Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Protein analysis using low-voltage electrophoresis
Low-voltage paper electrophoresis
Determination of protein content

Spectrophotometric determination of Proteins

Principle of the method. The extinction of proteins containing phenylalanine, Tyrosine, and Tryptophan at 280 nm is directly proportional to their concentration in solution. It should be noted that extinction coefficients vary among different proteins.

Procedure. A colorless, completely transparent protein solution is placed in an appropriate spectrophotometer cuvette, and the extinction is measured at a wavelength of 280 nm. The protein concentration is calculated based on the known extinction coefficient.

Notes. 1. The advantages of spectrophotometric protein determination are its simplicity, speed, and ease of execution. Using appropriate equipment (for example, the “Uvicord” instrument by LKB, Sweden), this method allows for continuous monitoring of protein concentration in solution.

2. A limitation of this method is that it cannot be applied with equal efficiency to determine different proteins, as their aromatic amino acid content varies widely. In addition, several non-protein substances can interfere with protein determination (for example, Nucleic Acids, colored prosthetic groups, etc.).

3. The extinction coefficient of different proteins at a wavelength of 280 nm ranges from 0.5 to 1.0 cm³ ∙ mg^-1.

4. The spectrophotometric method can detect protein at concentrations of no less than 10–20 µg/ml.

Quantitative determination of PROTEIN USING FOLIN–CIOCALTEU PHENOL REAGENT [8]

Principle of the method. The Folin–Ciocalteu phenol reagent yields a blue color with alkaline protein solutions. The intensity of the color depends primarily on the tyrosine and tryptophan content of the protein being analyzed.

Sensitivity of the method. The macromethod can determine 100–500 µg, and the micromethod 5–35 µg of protein nitrogen.

MACROMETHOD

Reagents. 1. Folin–Ciocalteu phenol reagent, preparation. In a 2 L flask, dissolve 100.0 g of Na₂WO₄∙Н₂О and 25.0 g of Na₂MoО₄∙H₂О in 700 ml of distilled Water, then add 50 ml of an 85% Н₃РО₄ solution and 100 ml of concentrated НСl. The mixture is refluxed for 10 h, then 150.0 g of Li₂SO₄, 50 ml of distilled water, and a few drops of bromine are added; the mixture is boiled again for 15 min without a reflux condenser to remove excess bromine. After cooling, the volume of the reagent is adjusted to 1000 ml with distilled water and filtered into a dark Glass bottle, as the reagent must be stored in the dark and protected from bacterial growth and contamination by reducing agents.

2. 20% Na₂CO₃ solution.

Procedure. Pipette 9 ml of the test solution into a centrifuge tube or a 50 ml Erlenmeyer flask, and slowly add 5 ml of 20% Na₂CO₃ solution while stirring constantly. Then, while gently mixing, add 1 ml of Folin–Ciocalteu reagent dropwise. Immerse the sample in a water bath at 37°C for 5 min, then leave it at room Temperature for 30 min and measure the optical density at 750 nm.

Control sample: add 5 ml of 20% Na₂CO₃ solution and 1 ml of Folin reagent to 9 ml of distilled water.

MICROMETHOD

Reagents. 1. Solution A: 2% Na₂CO₃ solution in 0.1 N NaOH.

2. Solution B: 0.5% CuSO_4∙5H₂O solution in 1% sodium tartrate solution.

3. Solution C: add 1 ml of Solution B to 60 ml of Solution A; prepare immediately before use.

4. Solution D: Folin–Ciocalteu reagent (see above). Immediately before use, the Folin–Ciocalteu reagent is titrated with a NaOH solution of known normality in the presence of phenolphthalein, and diluted to a concentration of 1 N based on the titration data (usually approximately twofold).

Procedure. Mix 0.2 ml of the test solution, which may contain 5 to 100 µg of protein, with 1 ml of Solution C in a test tube, leave for 10 min, and then very rapidly add 0.1 ml of Solution D to the mixture. Shake for 1–2 s, leave for 30 min, and measure the absorbance spectrophotometrically at 750 nm. Each determination must be performed with duplicate samples.

Notes. 1. The intensity of the color reaction can vary with different proteins; therefore, when performing either the macromethod or the micromethod, it is recommended to construct a calibration curve for each protein being studied.

2. The color reaction depends not only on the tyrosine and tryptophan content of the given protein, but also on the presence of SH- and other reducing groups, as well as on the duration of time the protein was kept in an alkaline medium prior to The addition of the Folin–Ciocalteu reagent.

3. Protein determination by the Folin–Ciocalteu method is relatively rapid and does not require prior Digestion of the sample material; thus, it is undoubtedly more convenient than the Kjeldahl protein determination. However, if results need to be expressed in units of protein nitrogen, a calibration curve should be constructed based on quantitative Kjeldahl protein nitrogen determination.

