Caseins, isoelectric points

Soybean Protein Isolates. Soybean protein isolates, having a protein content of >90 wt%, are the only vegetable proteins that are widely used in imitation dairy products (1). Most isolates are derived from isoelectric precipitation, so that the soybean protein isolates have properties that are similar to those of casein. They are insoluble at thek isoelectric point, have a relatively high proportion of hydrophobic amino acid residues, and are calcium-sensitive. They differ from casein in that they are heat-denaturable and thus heat-labile. The proteins have relatively good nutritional properties and have been increasingly used as a principal source of protein. A main deterrent to use has been the beany flavor associated with the product. Use is expected to increase in part because of lower cost as compared to caseinates. There has been much research to develop improved soybean protein isolates. 

At the pH = Jt there is a balance of charge and there is no migration in an electric field. This is referred to as the isoelectric point and is determined by the relative dissociation constants of the acidic and basic side groups and does not necessarily correspond to neutrality on the pH scale. The isoelectric point for casein is about pH = 4.6 and at this point colloidal stability is at a minimum. This fact is utilised in the acid coagulation techniques for separating casein from skimmed milk. 

Addition of acetic or mineral acid to skimmed milk to reduce the pH value to 4.6, the isoelectric point, will cause the casein to precipitate. As calcium salts have a buffer action on the pH, somewhat more than the theoretical amount of acid must be used. Lactic acid produced in the process of milk souring by fermentation of the lactoses present by the bacterium Streptococcus lactis will lead to a similar precipitation. 

Casein exists in milk in the form of a calcium derivative pH 4.6 is the isoelectric point of free casein, which is soluble to the extent of only 0.11 g. per liter.1... 

Difficulties in detecting nucleolin on the cell surface could be explained by its very low concentration in this compartment (Hovanessian et al, 2000). Moreover, this cell surface expressed nucleolin protein has a different isoelectric point and it is recognized by only one monoclonal antibody (mAb D3) in its native conformation (Hovanessian et al, 2000). This probably reflects specific post-translational modifications undergone by nucleolin on the cell surface. Consistent with this hypothesis, extracellular nucleolin is a substrate of ecto-protein kinases including casein kinase II (Dumler et al, 1999 Jordan et al., 1994). Interestingly, indirect evidence suggests... 

Kermack and Wright Biochem. J. XVII. 635,1923) have shown that gelatine at a of 4 T exerts but little protective power on a negative gum benzoin sol, acid gelatine precipitates the colloid in small concentrations whilst alkaline gelatine protects it. Similar observations have been made by Zsigmondy on the effect of casein on gold, but its exact isoelectric point was not established. 

Some like casein, salt free globulin and acid albumin are not heavily solvated in solution and are thus readily precipitated at the isoelectric point in a manner similar to the suspensions already considered. Others, e.g. glutin, gelatine and natural albumin, are solvated like silica which at the isoelectric point are not necessarily precipitated being maintained in the sol form by the solvent. On removal of the stabilising water however by the addition of alcohol or neutral salts precipitation will occur and this most readily at the isoelectric point. 

The self-assembly of caseins may be readily manipulated by processing methods that affect the integrity of native casein micelles and the character of the casein interactions in aqueous media. Examples of such procedures are (Dickinson, 2006) (i) acidification toward the isoelectric point (p/) (pH 4.6-4.8), leading to a neutralization of the net protein charge (ii) enzyme action, as exploited in the production of cheeses and fermented milks (iii) addition of divalent ions, especially, Ca2+ ions (iv) addition of sucrose or ethanol (v) temperature treatment and (vi) high-pressure treatment. 

Co-preclpltate is an insoluble milk protein product that is produced by heating skinimllk to high temperatures ( > 90 C) to denature the whey proteins and complex them with the casein micelles. The heated system is subsequently adjusted to isoelectric point conditions of pH 4.5-5 to precipitate the complexed whey protein-casein micelles, centrifuged or filtered to recover the precipitate, washed and dryed. The resulting product, which is virtually insoluble, exhibits only minor functionality in most typical emulsification applications. 

