Chemistry and Biology of Proteins - F. Haurowitz 1953

Methods for Protein Isolation, Purification, and Characterization
Quantitative Determination of Proteins

The precipitates obtained by one method or another (see the previous section) can be used for the Quantitative determination of their protein content via the Kjeldahl method. When employing this method, it is assumed that the precipitate contains solely protein nitrogen. However, this assumption is not entirely accurate, because other macromolecular compounds—such as polysaccharide acids (e.g., chondroitinsulfuric acid) and certain nitrogen-containing Lipids (e.g., lecithins or kephalins)—are co-precipitated with Proteins. The Kjeldahl method is based on converting the protein nitrogen present in a sample into ammonium sulfate by digesting the protein with concentrated sulfuric acid and potassium sulfate in the presence of a catalyst. It is usually assumed that the nitrogen content of all proteins is 16%. In reality, this value varies considerably. For instance, egg albumin contains 15.75% nitrogen, whereas edestin contains 18.7% [34]. Hence, it is clear that the exact nitrogen content in the protein under investigation must be known. Although the Kjeldahl Procedure is a standard method used in biochemical laboratories, the results obtained by it are often somewhat underestimated. The most accurate data are obtained when mercury is used as a catalyst [35, 36]. Heating the protein with sulfuric acid and potassium sulfate in the presence of a catalyst should be continued for 8 hours [34]. Adhering to these conditions has yielded precise data for A number of proteins whose nitrogen content was known.

Interestingly, under certain pathological conditions of the Organism, proteins with a nitrogen content deviating significantly from the norm can be detected in its Tissues. For example, it was found that in patients suffering from famine-induced edema, Serum proteins contain less nitrogen than normal [37]

In its original form, the Kjeldahl method is somewhat complicated due to the necessity of distilling off the generated ammonia. Several modifications of this method have been proposed in which ammonia distillation is eliminated. For instance, it was suggested to oxidize the ammonium sulfate resulting from Digestion with hypobromite (NaOBr) and to titrate the excess hypobromite iodometrically [38]. Ammonia content can also be determined colorimetrically using Nessler's reagent. Conway proposed conducting the ammonia distillation at room Temperature in closed dishes (Fig. 1) consisting of two concentric circular compartments [39, 40]. Despite all these simplifications, the Kjeldahl method remains rather labor-intensive. Attempts to replace the Kjeldahl method with nephelometric or colorimetric protein determination Procedures have been described in the literature. However, nephelometric Methods yield only approximate values, since the turbidity produced by precipitating agents depends partly on the concentration of salts and other impurities. Better results are achieved through the Colorimetric determination of protein via the biuret reaction [41–43].

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Fig. 1. Cross-section of Conway's dish [39].

1 — outer vessel; 2 — Glass lid; 3 — inner vessel containing acid; 4 — ammonium salt solution to which potash is added. Dish diameter 70 cm.

¹ The decrease in the nitrogen content of serum proteins in these cases is caused not by the appearance in the serum of proteins containing less nitrogen, but by a shift in The ratio of various proteins within it. Numerous studies have demonstrated that during starvation accompanied by edema, the albumin content drops particularly sharply. The amount of various globulins decreases less drastically and sometimes even remains unchanged. Since the nitrogen content in individual serum proteins is not uniform, A change in the quantitative proportions of these proteins must lead to corresponding alterations in the nitrogen content of total serum proteins. To date, however, there is no evidence that the nitrogen content in individual pure proteins changes during starvation or under any other pathological conditions. All data on this issue obtained by Schenck, Lang, Dirr, and other authors proved erroneous upon subsequent verification, stemming from the inaccuracy of the Analytical Methods employed in those authors' studies. — Ed. note.

Since the specific gravity of Blood serum largely depends on its protein concentration, the latter can be determined by measuring the specific gravity of the serum in test tubes containing a mixture of bromobenzene and kerosene [44]. Potassium sulfate solutions of varying densities or copper sulfate solutions are used as reference standards. However, neither of these methods is free from errors caused by the partial dependence of serum specific gravity on non-protein substances [45, 46].

From this Structure/133.html">Discussion, it is evident that the quantitative determination of proteins is a rather complex procedure. Large amounts of protein can be determined by direct weighing of the dried precipitate obtained either by boiling protein solutions or by precipitating the protein at its isoelectric point. The main difficulty here lies in removing salts and other soluble compounds, as protein precipitates often form colloidal solutions when washed with distilled Water. Sometimes this can be avoided by washing the protein precipitate with dilute acetic acid or a 1 : 1 mixture of methyl alcohol and water, followed by acetone and ether.

To determine very small amounts of protein, one can utilize the ability of Tyrosine to intensely absorb ultraviolet light at 280 mμ [47] or the ability of proteins to reduce Folin's reagent (phosphomolybdic acid) with The formation of a blue color [48]. Both methods are very simple and rapid. However, it must be kept in mind that ultraviolet absorbance and the blue color developed with Folin's reagent depend on the content of tyrosine and other aromatic Amino Acids, which varies from protein to protein. Therefore, the analyzed protein sample must be standardized against a known protein.