The proteins that remain in solution after removal of casein are by definition termed whey proteins. Table 2 gives the composition of the proteins in milk.
Table 2. Distribution of milk proteins.
The most prevalent protein in whey is b-lactoglobulin. It comprises 10% of the total milk protein or about 58% of the whey protein. It contains 162 amino acids with a molecular weight of about 18,300. There are two genetic variants, A and B, that differ in the substitution of a glycine in Variant B for an aspartic and in Variant A. The molecule contains two disulfide and 1 free sulfhydryl groups and no phosphorus.
The primary sequence of b-lactoglobulin is given in Figure 8. One of the disulfide groups is shown between CYS 66 and 160. The other seems to be a dynamic one that involves 106 and is sometimes found with CYS 121 and sometimes with CYS 119. Thus, 1/2 of the CYS 119 and 1/2 of the CYS 121 exist as free sulfhydryl groups.
|41||Gln in Varient D||51||Varient C His|
|61||Gly in Varients B, C||71|
|101||111||Varients B, C Ala|
Figure 8. Primary structure of bovine b-lactoglobulin A. The locations of the amino acid substitutions in the genetic varients are indicated. There is a disulfide bound between cys 66 and cys 160. Another disulfide bound is formed between cys119 and cys 119 and cys 121. There is a 50:50 distribution of the bond between positions 119 and 121. Cys 121 is always involved in the bond.
Below pH 3.0 and above pH 8.0, b-lactoglobulin exist as a monomer. Between pH 3.1 and 5.1 at low temperatures and high protein contents, it associates to form an octamer. This polymerization seems to be mediated through the action of carboxyl groups and thus the A Variant forms better octamers than does the B Variant. At other pH values, including the pH of milk, beta-lactoglobulin tends to be found as a dimer. These dimers are spherical with diameters of about 18A. The complex association-dissociation behavior of beta-lactoglobulin has been the subject of extensive study.
Beta-lactoglobulin is manufactured specifically in the mammary gland for inclusion in milk where its role is unknown. All ruminant milk contains b-lactoglobulin while the milk of almost all non-ruminants does not. While biological functions have been speculated to exist for b-lactoglobulin, to date none have been fully accepted. The molecule has a very hydrophobic area that is quite effective in binding retinol. Some speculate that the binding of Vitamin A may have a regulatory role in the mammary gland. Because of its prevalence in bovine milk, to a large extent the properties of whey protein concentrates, are in effect, the properties of b-lactoglobulin.
The secondary structure of b-lactoglobulin is homologous to that of retinol-binding proteins. It contains 9 strands of beta structure, 8 of them arranged to form a bbarrel. The lone ahelix is located on the surface of the molecule. The center of the barrle is hydrophobic and can be involed in the binding of hydrophobic molecules. The three deminsional structure of b-lactoglobulin is presented in figure 9.
Figure 9. The three-dimensional structure of bovine b-lactoglobulin. Original structure by Sawyer, reproduced from Swaisgood, 1996.
A rasmol version of the structure can be found here.
The second most prevalent protein in whey is a-lactalbumin which comprises about 2% of the total milk protein which is about 13% of the total whey protein. The molecule consists of 123 amino acids and has a molecular weight of 14,146. The molecule contains 4 disulfide linkages and no phosphate groups. Its primary structure is shown in Figure 10.
|1||Arg in Varient B|
Figure 10. Primary structure of bovine a-lactalbumin B. The position of the amino acid substitution that occurs in genetic varient A is indicated. Dissulfide bounds are formed between the following pairs of cys residues: 6 and 120, 28 and 111, 61 and 77 and 73 and 91.
Alpha-lactalbumin has been shown to modify the activity of the enzyme galactosyl transferase. In the absence of a-lactalbumin, this enzyme adds UDP-galactose to N-acetyl glucosomine groups that are attached to proteins. It can transfer the UDP galactose to glucose, but the Km for glucose is 1400mM and thus, the reaction proceeds slowly, if at all. Alpha-lactalbumin serves to lower the Km for glucose to 5mM and the enzyme complex now will add UDP-galactose to glucose to produce lactose and UDP. Thus, the milk of all mammals that contain lactose also contain a-lactalbumin. The a-lactalbumin of any species isolated so far will serve to modify bovine galactosyl transferase activity.
