Chemistry and Biology of Proteins - F. Haurowitz 1953

Methods for Isolation, Purification, and Characterization of Proteins
Criterion of Protein Purity

In organic chemistry, The formation of homogeneous crystals is regarded as definitive proof of compound uniformity. If crystals exhibit a reproducible melting point that remains unchanged following recrystallization, the purity of the compound is considered firmly established. This criterion of purity is inapplicable to protein crystals because Proteins decompose upon heating. Until recently, the ability of certain proteins to crystallize was viewed as an indication of their homogeneity. However, examining The behavior of crystalline proteins in an Electrophoresis apparatus demonstrated that this assumption was incorrect. It turned out that egg albumin, which is readily recrystallized and was long considered a uniform substance, actually represents a mixture of at least two components that can be separated electrophrophoretically [19]. Similarly, crystalline serum albumin [20] and crystalline ricin from the castor bean [21] were shown to be mixtures of two or more components. These findings leave no doubt that crystallization alone does not yet ensure the isolation of homogeneous, pure proteins.

Solubility is also utilized as a criterion of compound purity. It is well known that the solubility of a homogeneous substance is constant and remains unaffected by the presence of an excess of solid phase. This method is hardly suitable for determining the Solubility of proteins in pure Water, since Protein solubility in water is strongly dependent on traces of electrolytes and on the concentration of hydrogen or hydroxyl ions. METABOLISM/18.html">The Influence of these substances can be eliminated by using a concentrated salt solution as the solvent. When testing the solubility of crystalline egg albumin or carboxyhemoglobin in an ammonium sulfate solution, it was found that solubility depends to some extent on The amount of the solid phase [22]. On this basis, it was concluded that the molecules of these Proteins can be regarded as systems of reversibly dissociating components. Highly meticulous determinations of the solubility of crystalline chymotrypsinogen [23] and Ribonuclease [24] have shown that these proteins behave as homogeneous compounds. It is highly significant that these homogeneous proteins are Enzymes (see Ch. XII).

In recent years, electrophoresis at various pH values has been employed to determine protein homogeneity (see Ch. V). Electrophoretic analysis has demonstrated that crystalline egg albumin, bovine serum globulin, ß-lactoglobulin, and all other protein preparations investigated by this method represent mixtures of several proteins [25]. It is quite possible that perfectly uniform protein molecules do not exist at all, and that the so-called "pure" proteins are in reality mixtures of very similar protein molecules. To this day, however, it remains unclear whether the heterogeneity of proteins detected by electrophoresis or solubility studies is a consequence of alterations occurring during the isolation of proteins from their natural environment, or whether this heterogeneity is inherent to native proteins of Cells and Body Fluids.

Indeed, it was discovered that upon the action of Bacillus subtilis on egg albumin, the crystal form, electrophoretic properties, and Molecular Weight of this protein change significantly [26]. It was established that a small amount of nitrogenous substances is cleaved from egg albumin in the process, resulting in a new protein related to the original one, the so-called plakalbumin. Analogous changes in The properties of albumin are also observed during the Aging of its solution [27].

It is entirely plausible that the Methods of Protein isolation themselves bring about similar alterations in the PHYSICOCHEMICAL PROPERTIES OF the isolated proteins. Naturally, the answer to this question is of great importance, and the Structure/127.html">Interpretation of Results from chemical and physicochemical studies of proteins depends largely on resolving this issue.

While we can gauge the purity and homogeneity of soluble proteins to a certain extent through crystal formation and the determination of their physicochemical properties, we lack these possibilities for insoluble proteins such as keratin or Collagen. Consequently, it remains completely unknown whether these proteins are homogeneous or represent mixtures of various proteins.

¹ In 1947, V. N. Orekhovich, A. A. Tustanovsky, and K. D. Orekhovich isolated a readily crystallizable protein from the Skin of various animals, which the authors named procollagen (Dokl. Akad. Nauk SSSR, 57, 5, 1947). In acidic extracts from skin or tendons, this protein is present alongside collagen, and A number of authors (Nageotte, Fauré-Fremiet, et al.) regarded it as a special variety of collagen which they termed soluble collagen. In subsequent research by V. N. Orekhovich, A. A. Tustanovsky, K. I. Strachitskaya, K. D. Orekhovich, N. A. Brinsker, and N. E. Plotnikova (V. N. Orekhovich, Procollagens, Their Chemical Composition, Properties, and Biological Role, Publishing House of the USSR Academy of Medical Sciences, Moscow, 1952), however, it was established that procollagen differs significantly from collagen in its Amino Acid Composition and a number of physicochemical properties. These investigators thus demonstrated the erroneousness of Nageotte's and other authors' views and simultaneously elucidated the cause of many discrepancies existing in the literature regarding the properties of collagen.

These discrepancies in most cases depend on the presence of procollagen as an impurity in the studied collagen preparations. For more detailed data on the COMPOSITION AND PROPERTIES of procollagen, see the note on p. 210. — Ed.