Methods for identification and quantitative determination of proteins in agrochemistry

Harvest quality of grain and forage crops directly depends on their protein content. To assess the nutritional value of produce or monitor nitrogen plant nutrition, laboratories conduct qualitative and quantitative analysis of plant samples. These studies are based on chemical reactions and physicochemical methods of molecular separation.

Qualitative reactions: how to determine protein composition

For rapid identification of proteins in plant raw materials, color reactions and precipitation reactions are used. Color tests allow for the detection of both the protein itself and individual critical amino acids that determine the feed value of a crop. Precipitation is necessary to convert unstable proteins into a solid phase for further study.

The qualitative composition of proteins is determined using the following specific reactions:

• Biuret reaction: gives a blue-violet or red-violet coloration when alkali and copper sulfate are added to an aqueous protein solution, indicating the presence of CO–NH peptide bonds.
• Ninhydrin reaction: a universal test for α-amino acids.
• Xanthoproteic reaction: gives a yellow coloration upon interaction with concentrated nitric acid, confirming the presence of cyclic amino acids (tyrosine, phenylalanine, tryptophan).
• Millon's reaction: gives a yellow-red coloration when heated with a reagent of mercury nitrates and nitrites in nitric acid (Hg(NI3)2+HNO3+HNO2), indicating the phenyl group of tyrosine.
• Adamkiewicz and Voisinet reaction: blue-violet coloration upon the addition of glyoxylic acid in sulfuric acid, which proves the presence of tryptophan.
• Pauly reaction: red coloration with diazotized sulfanilic acid in an alkaline medium (test for histidine and tyrosine).
• Sakaguchi reaction: crimson-red color after treatment with sodium hypochlorite and β-naphthol, indicating arginine.
• Nitroprusside reaction: a specific test for cysteine.

The general scheme for isolating proteins from a plant sample includes three sequential steps:

1. Grinding and homogenization of the initial plant material.
2. Extraction of proteins with a liquid solvent.
3. Isolation, purification, and acquisition of the protein in an individual state.

Quantitative methods and separation of protein fractions

For accurate determination of protein fractions and total nitrogen content in plants, laboratories use chromatography, electrophoresis, and the classic Kjeldahl method. The choice of method depends on the analysis objectives: whether proteins need to be separated by molecular size, their charge, or to find the total protein percentage in dry matter.

Protein separation is conducted using the following methods:

• Adsorption chromatography: separates proteins by polarity on a column with an adsorbent while eluting with a buffer solution.
• Partition chromatography: uses a stationary aqueous layer on silica gel or paper to separate substances based on their migration speed.
• Ion-exchange chromatography: separates proteins by their net charge (in a neutral medium at pH 7, positively charged molecules bind to a cation exchanger, negatively charged molecules — to an anion exchanger).
• Affinity chromatography: based on the selective binding of proteins to specific ligands (e.g., glucose) immobilized on a carrier.
• Gel filtration (molecular sieve method): passing the mixture through a column with Sephadex gel, agarose, polystyrene, or through porous glass beads and quartz (Porasil) for separation by molecular size.
• Electrophoresis: separation of charged molecules in an electric field based on their migration speed, which depends on the charge-to-mass ratio.

To determine the total protein content in plants, the amount of nitrogen is measured using the Kjeldahl method. This method is standard in agrochemistry and includes strictly sequential chemical operations.

1. Boiling the plant material sample with concentrated sulfuric acid in the presence of a catalyst (copper sulfate or mercury sulfate) to convert protein nitrogen into ammonia.
2. Steam distillation of the formed ammonia from the reaction mixture.
3. Quantitative determination of ammonia by titration or colorimetry using Nessler's reagent.
4. Conversion of the amount of found nitrogen into protein using coefficients established for the specific crop.

Basic chemical references for conducting protein fraction analyses are provided below:

• Neutral medium for ion exchange — pH 7
• Millon's reagent — Hg(NI3)2+HNO3+HNO2
• Nessler's reagent — alkaline solution of potassium mercuric iodide

To accurately assess harvest quality and control the nitrogen nutrition of plants, agrochemical laboratories use various methods for nitrogen determination. These analyses allow for the calculation of protein content in the plant samples being studied. In addition to classic approaches, technologies that accelerate the acquisition of results are used in practice.

In laboratory practice, three alternative methods have proven effective. The first is the Dumas method, based on the decomposition of an organic compound in a carbon dioxide atmosphere to a gaseous state, followed by measuring the volume of nitrogen. The second is the colorimetric method for nitrogen determination using phenate-hypochlorite on a "Technicon" device. Here, the intensity of the blue-green coloration is measured, which is formed through the interaction of ammonium sulfate, released during the sample mineralization process, with an alkaline solution of phenol and hypochlorite.

Optical and rapid methods for protein determination

For the rapid analysis of plant samples, the infrared spectroscopy method has become widely used. It is based on the ability of proteins to absorb light at a specific wavelength. Analyzer devices measure the intensity of the reflection of this radiation from the sample. This non-contact method allows for the prompt assessment of product quality.

Other methods for the quantitative determination of protein in the laboratory are also known. They are based on changes in the optical and physical properties of solutions upon contact with protein molecules. Agronomists use the following approaches:

• Nephelometric method — based on recording the varying degree of solution turbidity.
• Adsorption method — uses the ability of proteins to adsorb dyes (Coomassie Brilliant Blue R-250, Amido Black).
• Refractive index determination — calculates concentration based on the ability of proteins to refract light rays.