DAIRY CHEMISTRY AND BIOCHEMISTRY Fraction number WCx1 X VIII VII VI V IV IⅡ 触学 Figure 4.5(a) Chromatogram of sodium caseinate n column of deae cellul anion exchanger. Buffer: 5 M urea in imidazole-HCI pH 7.0; gradient: 0-0.5 M NaCl. of sodium caseinate on a Pharmacia Mono Q HRS/5 anion exchange column Buffer: 6 M in 5 mM bis-tris-propane7 mM HCl, pH 7; gradient: 0-0.5 M NaCl. (d) Chromatogram of sodium caseinate on a Pharmacia Mono S Hr5/5 cation exchange column Buer: 8 M urea in 20 mM acetate buffer pH 5: gradient: 0-1.0 M NaCl
156 DAIRY CHEMISTRY AND BIOCHEMISTRY N X L Y &- 20 40 00 80 LOO I20 I40 160 180 2w 220 Fraction number Figure 4.5 (a) Chromatogram of sodium caseinate on an open column of DEAE cellulose anion exchanger. Buffer: S M urea in imidazole-HCI buffer, pH 7.0; gradient: 0-0.5 M NaC1. (b) Urea polyacrylamide gel electrophoretograms of the fractions from (a). (c) Chromatogram of sodium caseinate on a Pharmacia Mono Q HR5/S anion exchange column. Buffer: 6 M urea in 5 mM bis-tris-propane/7 mM HCI, pH 7; gradient: 0-0.5 M NaC1. (d) Chromatogram of sodium caseinate on a Pharmacia Mono S HRS/S cation exchange column. Buffer: 8 M urea in 20 mM acetate buffer, pH 5; gradient: 0-1.0 M NaC1
MILK PROTEINS 157 八 ELUTION VOLUME (ml igure 4.5( Continued The caseins may be quantified by densitometrically scanning pol acrylamide gel electrophoretograms(section 4.4.1) but more quantitative results are obtained by ion-exchange chromatography using urea-containing buffers.However, it should be realized that the specific absorbance of the individual caseins differs greatly(Table 4.2)
- v Q Absorbance 280 nrn 0 ? 2 R e ABSORBANCE 280nm h 0 v P 0 0 d
Table 4.2 Properties of some milk proteins(modified from Walstra and Jenness, 1984) Caseins whey proteins -B 8P 11P A2 5P b iP la.B Molecular weigh 23614 23983 19023° Residues/molecule 207 169 5.1 mol residues Net charge/residue Distributio Uneven Very Very un 10.5 95 66
Table 4.2 Properties of some milk proteins (modified from Walstra and Jenness, 1984) Property Caseins Whey proteins Serum u,,-B 8P GL.~-A 11P p-A’ 5P K-B 1P u-la-B P-10-B albumin Molecular weight 23 614 25 230 23 983 19 023” 14 176 18 363 66 267 Residues/molecule Amino acids 199 207 209 169 123 162 582 Proline 17 10 35 20 2 8 34 Cysteine 0 2 0 2 8 5 35 Intramolecular disulphide 0 0 0 0 4 2 17 0 0 0 Phosphate 8 11 5 1 0 Carbohydrate 0 0 0 Hydrophobicity (kJ/residue) 4.9 4.7 5.6 5.1 4.7 5.1 4.3 mol YO residues 34 36 23 21 28 30 34 Net charge/residue -0.10 - 0.07 -0.06 -0.02’ - 0.02 - 0.04 - 0.02 Distribution Uneven Uneven Very uneven Very uneven Even Even bonds b e d Charge A,,, 10.1 14.0’ 4.5 10.5 20.9 9.5 6.6 “Exclusive of carbohydrate residues. bVariable, see text. ‘A small fraction of the molecules. eAverage. except for a rare variant (Dr). ’A29l.l
MILK PROTEINS 4.4.1 Resolution of caseins by electrophoresis Zonal electrophoresis in starch gels containing 7 M urea was used by Wake and Baldwin in 1961 to resolve casein into about 20 bands(zones); the two principal bands were as - and B-caseins. Incorporation of urea was necessary to dissociate extensive intermolecular hydrophobic bonding. Electrophoresis in polyacrylamide gels(PAGE), containing urea or sodium dodecyl sulphate (SDS), was introduced in 1963; resolution was similar to starch gels (SGE) but since it is easier to use PaGe has become the standard technique for analysis of caseins; a schematic representation of a urea-PAGE elec trophoretogram of whole casein is shown in Figure 4.6. Owing to the presence of intermolecular disulphide bonds, K-casein resolves poorly on y 212P P Figure 4.6 Schematic diagram of an electrophoretogram of sodium caseinate in a polyacrylam ide gel containing 5 M urea in tris-hydroxymethylamine buffer, PH.9. 0 indicates origin
MILK PROTEINS 159 4.4. I Resolution of caseins by electrophoresis Zonal electrophoresis in starch gels containing 7 M urea was used by Wake and Baldwin in 1961 to resolve casein into about 20 bands (zones); the two principal bands were 01,~ - and p-caseins. Incorporation of urea was necessary to dissociate extensive intermolecular hydrophobic bonding. Electrophoresis in polyacrylamide gels (PAGE), containing urea or sodium dodecyl sulphate (SDS), was introduced in 1963; resolution was similar to starch gels (SGE) but since it is easier to use, PAGE has become the standard technique for analysis of caseins; a schematic representation of a urea-PAGE electrophoretogram of whole casein is shown in Figure 4.6. Owing to the presence of intermolecular disulphide bonds, Ic-casein resolves poorly on + -12 Yl Y3 - P I or asl -8P asl -9P Figure 4.6 Schematic diagram of an electrophoretogram of sodium caseinate in a polyacrylamide gel containing 5 M urea in tris-hydroxymethylamine buffer, pH 8.9. 0 indicates origin