MILK PROTEINS

The contribution of milk proteins to human nutrition can be appreciated when it is realized that on a per capita basis, about 25% of an American's daily protein comes on dairy products. Milk proteins are also responsible for the stability of the 3.5% fat that occurs in the aqueous phase of milk. Some very specific interactions of these proteins are also responsible for the low viscosity of milk even though it has a moderately high fat content and contains about 3.5% protein. The major proteins of milk can be divided into two classes on the basis of their structure and physicochemical behavior. These classes, casein and whey, will be considered separately due to the vast differences in their behaviors.

Casein

"Whole casein" has been operationally defined as a heterogeneous group of phosphoproteins precipitated from raw skim milk at pH 4.6 and 20C. Casein comprises about 83% of the total milk protein and bovine milk ranges from 2.5 to 3.2% casein. The caseins can been divided into four major and one minor group with the properties indicated in Table 1.

Table 1. Distribution and properties of caseins.

alpha s Casein

In 1956, the fraction of casein known until that time as a-casein was shown to be composed of two proteins, alpha s - and kappa casein. as was defined as the fraction of whole alpha-casein precipitating in 0.4M CaC1₂ at pH 7 and 0.4 C. Isolated a s-casein is very sensitive to calcium 2 and will begin to precipitate at 0.007M. Precipitation is virtually complete at 0.02M.

The amino acid sequence of a s-casein has been determined as is presented in Figure 1. From this data, it can be seen that the molecule contains 199 amino acids and 8 phosphate groups that are esterified to serine groups. The molecule has a net charge of about -24 at pH 6.7 and contains no cysteine residues.

The molecular weight of a s-casein B is 23, 644 and the molecule has an average hydrophobicity of 1170. One segment of the molecule (45-89) is highly charged. This segment contains all 8 phosphate groups, 12 carboxyl groups and 2 epsilon amino groups and has a net charge of close to -23 at pH 6.7. This means that the rest of the molecule can only have net negative charge of -1 at the pH of milk.

Alpha s1-casein has three very hydrophobic regions that include residues 1-44, 90-113 and 132-199. The molecule contains 17 proline residues which are almost randomly distributed within the hydrophobic portion of the molecule. This distribution of these structure breaking residues effectively precludes the presence of secondary structure in the molecule.

The native molecule will precipitate when eight Ca⁺⁺ ions are bound. The calcium binding reduces the charge of the molecules from -24 to -8 and this residual charge apparently is not enough to prevent aggregation. Recent evidence indicates that the binding of Ca⁺⁺ to a s1-casein causes a conformational change in the protein which causes more hydrophobic groups to become exposed to the solvent.

There are four genetic variants of alpha s1-casein known with variant B being by far the most common. Variants C and D differ by only one amino acid from B and probably arose from point mutations. Variant A contains 13 less amino acids than do the other caseins (amino acids 14-26) and gene deletion must have occurred.

Alpha s-casein comprises from 44 to 55% of the total casein and a s1 comprises approximately 80% of the total a s casein. The very uneven distribution of charged and hydrophobic amino acids must have an effect on the shape of the a s-casein molecules. The large hydrophobic regions will attempt to minimize as much as is possible its contact with water. This probably leads to a largely globular structure that is as compact as possible. The absence of charged groups and presence of larger number of proline residues effectively prevent this portion of the molecule from assuming much, if any, secondary structure.

The highly charged, 46 amino acid segment of the molecule will attempt to maximize its contact with water. The molecule thus has one end that is highly negatively charged and another end that contains little charge, but is largely apolar. The sequence of a s casein B is presented bellow:

Figure 1. Primary sequence of bovine a s1- casein B. The amino acids in brackets are the sites that are different in genetic varients A, C and D.

Isolated with the a s1 caseins are the a s2 family of caseins. These caseins contain 8 more amino acids, have from 10 to 13 phosphates and also 2 cysteines. The a s 2 caseins have an average molecular weight of about 25,100. The primary sequence for a s2 casein variant A is given below.

Figure 2. Primary sequence of bovine a s2- casein, varient A.

Beta Casein

Beta-Casein comprises from 25-35% of the total casein. It is made up of 209 amino acids and its primary sequence is given in figure 2. The molecule contains 5 phosphates each as a serine phosphate ester. b-casein contains no cysteine residues and contains 17% proline which is randomly distributed throughout the molecule. The protein has a molecular weight of 23,980 and a net charge of -13 at pH 6.7.

The N termed segment of the molecule, 1-42, contains all of the phosphate groups and all of the molecules net charge. The remaining 80% of the molecule contains no net charge and is very hydrophobic. The entire molecule has an average hydrohphobicity of 1335.

The molecule is even more asymmetric than alpha s1-casein and similar types of behavior can be postulated for it. b-casein will also precipitate in the presence of calcium, but the phenomenon is strongly temperature dependent. Removal of the very hydrophobic C terminal tripeptide -ILE-ILE-VAL will prevent the association of beta-casein.

At 4 C and the ionic environment of milk, b-casein is soluble, unlike a s-casein which will be precipitated by the calcium present. As the temperature is increased, the b-casein will become less soluble and will tend to associate with other casein molecules.

Intrinsic viscosity data suggests that at 4 C, b-casein is highly asymmetric and may even exist as a random coil. The viscosity doesn't increase when the molecule is placed into 6M guanidine HC1 suggesting a lack of secondary structure. At temperatures above 13 C, there is evidence for the existence of small amounts of a helix (10%) in the structure of beta-casein. There are five genetic variants of b-casein, all of which are the result of one amino acid change. The primary sequence of b-casein is given in figure 3.

