MILK PROTEINS 4.3. 10 Other methods for the preparation of whey proteins Highly purified whey protein preparations, referred to as whey protein isolates(containing 90-95% protein), are prepared industrially from whey by ion exchange chromatography. Denatured (insoluble) whey proteins, referred to as lactalbumin, may be prepared by heating whey to 95C for 10-20 min at about pH 6.0 the coagulated whey proteins are recovered by centrifugation. The whey proteins may also be precipitated using FeCl3or polyphosphates( section 4. 15.6) 4.4 Heterogeneity and fractionation of casein Initially, casein was considered to be a homogeneous protein Heterogeneity was first demonstrated in the 1920s by Linderstrem-Lang and co-workers, using fractionation with ethanol-HCl, and confirmed in 1936 by Pedersen using analytical ultracentrifugation, and in 1939 by Mellander, using free boundary electrophoresis. Three components were demonstrated and named a-, B- and 2-casein in order of decreasing electrophoretic mobility and represented 75, 22 and 3%, respectively, of whole casein. These caseins were successfully fractionated in 1952 by Hipp and collaborators based on differential solubilities in urea at c. pH 4.6 or in ethanol /water mixtures; the former is widely used although the possibility of forming artefacts through interaction of casein with cyanate produced from urea is of concern n 1956, Waugh and von Hippel showed that the a-casein fraction of Hipp et al. contained two proteins, one of which was precipitated by low ncentrations of Ca*and was called a- casein(s= sensitive) while the other, which was insensitive to Ca2+, was called K-casein. a-Casein was later shown to contain two proteins which are now called x,,.and as2-caseins. Thus, bovine casein contains four distinct gene products, desig nated &, 1", %s2"B- and K-caseins which represent approximately 37, 10, 35 and 12% of whole casein, respectively Various chemical methods were developed to fractionate the caseins but es homogeneous preparations. Fractionation is now usually achieved by ion-exchange chromatography on, for example, DEAE-cellu lose, using urea-containing buffers; quite large (e.g. 10 g)amounts of caseinate can be fractionated by this method, with excellent results(Figure 4.5a, b). Good results are also obtained by ion-exchange chromatography using urea-free buffers at 2-4C. High performance ion-exchange chromatography(e.g Pharmacia FPLC M on Mono Q or Mono S)gives excellent results for small amounts of sample(Figure 4.5c, d ). Reversed phase HPLC or hydrophobic interaction chromatography may also be used but are less effective than ion-exchange chromatography.MILK PROTEINS 155 4.3.10 Highly purified whey protein preparations, referred to as whey protein isolates (containing 90-95% protein), are prepared industrially from whey by ion exchange chromatography. Denatured (insoluble) whey proteins, referred to as lactalbumin, may be prepared by heating whey to 95°C for 10-20 min at about pH 6.0; the coagulated whey proteins are recovered by centrifugation. The whey proteins may also be precipitated using FeCl, or polyphosphates (section 4.15.6). Other methods for the preparation of whey proteins 4.4 Heterogeneity and fractionation of casein Initially, casein was considered to be a homogeneous protein. Heterogeneity was first demonstrated in the 1920s by Linderstrsm-Lang and co-workers, using fractionation with ethanol-HC1, and confirmed in 1936 by Pedersen, using analytical ultracentrifugation, and in 1939 by Mellander, using free boundary electrophoresis. Three components were demonstrated and named a-, ,!?- and y-casein in order of decreasing electrophoretic mobility and represented 75, 22 and 3%, respectively, of whole casein. These caseins were successfully fractionated in 1952 by Hipp and collaborators based on differential solubilities in urea at c. pH 4.6 or in ethanol/water mixtures; the former is widely used although the possibility of forming artefacts through interaction of casein with cyanate produced from urea is of concern. In 1956, Waugh and von Hippel showed that the a-casein fraction of Hipp et al. contained two proteins, one of which was precipitated by low concentrations of Ca2+ and was called a,-casein (s = sensitive) while the other, which was insensitive to Ca2+, was called k--casein. a,-Casein was later shown to contain two proteins which are now called uSl- and a,,-caseins. Thus, bovine casein contains four distinct gene products, desig￾nated ctsl-, rs2-, /I- and K-caseins which represent approximately 37, 10, 35 and 12% of whole casein, respectively. Various chemical methods were developed to fractionate the caseins but none gives homogeneous preparations. Fractionation is now usually achieved by ion-exchange chromatography on, for example, DEAE-cellu￾lose, using urea-containing buffers; quite large (e.g. 10 g) amounts of caseinate can be fractionated by this method, with excellent results (Figure 4Sa, b). Good results are also obtained by ion-exchange chromatography using urea-free buffers at 2-4°C. High performance ion-exchange chromatography (e.g. Pharmacia FPLCTM on Mono Q or Mono S) gives excellent results for small amounts of sample (Figure 4.5c, d). Reversed￾phase HPLC or hydrophobic interaction chromatography may also be used but are less effective than ion-exchange chromatography