When the sequences of a-lactalbumin and lysozyme are compared, about 40% of the residues are found to be the same, including all the cysteine residues. Another 20% of the residues have similar structures. This information coupled with the fact that a-lactalbumin helps to synthesize the same linkage that lysozyme cleaves, suggests that the molecules are closely related. In fact, knowledge of the three-dimensional structure of lysozyme has been utilized to predict the three-dimensional structure of a-lactalbumin.
Despite their similarity, they do not work on the same substrates and are not related antigenetically. The site of synthesis of a-lactalbumin like beta-lactoglobulin is the mammary gland. Alpha-lactalbumin is unusual in that the molecule is more stable to heat in the presence rather than the absence of calcium. Most proteins show increased heat sensitivity in the presence of calcium. This is probably due to the ability of calcium to promote the formation of ionic intermolecular cross links with most proteins. These crosslinks hold the molecules in proximity and increase the likelihood of aggregation upon heating . Alpha-lactalbumin, on the other hand, uses calcium to form intramolecular ionic bonds that tend to make the molecule resistant to thermal unfolding. Under favorable conditions of calcium and pH, a-lactalbumin can remain soluble after exposure to 100C. The structure of a-lactalbumin is presented in figure 11.
Figure 11. Three-dimensional structure of baboon a-lactalbumin for Swaisgood, 1996.
A rasmol version of the structure can be found here.
Bovine Serum Albumin
The Bovine Serum Albumin (BSA), isolated from milk, is
identical to the blood serum molecule. Thus, BSA is not
synthesized in the mammary gland, but rather into the milk
through passive leakage from the blood streams. The protein has a
molecular weight of 69,000. It contains no phosphorus, 17
disulfides and 1 free sulfhydryl group. In blood plasma albumin
is a carrier of free fatty acids. The molecule has specific
binding sites for hydrophobic molecules and may bind them in milk
as well. The rasmol version of the human enzyme with attached
myristic acid can be found here.
To see the fatty acids set the color scheme to chain. The albumin
will be in blue and the myristic acid in red.
The immunoglobulins comprise at least 2% of the total milk protein. There are four classes of immunoglobulins found in milk: 1gG1, lgG2, lgA and lgM. All of these molecules have a similar basic structure being composed of 2 light chains with molecular weights of 20,000 - 25,000 and two heavy chains, having molecular weights of 50,000 - 70,000.
These molecules are not synthesized in the mammary gland and
thus must first enter into the gland and then be transported
through it to be able to enter the milk. In the case of at least
one class of antibodies, lgG1, a specific receptor site has been
located on the membrane of the cells of the mammary gland that
facilitates the entry of this protein into the gland. The
immunoglobulins supply passive immunity to the calf when supplied
in the colostrum. This protection lasts until the animal is old
enough to begin synthesis of its own antibodies.
This fraction of milk has been defined as those proteins that remain in solution after milk has been heated at 95C for 20 minutes and then acidified to pH 4.7. These proteins are precipitated by 12% trichloroacetic acid.
This fraction can be divided into 4 major components while other minor components are recognized. Proteose peptone component 3 is found only in whey and is not associated with casein. This protein contains over 17% carbohydrate and has a molecular weight of 20,000. Antibody to proteose peptone component 3 will cross react with fat globule membrane and it has been suggested that this component is of membrane origin.
Proteose peptone component 5 has a molecular weight of 13,000 and is associated with both the whey and casein fractions of milk. The molecule contains phosphorus and has been shown to consist of the N-terminal 107 amino acids of b-casein that arrive from the proteolytic cleavage that yields the g-caseins.
In a like manner, proteose peptone component 8 fast with a
molecular weight of 3,900 represents the N terminal 28 amino
acids released from the cleavage of b-casein.
The other major proteose peptone component, 8 slow, has not yet
been shown to have been derived by the proteolysis of any milk
proteins. In time, however, this will probably occur. The protein
has a molecular weight of 9,900. As a group, the proteose
peptones are by definition resistant to heating. They are also
very surface active due in part to their low molecular weights
and also to the carbohydrate associated with component three.
About 1.1% of the total milk protein consist of proteose peptone.
As some of these molecules are derived from the proteolysis of
beta-caseins, their concentration in any given milk can be
expected to increase with time.