( K. H. Valkonen* N. Mart...)Purification and Characterization of beta-Lactoglobulin Genetic Variants A and B Using Preparative Electrophoresis and Isoelectric Focusing

K. H. Valkonen*, N. Marttinen*, H.-L. Malinen*, V.-P. Jaakola** and T. Alatossava*

* Biotechnology Laboratory, REDEC of Kajaani, University of Oulu, FIN-88600 Sotkamo, Finland
** Department of Biological and Environmental Science, University of Jyvskyl, PL 35, FIN-40351 Jyvskyl, Finland

Introduction
β-lactoglobulin (βLG) is a major whey protein found in the milk of cows and other ruminants, deer, bison, and buffalo. βLG is also found in some nonruminants, such as pigs,¹ horses,² dogs, dolphins,³ cats,⁴ and whales. However, βLG is not found in human milk.^{5, 6, 7, 8} βLG is a glycoprotein which exists at the normal pH of bovine milk as a dimer with a molecular weight of 36,000, and consists of two monomeric subunits with molecular weight of 18,000 (162 residues). Several genetic variants of βLG have been detected,⁹ of which the bovine phenotype A and phenotype B are most predominant. The bovine βLG A variant differs from the βLG B variant by only two amino acids: aspartate-64 (Asp) and valine-118 (Val). These amino acids are substituted by glycine (Gly) and alanine (Ala) in the B variant. All variants contain five cysteine residues, four of which are involved in forming intrachain disulfide bridges.

The function of βLG is not yet clear, although binding and transport of retinol, small hydrophobic ligands and fatty acid in postnatal animals, have been suggested as possible functions, since its three-dimensional structure is essentially similar to that of human retinol-binding protein in serum.^{10, 11, 12, 13, 14, 15} βLG is considered as one of the main allergenic components in bovine milk,^{16, 17, 18} and therefore the modification of βLG is considered as a promising treatment for milk allergy.¹⁹ βLG also has good emulsifying and foaming properties, and therefore offers a good model for elucidation of the adsorption characteristics of proteins at a surface. βLG is also a model protein for studying the denaturation mechanism.^{20, 21} These properties are important in the milk industry and bovine milk allergy.

Purification and analytical separation of the genetic variants of bovine βLG have been carried out by using different chromatographic methods, e.g. ion exchange chromatography with DEAE cellulose and recently HPLC, ^{22, 23, 24, 25, 26} and also by different electrophoretic methods.²⁷ Many of the above separation methods are either uneconomical or too timeconsuming to be used routinely for large scale purification and phenotyping of dairy cow populations.

We report here a novel method which can be used for both preparative and analytical scale purification of βLG A and βLG B variants with native PAGE by using continuous elution electrophoresis.

Methods
SAMPLE PREPARATION; NATIVE BLG
A mixture of the phenotypes A and B from bovine milk (approximately 80%, lyophilized powder, Sigma Chemical Co., St. Louis, MO, USA) was dissolved in deionized water, pH 7.0 (20 mg/300 l), and the protein concentration of the solution was measured both at 280 nm (Shimadzu UV-1201 spectrophotometer) Ε^1%1 cm=9.5²⁸ and by using Bio-Rads Protein Assay (Bio-Rad, Richmond, CA, USA) modifying the method of Bradford²⁹ at 595 nm. For preparative electrophoresis (PE) 0.0625 M Tris-HCl (100 l), pH 6.8, containing 25% glycerol and bromophenol blue 0.012% (final concentration) were added to the βLG solution.

STANDARDS
βLG variants A, B and A/B from bovine milk (Sigma Chemical Co., St. Louis, MO, USA), bovine a-lactalbumin (Sigma Chemical Co., St. Louis, MO, USA), a mixture of caseins (Sigma Chemical Co., St. Louis, MO, USA), and bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA), 10 mg each were dissolved in 10 ml deionized water. Protein concentrations of the standards were estimated, and the EF samples were prepared as described above. Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) low molecular weight standards, kaleidoscope polypeptide standards, and isoelectric focusing (IEF) standards were from Bio-Rad (Bio-Rad, Richmond, CA, USA).

PREPARATIVE CONTINUOUS ELUTION PAGE ELECTROPHORESIS
Buffers used in preparative native PAGE were those described by Ornstein-Davis.³⁰ The electrophoresis equipment used was Bio-Rads Model 491 Prep Cell. The separating gel (110 x 37 mm) was 15% acrylamide/0.4% bisacrylamide in 0.375 M Tris-HCl (final concentration), pH 8.8 (catalyst concentrations in resolving gel were 0.025% APS/0.025% TEMED). The stacking gel (10 x 37 mm) was 3% acrylamide/0.08% bisacrylamide in 0.125 M Tris-HCl (final concentration), pH 6.8. The running buffer was 0.025 M Tris-base, 0.19 M glycine, pH 8.3. Before sampling loading, an electrophoresis field was applied at constant power at 5 W for 30 minutes Thereafter 400 l sample was injected and PE was run at constant power of 12 W for 4,000 Vh for 19 h at 4 C. Elution flow rate was 0.751.0 ml/min and fractions of 5 ml were collected. Elution of βLG variants was monitored at 280 nm with a UV detector (Bio-Rad BioLogic LP workstation, Model 2128).

