Module 3. Milk proteins

Lesson 6

6.1 Introduction

It is difficult to define caseins in a way that includes all proteins belonging to this class and excludes all others. Their common property of low solubility at pH 4.6 serves as the basis for a rather convenient operational definition (at least for bovine milk). At this pH, all of the caseins except some of the proteolytic derivatives precipitate. As the solubility of caseins is much less and the whey proteins are having better solubility than casein the separation of the casein has become possible by lowering the pH value to 4.6.

As mentioned earlier obtaining molecular components individually from a mixture of casein is difficult. The ion exchange chromatography using DEAE-cellulose or hydroxy­apatite columns would give satisfactory fractions. However, it is necessary to control the interaction of casein molecules. The unique feature of the caseins is their ester-bound phosphate. All of the casein polypeptide chains have at least one such group per mol­ecule; whereas, none of the whey proteins have any ester bound phosphate. The αs1 and β-caseins contain no cysteine residues,while αs2 and κ-caseins each have two cysteine residues. Proline contents of caseins are rather high ( αs1-, αs2- , β - ,and κ- caseins contain 17,5, 17 and 12 mol %, respectively). There is no organized secondary structure for the various fractions of casein. From the various analytical studies carried out on this protein revealed that they have short lengths of α -helix or β -sheet structure in them. Their ionizable groups of the amino acids are accessible to titration and are also involved in several other side chains to reaction. Denaturing agents and heating seems to have no effect on the secondary structure of these proteins. Thus their conformation appears to be much like that of denatured globular proteins. As the protein is having large proportion of proline residues, closely packed orderly secondary conformation is being prevented in this protein molecule. The four caseins differ greatly from each other in charge distribution and the tendency to aggregate in the absence and presence of Ca2+ ions.

6.2 αs1-Caseins

The polypeptide chain of αs1-casein consists of two predominantly hydrophobicregions (residues 1-44, and 90-199) and a highly charged polar zone (residues 45-89).All but one of the phosphate groups is in the 45-89 residues segment, and the prolines are distributed at intervals in the hydrophobic segments. Thus, this protein can be visualized as a rather loose flexible polypeptide chain.Self-association of αs1-casein depends markedly on its concentration and on the pH, ionic strength, and kind of ion in the medium, but it is relatively independent of temperature. Physical measurements such as lights cattering, sedimentation, and viscosity indicate that αs1- casein is completely dissociated to a flexible random chain monomer of MW = 24,000 at pH 12, in 3 M guanidinium chloride and at neutral pH and 0.01 ionic strength, it associates at neutral pH and higher ionic strength, the extent of association depending on protein concentration, it binds about 8 moles Ca2+ per mole near pH 7, probably to the ester phosphate groups. It aggregates and precipitates at very low concentrations of Ca2+ (7 mM Ca2+,28 mM NaCl). A small amount of peptides, sometimes called A-casein, is present in milk; these appear to originate from proteolysis of αs1-casein.

6.3 αs2-Caseins

αs2-casein has a remarkable dipolar structure with a concentration of negative charges near the N-terminus and positive charges near the C-terminus. Its properties have not been investigated as thoroughly as those of the other caseins, but certainly it binds Ca strongly and is even more sensitive to precipitation by Ca2+ than αs1-casein. It self-associates at neutral pH in the absence by Ca2+, and the association depends markedly on ionic strength and is at a maximum at an ionic strength of about 0.2.

6.4 β-Caseins

β-casein has a strong negatively charged N-terminal portion. The net charge of the 21-residue N-terminal sequence is 12 at pH 6.6, and the rest of the chain has virtually nonet charge. The large number of Pro residues effectively precludes extended helix formation. Thus, the β casein molecule is somewhat like that of an anionic detergent with a nega­tively charged head and an uncharged essentially hydrophobic tail. The outstanding characteristics of the association ofβ-casein in both the absence and the presence of Ca2+ are its strong dependence on temperature. In the absence of Ca2+, only monomer is present at 4°C, but large polymers (20-24 monomers) are formed at room temperature. Removal of the 20-residue C-terminal sequence destroys the ability of β-casein to associate, suggesting that specific hydrophobic interactions maybe involved. β -casein tightly binds about 5 Ca2 + per mole, consistent with its ester phos­phate content.

6.5 γ-Caseins

Group of caseins designated as γ-caseins have been known for some time to correspond to C-terminal portions of the β -casein sequence. These are formed by cleavage of β-casein at positions 28/29, 105/106, and107/108 by the enzyme plasmin. The fragments 29-209, 106-209, and 108-209constitute the y-caseins. The smaller fragments resulting from the cleavage appear in the whey when casein is precipitated by acid and constitute part of what has long been designated as the proteose -peptone fraction of the whey. Thus, fragments 1-105 and 1-107 were called as whey component 5, fragment 1-28was whey component 8-fast, and fragments 29-105 and 29-107 were named as whey component8-slow. However the revised nomenclature for the fragments of β-caseins in milk is given here under (Table 6.1).

Table 6.1
Revised nomenclature for the fractions of β-caseins


6.6 κ – Caseins

About one-third of the κ casein molecules are carbohydrate-free and contain only one phosphate group (SerP-149). At least six other components differing in charge also occur. They have varying numbers of N-acetyl neuraminic acid (NANA) residues and one, at least, appears to have a second phosphate (SerP-127). Three different glycosyl oligomers linked to Thr-133 have been identified. The N-terminal residue of κ-casein is glutamic acid. In the isolated protein it is present as the cyclic derivative pyroglutamic acid. κ-casein as isolated from milk consists of a mixture of polymers probably held together by intermolecular disulfide bonds; these polymers range in molecular weight from about 60,000 (trimers) to more than 150,000. κ-casein is rapidly hydrolyzed at the Phe (l05)-Met (l06) bond by the enzyme chymosin (EC, and by other proteases, yielding N-terminal fragment called para- κ casein, which contains the two cysteine residues, a C-terminal fragment of 64 residues called the macropeptide,containing all of the carbohydrate and phosphate groups as well as the genetic substitu­tions. κ-casein binds about 2 moles Ca2+per mole of protein at neutral pH but differs markedly from the other caseins in its solubility over a wide range of Ca2++ concentration.

The naturally occurring mixture of bovine κ-casein variants polymerizes, as previously mentioned, through -S-S- linkages to subunits containing three to eight monomers. These further polymerize by no covalent association to particles of about 6,50,000 daltons. This polymerization is insen­sitive to concentration of Ca 2+ and to temperature.
Last modified: Tuesday, 6 November 2012, 4:47 AM