Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin, I. P. 1968

Isolation and Purification of Proteins
Assessment of Protein Purification Efficiency
Quantitative Determination of Proteins

Reliable Methods for Quantitative Protein Determination are primarily required to monitor the degree of purification and assess the quality of the final preparation. A widely used approach is protein assay based on nitrogen content in the precipitate formed upon The addition of trichloroacetic acid (TCA). In 10% TCA, the vast majority of Proteins undergo complete precipitation. It should only be borne in mind that at very low protein concentrations (e.g., less than 1 mg/mL), quantitative determination and loss-free washing of the precipitate are not always feasible. Protein nitrogen can be determined either directly in the precipitate or by the difference in nitrogen content in the solution before and after protein precipitation with TCA. The latter, "indirect" option is more popular as it avoids the difficulties associated with collecting and washing small-volume precipitates. However, it is poorly suited when the solution contains high concentrations of nitrogen-containing substances that are not precipitated by TCA. The nitrogen determination itself is carried out using the classical Kjeldahl method or its modifications (micro-methods, Conway's method, etc.). The Procedure boils down to digesting the protein with concentrated sulfuric acid and potassium sulfate in the presence of catalysts (copper sulfate, selenium, or mercury salts) until nitrogen is completely converted into ammonium sulfate, followed by its conversion to ammonia (by adding alkali), distillation, and Quantitative determination of the latter (titrimetrically or using color reactions). A detailed Description of the method can be found in many practical guides to biological chemistry. Here, we only note that caution is required when calculating The amount of protein from the detected nitrogen. The conversion factor of 6.25 used for this purpose is an average value. As mentioned above, A number of proteins exhibit significant deviations from the average nitrogen content. These deviations are particularly large for basic Nuclear Proteins, where discrepancies can be more than twofold. As a rule, however, deviations do not exceed 5–10%.

Determining protein content via nitrogen quantification is a relatively cumbersome procedure. For assays during purification, simpler, albeit less reliable, methods can be successfully employed. Good results are yielded by techniques based on the so-called biuret reaction. The Essence of this color reaction will be discussed later. Here, we merely point out that the intensity of the developed color is a function of the number of peptide bonds in the protein and is practically independent of its other components. Therefore, the reaction efficiency is more or less constant when transitioning from one protein to another. Micro-variants of this method have been described, enabling the quantitative Determination of protein at concentrations of 0.15 mg/mL and higher. Special attention is merited by the Itzhaki and Gill modification, in which the colorimetry of the reaction products is performed in ultraviolet light at λ = 310 mµ.

The Lowry Method has also gained widespread use, combining two reactions—one for aromatic Amino Acids (using Folin's reagent) and the biuret reaction. Its sensitivity is significantly higher, reaching down to 0.01–0.05 mg/mL. However, its efficiency is not uniform for proteins that differ substantially in their aromatic amino acid content. Furthermore, the accuracy of Lowry assay results is noticeably affected by many impurities, such as Nucleic Acids.

For rapid preliminary and comparative estimates, protein determination based on ultraviolet Light absorption at λ = 280 mµ—caused by its constituent aromatic amino acids—can be useful. Its sensitivity is quite high, at about 0.2 mg/mL. However, in the presence of nucleic acids, the results are significantly distorted. These distortions can be partially corrected by measuring absorption not only at 280 but also at 260 mµ, the region of maximum light absorption by nucleic acids. Special tables and formulas are available to estimate The ratio of protein to nucleic acid content from these data. Nevertheless, this method cannot be recommended for precise quantitative determinations of poorly studied proteins.

We have examined only those methods of quantitative protein determination that can be particularly useful for monitoring the course of Isolation and Purification. Numerous other, less universal methods are known and applied to solve different problems. These include, for instance, determining Blood serum protein concentration via its specific gravity—a very valuable method for clinical analyses. Also deserving special mention is the highly sensitive method of protein determination based on ultraviolet light absorption at λ = 200-:-220 mµ (the absorption region of protein peptide groups). Its widespread adoption is currently hindered by the extreme scarcity of specialized equipment.