Determination of Protein BY THE BIURET REACTION [4]

Principle of the method. In alkaline protein solutions, four nitrogen atoms involved in peptide bonds can form a complex with a single copper atom; this reaction produces a blue-violet coloration.

Sensitivity of the method. The biuret reaction allows for the quantitative determination of 20 to 400 µg of protein nitrogen.

Reagents. Biuret reagent contains, per 1 L of solution: sodium potassium tartrate 9.0 g; CuSO₄∙ 5Н₂O 3,0 g; KI 5,0 g; 0.2 N NaOH (carbonate-free).

Sodium potassium tartrate is dissolved in 400 ml of 0.2 N NaOH, and copper sulfate is added. Once the components are dissolved, potassium iodide and 0.2 N NaOH are added, and the solution is then made up to 1000 ml.

Procedure. 1 ml of the protein test solution is mixed with 1.5 ml of biuret reagent, incubated for 30 min at 37°C, and the optical density is measured at 555 nm.

Control sample. 1 ml of distilled water is mixed with 1.5 ml of biuret reagent. Each determination is carried out in duplicate.

Note. When using the biuret reagent in a quantitative precipitation reaction (see p. 125), it is convenient to perform the reaction in 8 ml centrifuge tubes graduated at 2.5 ml. After washing, the precipitate is dissolved in 1.5 ml of biuret reagent, and the volume is made up to the mark (2.5 ml) with distilled water. After incubation, the optical density is determined.

DETERMINATION OF PROTEINS BY THE NINHYDRIN REACTION [13]

Principle of the method. Ninhydrin reacts to form a colored product with terminal a-NH₂ groups of proteins, as well as with ε-NH₂ groups of Lysine residues.

Sensitivity of the method. The method is applicable when the test solution contains 1–20 µg of protein nitrogen.

Reagents. 1. Preparation of 0.2 M citrate buffer solution, pH 5.0: 2.101 g of citric acid monohydrate С₆Н₈О₇ ∙ Н₂О is dissolved in 20 ml of 1 N NaOH, and 50 ml of distilled water is added. After a two-fold dilution with distilled water, the pH of the buffer solution should be 5.0±0.1. The solution can be stored in a refrigerator after adding a few crystals of thymol.

2. Methyl cellosolve (Ethylene glycol monomethyl ether).

3. Propanol-water mixture (a mixture of equal volumes of n-propanol and distilled water).

4. Ninhydrin reagent. Preparation of ninhydrin reagent: 40 mg of SnCl₂ ∙ 2Н₂О is dissolved in 25 ml of citrate buffer solution, and 4.0 mg of ninhydrin, previously dissolved in 12.5 ml of methyl cellosolve, is added. The reagent is prepared immediately before the experiment.

Procedure. 0.1 ml of the protein test solution is pipetted into a 5 ml ground-glass stoppered tube, and 0.5 ml of ninhydrin reagent is added; the tube is closed with a glass stopper, mixed thoroughly, and boiled in a water bath for 20 min. Then, 2 ml of the propanol-water mixture is added, the solution is mixed, centrifuged, and the optical density of the supernatant is determined at 570 nm.

In the control sample, all operations are carried out with 0.1 ml of distilled water.

QUANTITATIVE DETERMINATION OF PROTEIN USING AMIDO BLACK [9, 10]

Principle of the method. Amido black binds to proteins proportionally to The amount of protein.

Sensitivity of the method. The method is applicable for analyzing samples containing at least 0.01 mg of protein. It is quite convenient for determining protein concentrations in low-protein solutions, such as CEREBROSPINAL FLUID, aqueous humor of the eye, etc.

Reagents. 1. Dye solution: a saturated solution of Amido Black 10B in a chilled mixture of glacial acetic acid and methanol (1:9) (see p. 49).

2. Washing solution: a mixture of glacial acetic acid and methanol (1:9).

3. Solvent: 0.1 N NaOH.

Procedure. To 0.2 ml of the protein test solution (in a centrifuge tube), 1.0 ml of distilled water is added, followed by pipetting first 1 ml and then, after thorough mixing, another 4 ml of the dye into the tube. Shake several times and leave the mixture for 20 min at room temperature; centrifuge at 2000 rpm for 10 min, and carefully decant or aspirate the supernatant. The precipitate is washed with the washing solution until the supernatant after subsequent centrifugation remains colorless. The washed precipitate is dissolved in 5 ml of the solvent, and the optical density is measured in a 1 cm cuvette using an S-61 filter at 595 nm against the control. The extinction value multiplied by 105 gives the protein concentration in mg%.