They aggregate and precipitate from solution when the pH is adjusted to the isoelectric point of caseins (c. pH4.6). Precipitation at this pH, which is temperature-dependent (i.e. does not occur at temperatures below 5-8°C and occurs over a wide pH range, perhaps 3.0-5.5, at higher temperatures, e.g. 70°C), occurs owing to the loss of net positive or negative charge as the pH approaches 4.6. 

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

Commercial casein is usually manufactured from skim milk by precipitating the casein through acidification or rennet coagulation. Casein exists in milk as a calcium caseinate-calcium phosphate complex. When acid is added, the complex is dissociated, and at pH 4.6, the isoelectric point of casein, maximum precipitation occurs. Relatively little commercial casein is produced in the United States, but imports amounted to well over 150 million lb in 1981 (USDA 1981C). Casein is widely used in food products as a protein supplement. Industrial uses include paper coatings, glues, plastics and artificial fibers. Casein is typed according to the process used to precipitate it from milk, such as hydrochloric acid casein, sulfuric acid casein, lactic acid casein, coprecipitated casein, rennet casein, and low-viscosity casein. Differences... 

The physical and chemical characteristics of cheese curd depend on the method used to form the curd matrix. The curd is formed in basically one of two ways acid or enzymatic coagulation. In acid curd cheeses (cottage, baker s, cream), the curd is formed by direct addition of acid to the milk or by lactic acid produced by the fermentation of lactose. As the pH of the milk approaches the isoelectric point of casein (pH... 

In the first part of this experiment, you are going to isolate casein from milk which has a pH of about 7. Casein will be separated as an insoluble precipitate by acidification of the milk to its isoelectric point (pH = 4.6). The fat that precipitates along with casein can be removed by dissolving it in alcohol. 

Casein has an isoelectric point at pH 4.6. What kind of charges will be on the casein in its native environment, that is, in milk ... 

Proteolytic modification has special importance for the improvement of solubility of proteins. This effect becomes significant even after very limited proteolysis. Hydrolysis of casein to DH of 2 and 6.7% with Staphylococcus aureus V8 protease increased the isoelectric solubility to 25 and 50%, respectively (Chobert et al., 1988a). However, it should be noted that the solubility profiles were not identical, due to a shift of the isoelectric point of the modified proteins. Solubility of a protein hydrolysate depends on the enzyme used (Adler-Nissen, 1986a). Protamex (a Bacillus proteinase complex) hydrolysates of sodium caseinate (DH 9 and 15%) displayed 85-90% solubility between pH 4 and 5 (Slattery and FitzGerald, 1998). 

Fischer and Schmitz were also interested in the interaction of heparin with proteins, such as casein and serum albumin. They reported that heparin appeared to shift the isoelectric point of the proteins to the acid side, probably by the formation of a molecular complex, and that combination with the protein occurred only near the isoelectric point and on the acid side." The complex was reversibly dissociated by the addition of alkali. These results are of irnportance in selecting conditions for the purification of crude heparin preparations, and constitute the basis for the extraction procedure developed by Charles and Scott, which has subsequently been used extensively. 

A stomach (pH 1.5-3.5), where many proteins are brought below their isoelectric point (e.g., the curdling of casein), proteolytic enzymes are introduced, and protein-based matrixes are further reduced by churning gastric lipases may also be introduced into the digesta. 

However, the procedures used in the dairy industry, rennet and acid casein, have the disadvantages to produce large volume of chemical effluents due to the addition of bases and acids during treatments, and to generate inherent risk linked to handling, stocking, and transportation of concentrated bases and acids. In acid casein production, milk pH is decreased to the isoelectric point of the casein by addition of strong acid (hydrochloric, sulfuric, nitric, lactic, etc.). H" " concentration of milk is increased... 

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