Figure 3. Primary sequence of bovine b-casein A. Amino acid substitutions are indicated for genetic varients B, C and E. Arrows indicate points of hydrolysis to yield g-caseins.

Gamma Casein

About 5% of the casein is made up of a heterogeneous group of proteins previously known as g-caseins. It has now been demonstrated that the gamma-caseins occur as the result of limited proteolysis of b-casein. The proteolysis results from the activity of the enzyme plasmin which apparently is transmitted from the blood into the milk in small quantities.

Figure 4 shows the relationship between the g-casein and b-caseins. The committee on nomenclature of the American Dairy Science Association has recommended the following nomenclature for the g-caseins: g-casein (b-casein segment 29-209, ~ 20,600 daltons); g-casein (b-casein segment 106-209 ~ 11,800 daltons); g-casein (b-casein segment 108-206 ~ 11,600 daltons).

The g-caseins, being derived from the C terminal portion of b-casein, have very low net charges and are extremely hydrophobic. These molecules can be solubilized by ethyl alcohol and other solvents of low dielectric constant. The N terminal portion of the beta-casein is the source of proteose peptones 5, 8-fast and 8-slow (see below).

Figure 4. The relationship of gcaseins and proteose peptones 8 and 5 to b casein.

Kappa-Casein

Kappa-casein consists of 169 amino acids and has a molecular weight of 19,005. The protein can contain from 0 to 5 trisaccharide units composed of a N-acetylneuraminyl (2-->6) b-galactosyl (1-->3 or 6) N-acetylgalactosamine. It contains one serine phosphate group and two cysteine residues. There are two genetic variants of kappa-casein. The primary sequence of kappa-casein is presented in Figure 5.

Theaverage hydrophobicity of k-casein is 1205. Unlike a s and b-caseins, k-casein has charged sections at both ends of the molecule. The 53 C-terminal amino acids have a net charge of -11, contain the 1 phosphate group and all of the carbohydrate associated with k-casein. This carbohydrate further increases the net negative charge of this portion of the molecule. The remainder of the molecule is very hydrophobic and contains net positive charge at pH 6.7.

Most of the molecules net charge is not derived from serine phosphate groups and thus k-casein is soluble in the presence of Ca⁺⁺. It can also interact with a s and b-caseins and stabilize them in the presence of calcium ions. Extensive studies with model systems have demonstrated that while the complex is stable to the presence of Ca⁺⁺. In fact, Ca⁺⁺ must be present for the complex to form.

It has also been observed that there is a relationship between the amount of k-casein present and the size of casein micelles. Large micelles tend to have less k-casein than do small micelles and when k-casein is added to a suspension of micelles, the average micelle diameter decreases. This suggests that k-casein limits the size of casein micelles by interacting at the surface of these units.

When milk is exposed to the enzyme rennin, the casein micelles are destabilized and precipitated. It has been demonstrated that rennin cleaves a bond between PHE 105 and MET 106 in the k-casein molecule. This results in the liberation of the soluble C terminal portion of the molecule. This (glyco) macropeptide has a molecular weight of ~6,800 and contains the serine phosphate and carbohydrate groups of the molecule. The remainder of the k-casein molecule is called para-kappa-casein. It has a molecular weight of 12, 271 and a net positive charge at pH 6.7. This molecule is extremely hydrophobic and is insoluble. The structure of k casein is presented in figure 5.

Figure 5. Primary sequence of bovine k-casein B. Substitutions for genetic varient A, point of attack by rennin and point of attachment of carbohydrate are indicated.

Phosphorylation of Casein

Many of the important properties of casein are related to its ability to exist in the micelle form. The micelles exist, in part, due to the calcium sensitivity of as and b-caseins. This sensitivity to precipitation due the the binding of calcium is due in large part to the presence of serine phosphate residues in these molecules.

The genetic code does not provide for the incorporation of serine phosphate residues directly into proteins, so that regular serines must be modified after the synthesis of the protein. This modification occurs in the Golgi apparatus and is mediated by the action of a specific casein kinase.

Examination of the Sequence of a s and b-casein as well as the location of serine phosphate groups has led to the suggestion that the casein kinase recognizes the following sequence:

Where 1 is the serine or in one genetic variant threonine that will be phosphorylated. Residue two, designated by X can be any amino acid and residue three appears to be a recognition site for the enzyme.

If the enzyme finds a glutamic acid or serine phosphate residue, that is 2 residues removed from a serine or threonine residue, that serine or threonine will be phosphorylated. This suggested rule holds in all cases for as-casein, not quite as much for the kappa-casein and most of the beta-casein of milk.

At reduced temperatures as much as 30% of the b-casein and a lesser amount of the k-casein can be found in the free state in the milk serum. The micelles remain intact, however. Milk contains far more calcium phosphate than would be soluble in water at pH 6.7. Much of this is found associated with the casein micelles in an undissociated form. The micelles in milk can be destabilized by the action of the enzyme rennin, the addition of acid to pH 4.6 or by the addition of ionic calcium.

Rennin causes cleavage of the kappa-casein molecules and thus removes the factor that protects alpha s-casein from calcium precipitation. The additional ionic calcium seems to overload the kappa-casein stabilization of the alpha s-casein and causes precipitation to occur.

The reduction of the pH to 4.6 has two effects that lead to instability. As the pH decreases, there is a shift in the equilibrium constant for the dissociation of colloidal calcium phosphate to the ionic species. This causes the calcium ion activity to increase which leads to insolubility. More importantly, the casein have an isoelectric point of pH 4.6 and those proteins are insoluble at this pH. Since the discovery of k-casein , a number of models have been prepared to describe the structure of casein micelles.

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