Determination of protein content in solutions

FIELD: biochemistry, biotechnology.

SUBSTANCE: claimed method includes sample treatment with alkali copper-containing reagent comprising 49 pts of 2 % sodium carbonate solution in 2 N sodium hydroxide (A) and 1 pts of 0.5 % blue copper in 3.33 % sodium or potassium tartrate followed by addition of Folin's reagent and mixture conditioning in ultra-thermostat at 50°C for 10 min. Method of present invention allows reducing analysis time to 20 min and increasing sensitivity and reproducibility protein content determination by Lowry method.

EFFECT: accelerated method for determination of protein content in biological liquids and enzyme solutions.

2 dwg, 1 tbl, 3 ex

 

The invention relates to the field of biochemistry and biotechnology and can be used for determination of protein in biological fluids and enzyme solutions.

The known method of protein determination by the method of Lowry (prototype), based on a color reaction with tyrosinase and cysteine radicals protein molecule, resulting in the recovery of the mixture of phosphorus-tungsten and phosphorus molybdenum acid with the formation of complex compounds of the blue color. The flow of specified reactions contribute to complex compounds of copper caused by interaction of the protein with an alkaline solution of copper sulphate. For the analysis of mixing 49 parts of solution a (2%sodium carbonate in 0.1 G. of the sodium hydroxide) with 1 part of solution B (0.5%copper sulphate at 3.33%sodium tartrate, or potassium). The obtained alkaline copper reagent is added in a ratio of 4:1 in the sample containing 10-100 µg any protein shake and leave for 10 minutes at room temperature. Then add the reagent Polina in a ratio of 1:10 by volume of alkaline copper reagent. The ratio of the volumes of sample and reagents is 1:4:0,4. The reaction mixture is vigorously stirred and leave for 30 to 90 minutes to develop color. The optical density is determined at a wavelength of 750 nm. Sod is neigh protein in the test set via a calibration curve, built in solutions of the same protein with a precisely known concentration (Philipovich SHE and other Workshop on General biochemistry. - 2nd ed., Rev. - M.: Education, 1982. - P.75-77.).

The disadvantage of this method is the long duration (up to 100 minutes), and not enough high reproducibility, sensitivity, precision, analysis, and narrow linear range of the calibration graph (0-100 µg/cm3). This is due to the fact that in the conditions of the prototype method color reaction proceeds slowly and when uncontrolled by the exposure time measurements (30-90 minutes) may be incomplete development of the staining solution. In terms of the prototype method, i.e. at a ratio of 10:1 volume of alkaline copper reagent and reagent Polina, achieved a pH of about 9.0, as shown in figure 1, there is a minimum speed of color development solution and its intensity.

The technical result of the present invention is to increase expressnet, sensitivity, reproducibility and accuracy of the definitions of the protein in solution, as well as the extension of the linear range of the calibration graph.

The technical result is achieved by the fact that the definitions of increasing concentration of sodium hydroxide in solution And 0.2 N. while maintaining the same volume all dosing solutions or increases the t amount of alkaline copper reagent to the volume ratio of sample and reagents 1:8:0,4, and the optical density of the solution was determined after exposure for 10 minutes at a temperature of 50°C.

Increasing the concentration of sodium hydroxide in the solution or the amount of used alkaline copper reagent leads to an increase in the pH of the reaction mixture up to 11-12. This contributes to a more complete development of the painting (see Figure 1) and, consequently, increase the sensitivity and accuracy of definitions, as well as the extension of the linear range of the calibration graph. Heating of the solution after mixing of the reagents for 10 minutes at a temperature of 50°increases the flow speed of the color reaction that increases expressnet and reproducibility of the analysis (see Figure 2).

The method is as follows: the test sample containing 10-300 µg of a protein, add alkaline copper reagent (a+b) in a ratio of 8:1 to the sample volume, shake and leave for 10 minutes at room temperature, then add the reagent Polina in a ratio of 1:20 by volume of alkaline copper reagent. The ratio of the volume of samples and reagents is 1:8:0,4. The reaction mixture is vigorously stirred and set in ultraharmonic at a temperature of 50°10 minutes (stopwatch). The optical density was determined after cooling the reaction mixture at the line wavelength 750 nm relative to a reference solution, containing all of the reagents in the same volumes, in addition to the samples, instead of which add distilled water. The calibration curve is built similarly to solutions of the same protein with a precisely known concentration.

When using the solution with a higher concentration of sodium hydroxide (0,2 N.) method works similarly, but alkaline copper reagent (a+b) add in a 4:1 ratio to the volume of sample and reagent Polina add in a ratio of 1:10 by volume of alkaline copper reagent. The ratio of the volume of samples and reagents is 1:4:0,4.

