Protein analysis

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 (K 2 SO 4 ) (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 (NH 4 + ) which binds to the sulfate ion (SO 4 2- ) and thus remains in solution: The general equation for the digestion of an organic sample is shown below: Protein + H 2 SO 4 → (NH 4 )SO 4 + H 2 O + CO 2

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(NH 4 + ) ions (which are dissolved in the liquid) to ammonia (NH 3 ), which is a gas as indicated in the following equation. (NH 4 )2SO 4 + 2NaOH 2NH 3 + Na 2 SO 4 + 2H 2 O 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 μ g). 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: 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.

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