Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Protein Purification Methods
Purity Control of the Isolated Protein

To monitor protein purity, quantitative protein assays are employed. The earliest method relies on determining protein content via nitrogen quantification, featuring two main approaches: Direct and Indirect. In the direct method, Proteins are precipitated from solution using trichloracetic acid, after which the precipitate is washed and its nitrogen content is measured. The indirect method involves determining the total nitrogen content in a protein solution; the protein is precipitated, separated, and the nitrogen remaining in the supernatant is quantified. The difference between these values yields the protein nitrogen content. When the protein concentration in the solution is very low and low-molecular-weight impurities are rich in nitrogen, the measured nitrogen values (with and without protein) will be extremely close, rendering the indirect method unsuitable due to significant error margins.

Nitrogen content is determined using the Kjeldahl method or its micro-scale variation, the Conway method. The Procedure involves digesting the protein by boiling it with concentrated sulfuric acid and potassium sulfate, typically using copper sulfate as a catalyst. This reaction yields ammonium sulfate, which is then converted into ammonia under alkaline conditions. Finally, the liberated ammonia is titrated with Hydrochloric acid. The resulting nitrogen quantity is multiplied by a factor of 6.25 (the average nitrogen content in proteins). Because this conversion factor is largely conventional, a certain degree of caution is always required. In some proteins—such as core Histones—the nitrogen content can deviate significantly from the average, showing variations of twofold or more. As a rule, however, in routine cases these discrepancies do not exceed 5-10%.

Protein determination via nitrogen analysis is a labor-intensive procedure requiring relatively large sample volumes. Consequently, alternative Methods are more commonly employed, including:

> Biuret test. Biuret (NH₂CONHCONH₂) and Other Compounds containing peptide bonds develop a blue-violet coloration upon reaction with copper salts in an alkaline environment. When protein solutions are treated with Cu²⁺ ions, a purplish-blue color appears with λ_mах = 550 nm. This coloration stems from The formation of Cu²⁺ complexes with peptide bonds. The intensity of the color reaction is proportional to the number of peptide bonds and is virtually independent of other components. Micro-modifications of this method are available, enabling protein analysis at concentrations of 0.15 mg/mL or lower.

> Lowry assay. This is the most widely adopted method, combining two distinct reactions: one targeting aromatic Amino Acids and the other being the biuret reaction. Color development is achieved using the Folin-Ciocalteu reagent, which contains Cu²⁺ ions that react with proteins in the presence of strong bases and phosphomolybdotungstic acid to produce a purple-blue color. This coloration results from the formation of copper-peptide complexes and the reaction of phosphomolybdotungstic acid with Tyrosine residues, with Tryptophan, Histidine, and Cysteine residues also thought to contribute to some extent. The Lowry Method offers significantly higher sensitivity than the biuret test, allowing for the detection of 0.01 - 0.05 mg/mL of protein. However, its sensitivity heavily depends on the aromatic amino acid content of the protein, rendering it practically useless for analyzing collagens, which have an exceptionally low proportion of aromatic residues.

A major drawback of this method is the lack of a linear relationship between optical density and protein concentration across a broad concentration range. Linearity is typically observed only within a narrow protein range of 15-40 µg. To overcome this limitation, several modifications of the assay have been developed. Optical density measurements are most frequently performed at 500, 650, 750 nm, or other specific wavelengths to establish a linear calibration curve.

A second disadvantage of the method is its relatively high susceptibility to Interference from various contaminants, Nucleic Acids in particular.

> Bradford Assay. The method proposed by Bradford is based on the non-specific binding of Dyes to proteins—specifically, the capacity of proteins to readily adsorb certain dyes. In many cases, this approach yields reproducible results with remarkably high sensitivity. Generally, however, the relationship between the degree of dye sorption and protein concentration is non-linear, and the slope of the calibration curve varies considerably among different proteins.

The most commonly used dyes are Coomassie Brilliant Blue G-250 and Coomassie R (from Sigma) or the BIO-RAD reagent (manufactured by Bio-Rad Laboratories). Following incubation with the dye, the protein solution is photocolorimetered at 595 nm (across all modifications of the Bradford assay).

Rigorous evaluation of the Bradford assay has demonstrated that a linear calibration curve is typically maintained within the range of 0.5-50 µg of protein per assay (0.5-10 µg/mL). A micro-assay version of the Bradford method has also been described.

> If a UV spectrophotometer is available in the laboratory, protein analysis can be performed by exploiting their inherent Light absorption in the UV region at λ_mах = 280 nm. Tyrosine, tryptophan, and phenylalanine absorb light at this wavelength. The method exhibits high sensitivity, approximately 0.2 mg/mL. However, results are significantly skewed in the presence of nucleic acids, which display a maximum absorption peak around 260 nm. Consequently, optical density is routinely measured at two distinct wavelengths: 260 and 280 nm. This dual-wavelength approach helps minimize inaccuracies caused by nucleic acid contamination in the sample. Special correction tables and formulas are available to estimate the protein-to-nucleic acid ratio in the sample based on spectroscopic data.