Chemistry of Milk

Module 3. Milk proteins

Lesson 9
WHEY PROTEINS: α-LACTOLBUMIN AND β-LACTOGLOBULIN, THEIR FRACTIONATION AND PHYSICO-CHEMICAL PROPERTIES

9.1 Introduction

Several procedures have been suggested for isolating the whey proteins from other whey components. The study conducted by Hansen and co- workers (1971) has revealed that the whey proteins could be separated by complexing the whey proteins with carboxymethyl cellulose. Similarly ferripolyphosphate, hexameta phosphate and polyacrylic acid were also used to isolate whey proteins by forming a complex. Gelfiltrationwith Sephadex G-25, ultrafiltration using cellulose acetate membranes, reverse osmosis and electro dialysis are some of the methods available for isolation of whey proteins from whey. As such it is necessary to know about the separation of whey proteins and also to study the properties of these proteins is also necessary for proper understanding of chemistry of milk and applying them to study in detail their behaviour in milk. This information could be used to develop suitable procedure in the manufacture of several dairy products and also to prevent some important and undesirable changes which are likely to interfere in developing some new products.

9.2 Fractionation of Whey Proteins

Separation of individual components of whey protein mixture was origi­nally accomplished by fractionations based on differences in solubility and by crystallization. At present the preferred methods of fractionation for both analytical and preparative purposes make use of gel filtration (which fractionates on the basis of molecular size) and ion exchange(which separates on the basis of net charge on protein). Gel filtration technique is particularly useful for fractionation of bovine whey proteins because of their great variation in their molecular size. The fact that β-lactoglobulin (MW = 18,277) forms a noncovalent dimer makes it readily separable from α-lactalbumin (MW = 14,175), as the latter does not aggregate.

9.3 α-Lactoglobulin

This family of proteins consists of a major component and several minor components. Three genetic variants of α-lactalbumin have been identified.

Two genetic variants, A and B, of this protein exist. They differ by a single substitution,A having Gln and B having Arg at position 10. In the milk of European breeds and yaks only B variant is observed while both A and B variants occur in the milk of Indian cattle. Some minor forms of bovine α-lactalbumin are revealed by electrophoresis. Some of these contain covalently bound carbohydrate groups;the major component of bovine α-lactalbumin is devoid of carbohydrate. Other minor components seem to have fewer amide groups than the major ones, and one minor α-lactalbumin containing three instead of four disulfides has been reported. In total, the major components do not account for more than 10% of the α-lactalbumin.

The complete primary structure of the major α-lactalbumin has been determined . The B variant consists of123 amino acid residues with a calculated molecular weight of 14,178 and the A variants differ from it only in having Gln instead of Arg at position 10.

The amino acid sequence of α-lactalbumin is similar to that of lysozyme. Indeed, bovine α-lactalbumin B and chicken egg white lysozyme have iden­tical amino acid residues at 49 positions, and the four disulfide groups are located identically ( positions between 6and 120; 28 and 111; 61 and 77; and 73 and 91, respectively) in α-lactalbumin. The two proteins have different biological activities without mutual interference.The biological activity of α-lactalbumin is its interaction with galactosyltransferase to promote the transfer of galactose from uridine diphosphate galactose (UDP - galactose) to glucoseto form lactose. It has been shown that α-lactalbumin binds two atoms of Ca ²⁺ very tenaciously. In fact it is probable that all preparations of this protein have carried this Ca undetected. Removal of the bound Ca with ethylenediamine tetraacetate renders α-lactalbumin more susceptible to denaturation by heat or by addition of guanidine.

9.4 β-Lactoglobulin

As chaffenburg and Drewry (1957) demonstrated that there are two components of β-lactoglobulin in the electrophoretic pattern of this protein in the western cattle. However, two more variants have also been identified by other workers. These genetic variants differ in their electrophoretic mobilities in starch or poly acrylamidegel in the ascending order as A > B > C > D. Bovine β-lactoglobulin‘B’ consists of 162 amino acid residues. Their calculated molecular weight for monomer is 18,227 and dimer is 36000 respectively . The dimer contains five cysteine residues per mole, of which four are involved in disulfide linkages. Location of one disulphide bond always occurs between Cys residues at 66 to 160 positions and the other link is between 106 and119 or 121. The single free thiol appears to be equally distributed between Cys119 and Cys 121. The exist­ence of this thiol group is of great importance for changes occurring in milk during heating, as it is involved in reactions with other proteins, notably κ-casein and α-lactalbumin. It is likely that considerable portions of the sequence of β-lactoglobulin exist in the α-helix and β-sheet structures. Regions that are most likely helical are residues21-37, 51-63, 127-143, and 154-159 respectively. β-sheet structures are likely in 2-19, 39-43,76-88, 91-99, and 101-107, while the structure of the sequence 114-125 is not clearly established. β-Lactoglobulin exists naturally as a dimer of two monomeric subunits which is covalently linked. When more than one genetic variant is present, hybrid dimers are formed. Dissociation to the monomer occurs below pH 3.4. β-Lactoglobulin Aassociates to form an octamer at pH 4.5 and low temperature. The B variant (predominant in Western cattle) octamerizes to a smaller extent.