent, which contains BCA sodium salt, sodium
carbonate, NaOH, and copper sulfate, pH 11.25.
2. Incubate at 37◦C for 30 min, or room temperature
for 2 hr, or 60◦C for 30 min. The selection of the
temperature depends upon sensitivity desired. A
higher temperature gives a greater color
response.
3. Read the solution at 562nm against a reagent
blank.
4. Construct a standard curve using BSA.

24. • Advantages:-
1. Sensitivity is comparable to that of the Lowry method
(0.5–10 μg) is better than that of the Lowry method.
2. One-step mixing is easier than in the Lowry method.
3. The reagent is more stable than for the Lowry reagent.
• Disadvantages:-
1. Color is not stable with time. The analyst needs to
carefully control the time for reading absorbance.
2. Any compound capable of reducing Cu+2 to Cu+ will
lead to color formation.

25. Ultraviolet 280nm Absorption Method
• Principle :-
• Proteins show strong absorption in the region at
ultraviolet (UV) 280nm, primarily due to tryptophan
and tyrosine residues in the proteins. Because the
content of tryptophan and tyrosine in proteins from
each food source is fairly constant, the absorbance at
280nm could be used to estimate the concentration of
proteins, using Beer’s law.
• Since each protein has a unique aromatic amino acid
composition, the extinction coefficient (E280) or molar
absorptivity (Em) must be determined for individual
proteins for protein content estimation.

26. Procedure
1. Proteins are solubilized in buffer or alkali.
2. Absorbance of protein solution is read at 280nm
against a reagent blank.
3. Protein concentration is calculated according to the
equation
A = abc
where:-
• A = absorbance
• a = absorptivity
• b = cell or cuvette path length
• c = concentration

27. • Advantages:-
1. Rapid and relatively sensitive; At 280 nm, 100 μg or more protein is
required; several times more sensitive than the biuret method.
2. No interference from ammonium sulfate and other buffer salts.
• Disadvantages:-
1. Nucleic acids also absorb at 280 nm.
2. Aromatic amino acid contents in the proteins from various food sources
differ considerably.
3. The solution must be clear and colorless. Turbidity due to particulates in
the solution will increase absorbance falsely.

28. Dye-Binding Methods
1 Anionic Dye-Binding Method
Principle:- The protein-containing sample is mixed with a
known excess amount of anionic dye in a buffered
solution. Proteins bind the dye to form an insoluble
complex. The unbound soluble dye is measured after
equilibration of the reaction and the removal of
insoluble complex by centrifugation or filtration.
• Protein + excess dye → Protein −dye insoluble complex
+ unbound soluble dye
• The amount of the unbound dye is inversely related to
the protein content of the sample.

29. Procedure
1. The sample is finely ground (60mesh or smaller sizes) and added to
an excess dye solution with known concentration.
2. The content is vigorously shaken to equilibrate the dye binding
reactions and filtered or centrifuged to remove insoluble
substances.
3. Absorbance of the unbound dye solution in the filtrate or
supernatant is measured and dye concentration is estimated from a
dye standard curve.
4. A straight calibration curve can be obtained by plotting the unbound
dye concentration against total nitrogen (as determined by Kjeldhal
method) of a given food covering a wide range of protein content.
5. Protein content of the unknown sample of the same food type can
be estimated from the calibration curve or from a regression
equation calculated by the least squares method.

30. • Advantages:-
1. Rapid (15 min or less), inexpensive, and relatively
accurate for analyzing protein content in food
commodities.
2. No corrosive reagents.
3. Does not measure non-protein nitrogen.
4. More precise than the Kjeldahl method.
• Disadvantages:-
1. Not sensitive; milligram quantities of protein are
required.
2. Not suitable for hydrolyzed proteins due to
binding to N-terminal amino acids.