Example 1. For the analysis of mixed 49 cm3solution a (2%sodium carbonate in 0.1 G. of the sodium hydroxide) with 1 cm3solution (0.5%copper sulphate at 3.33%sodium tartrate, or potassium). To 1 cm3samples containing 10-300 µg bovine serum albumin (BSA), add 8 cm3alkaline copper reagent, shake and leave for 10 minutes at room temperature. Then add 0.4 cm3reagent Polina, vigorously stirred and set the mixture in ultraharmonic at a temperature of 50°10 minutes (stopwatch) to develop color. The reaction mixture is cooled to room temperature and determine the optical density at a wavelength of 750 nm relative to a reference process is and, containing all of the reagents in the indicated amounts and distilled water instead of sample. The protein content in the sample set by a calibration curve, constructed similarly to solutions of BSA with accurately known concentration. The coefficients of the calibration graph and the results of the analysis of BSA solution with an unknown concentration are presented in table 1.

Example 2. For the analysis of mixed 49 cm3solution a (2%sodium carbonate 0.2 called sodium hydroxide) with 1 cm3solution (0.5%copper sulphate at 3.33%sodium tartrate, or potassium). To 1 cm3samples containing 10-300 μg BSA, add 4 cm3alkaline copper reagent, shake and leave for 10 minutes at room temperature. Then add 0.4 cm3reagent Polina, vigorously stirred and set the mixture in ultraharmonic at a temperature of 50°10 minutes (stopwatch) to develop color. The reaction mixture is cooled to room temperature, the volume was adjusted to 9.4 cm3(analogously to example 1) by adding 4 cm3distilled water and determine the optical density at a wavelength of 750 nm relative to a reference solution containing all reagents in the indicated amounts and distilled water instead of sample. The protein content in the sample set for calibration Gras is the IR, built similarly to solutions of BSA with accurately known concentration. The coefficients of the calibration graph and the results of the analysis of BSA solution with an unknown concentration are presented in table 1.

Example 3 (the prototype). For the analysis of mixed 49 cm3solution a (2%sodium carbonate in 0.1 G. of the sodium hydroxide) with 1 cm3solution (0.5%copper sulphate at 3.33%sodium tartrate, or potassium). To 1 cm3samples containing 10-100 µg BSA, add 4 cm3alkaline copper reagent, shake and leave for 10 minutes at room temperature. Then add 0.4 cm3reagent Polina, vigorously mix and leave for 90 minutes to develop color. Bring the volume of the reaction mixture to 9.4 cm3(similar to examples 1-2) by adding 4 cm3distilled water and determine the optical density at a wavelength of 750 nm relative to a reference solution containing all reagents in the indicated amounts and distilled water instead of sample. The protein content in the sample set by a calibration curve, constructed similarly to solutions of BSA with accurately known concentration. The coefficients of the calibration graph and the results of the analysis of BSA solution with an unknown concentration are presented in table 1.

As can be seen from table is s 1, the proposed method allows to reduce the duration of the analysis in 5 times and increase the range of linear dependence of optical density on the concentration of protein in solution 3 times. The increase in the coefficient of proportionality k in the equation of a straight line calibration curve increases the sensitivity of the definitions, and lower values of relative standard deviation Snand confidence interval n increases the reproducibility and accuracy of definitions in comparison with the method of the prototype.

Table 1
TimeFactorsRangeResultsSn*,n**,
№ p/panalysis, minutesthe calibration graphlinearity, mg/cm3definitions µg/cm3%ág/cm3
kb
Example 157,9
57,6
201,95×10-300-30057,40,40±0,3
57,9
57,9
Example 257,4
of 57.5
202,21×10-300-30057,30,31±0,2
57,7
57,7
Example 360,4
(prototype)57,7
1001,47×10-300-10061,13,23±2,4
57,7
61,8
*Snthe relative standard deviation

**n - the confidence interval when f=0,95

Method for determination of protein concentration in solutions, including processing the analyzed samples of alkaline copper reagent, consisting of 49 parts of solution a: 2%sodium carbonate in sodium hydroxide and 1 part of solution: 0.5%copper sulphate at 3.33%tartrate of sodium or potassium hydroxide followed by the addition of reagent Polina when the ratio of the volume of samples and reagents 1:4:0.4-definition wide-angle is m its optical density at 750 nm, wherein the use solution And 0.2 N. the concentration of sodium hydroxide, and determining the optical density is carried out after keeping the reaction mixture at a temperature of 50°C for 10 minutes



 

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