Chemistry and Biology of Proteins - F. Haurowitz 1953

Conjugated Proteins
Phosphoproteins

Phosphoproteins differ from Nucleoproteins in that they lack purine and pyrimidine bases; the phosphoric acid residues within their Structure are bound by ester linkages. The most thoroughly studied phosphoprotein is milk casein, which has a phosphorus content of approximately 0.9%. Upon Hydrolysis with acids or alkalis, phosphoric acid is split off from casein. This phosphoric acid imparts acidic properties to the protein, which has an isoelectric point at pH 4.6–4.7. Casein precipitates from milk upon The addition of acetic or other acids, although it redissolves in the presence of excess strong acids. Casein is not a homogeneous protein; fractional precipitation using aqueous or alcoholic acid solutions allows it to be separated into several fractions [72, 73] with molecular weights ranging from 75,000 to 375,000 [74]. The principal fractions, designated as a- and ß-casein, differ in their Amino Acid Composition (see Table 1) [75] and electrophoretic mobility [73].

Phosphoric acid is linked via an ester bond to the hydroxyl groups of Serine residues. Enzymatic hydrolysis of casein with Trypsin releases phosphoserylglutamic acid [76, 77]. Partial hydrolysis of casein using dilute Hydrochloric acid yields serine phosphoric ester [78].

Phosphoproteins can be synthesized in vitro by phosphorylating Proteins with phosphorus oxychloride (POCl₃) [79, 80]. However, these synthetic phosphoproteins differ from natural ones in that a significant portion of their phosphoric acid is bound to amino groups and Tyrosine hydroxyl groups. Furthermore, they exhibit greater resistance to alkaline hydrolysis than natural phosphoproteins.

Casein precipitates from milk not only upon the addition of acids, but also through the action of chymosin (rennin), an enzyme produced by the gastric mucosa. Clotting of milk in The Stomach is a key factor enabling the assimilation of milk proteins. If milk did not coagulate in the stomach, it would pass through at the same rate as Water or other liquids, leaving the proteins undigested. The exact mechanism of coagulation remains unclear. It has been suggested that the insoluble curd consists of a casein dimer or polymer, or perhaps a complex of casein and calcium phosphate. Milk clotting has also been attributed to The conversion of caseinogen into casein, or casein into paracasein. However, the terms caseinogen and paracasein are rarely used today, as nothing definitive can be said about these compounds or their precise role in milk coagulation. It is possible that the enzyme induces a degree of unfolding in the peptide chains of casein, enabling polar and ionic groups to interact and form intermolecular salt-like bonds. This process occurs exclusively in the presence of Calcium Ions. The ability of casein molecules to unfold their chains and form fibers has been utilized in the manufacture of artificial wool from casein. By forcing alkaline casein solutions through fine orifices into an acidic bath, filaments are formed which, after Treatment with formaldehyde, harden and can be processed into yarn.

¹ Regarding the coagulation of milk by chymosin, see the note on page 292. — Ed.

While casein has been the subject of numerous investigations, egg and roe phosphoproteins remain comparatively poorly studied. The phosphoproteins of egg yolk are collectively known as vitellins. To isolate vitellin from chicken eggs, the yolk is extracted with a 10% sodium chloride solution, Lipids are removed from the extract using diethyl ether, and the lipid-free proteins are precipitated by diluting the solution with water [82]. Vitellins contain 0.92% phosphorus [82]. Treatment of vitellin with a 12% ammonia solution releases vitellinic acid. Hydrolysis of this acid with 2 N hydrochloric acid results in the Cleavage of serine phosphoric ester [83].

These findings demonstrate that phosphoric acid is linked to the protein moiety in vitellin in the same manner as in casein. The Biological Role of vitellin is likewise analogous to that of casein, serving as a vital source of phosphorus and Essential Amino Acids for the developing Organism.

In addition to vitellin, two other phosphoproteins have been isolated from egg yolk. One of these, termed livetin, is obtained by heating the filtrate remaining after the precipitation of vitellin [84]. The second phosphoprotein, designated phosvitin, is precipitated by copper salts from a yolk extract in which other proteins have been pre-precipitated with sodium sulfate. Phosvitin contains 10% phosphorus, 12% nitrogen, and lacks sulfur entirely. Its molecular weight is 21,000. Each phosvitin molecule contains 31 phosphorus atoms and 33 hydroxy amino acid residues [85]; the phosphoric acid in phosvitin is apparently bound to serine [85].