Protein concentrations of the eluted fractions were estimated by measuring the optical absorption of the protein fractions at 280 nm (Shimadzu UV-1201). The eluted fractions were analyzed by analytical SDS-PAGE and isoelectric focusing (IEF). According to these results, two combined fractions were each pooled and dialyzed against deionized water overnight at 4 C. After dialysis, the two fractions were concentrated by lyophilization (Christ LMC-1, model beta 216), and then analyzed further by using analytical SDS-PAGE, isoelectric focusing (IEF), and immunoblotting (IB).

ANALYTICAL ELECTROPHORESIS
The molecular weights of the proteins in the two fractions, separated with the Model 491 Prep Cell, were estimated by SDS-PAGE under reducing conditions modifying the method of Laemmli.31 Briefly, gels containing a 1220% acrylamide separating gel and a 3% stacking gel were loaded with the samples (0.55 g protein/well), mixed with sample buffer (1:1), and 10 l sample was injected per well. The gels were run (PROTEAN II electrophoresis cell) at 20 mA constant current, 3 W, 185 Vh at RT with 0.024 M Tris/0.192 M glycine/0.1% SDS, pH 8.3 as a running buffer. Bovine βLG containing the A and B variants as also the A and the B variant (Sigma) and molecular weight standards (Bio-Rad SDS-PAGE low range and kaleidoscope polypeptide standards) were run simultaneously. The gels were stained with 0.025% Coomassie Brilliant Blue R-250 (B-0149, Sigma Chemical Co., St. Louis, MO, USA) in 10% isopropanol/7% acetic acid, and were destained with 50% isopropanol/10% acetic acid.

ANALYTICAL IEF
The isoelectric points of the two βLG fractions separated on the Model 491 Prep Cell, were analyzed by modifying the method obtained from Bio-Rad (IEF Ready Gel Application Guide). IEF was performed on Bio-Rad IEF Ready Gels (pH 58) with 20 mM NaOH as a cathode buffer and 7 mM phosphoric acid as an anode buffer on Bio-Rads Mini Prep Cell apparatus. The two βLG fractions and the standards were dissolved in distilled water containing 10% glycerol (1 mg/ml). The run was performed at 5 W constant power and 500 V_max. for 2.5 h. The gels were stained and destained as described above.

IMMUNOBLOTTING
The two βLG fractions, from preparative electrophoresis, were also characterized by polyclonal antiserum to βLG (Nordic Immunologic, Tilburg, Netherlands). Standards were as described above. Sample proteins, separated by PAGE or IEF, were electrophoretically blotted into nitrocellulose (0,45 m nitrocellulose membrane, Hybond-C, Amersham) by the method of Towbin.³² The electrophoretic blotting was performed with 25 mM Tris, 192 mM glycine, pH 8.3, at 30 V overnight. Residual binding sites were blocked with 1.0% bacto-gelatin for 2 h at RT, and the nitrocellulose sheet was then washed three times with PBS Tween 20 (0.05%), 10 minutes each. To detect the βLG specific proteins, the nitrocellulose sheet was incubated with the polyclonal antiserum to βLG (5 l/50 ml PBS/0.05% Tween 20) for 2 h at RT, washed three times as above, and then incubated with horseradish peroxidase-coated second antiserum (10 l/50 ml PBS Tween 20 (0.05%) for 2 h at RT and washed as above. The sheet was stained with 4CN (4-Chloro-1-Naphthol) according to Bio-Rad's instructions.

Results
SEPARATION OF BOVINE BLG PHENOTYPES BY CONTINUOUS ELUTION ELECTROPHORESIS
Figure 1 shows an elution profile of bovine LG A/B separated by preparative continuous electrophoresis as described in Methods. The fractions were analyzed by analytical SDS-PAGE, and then pooled: peak I (fractions 3543) and peak II (fractions 4755) and concentrated. Total protein recovery of the combined fractions was 78% (15.6 mg/20 mg) while protein concentration of peak I (fractions 3543) was 0.190 mg/ml (total 7.05 mg) and that of peak II (fractions 4755) was 0.160 mg/ml (total 8.5 mg).

CHARACTERIZATION OF THE TWO COMBINED FRACTIONS SEPARATED BY THE MODEL 491 PREP CELL
Molecular weights of the pooled fractions analyzed by SDS-PAGE under reducing conditions are shown in Figure 2A. The two eluted fractions each have the same molecular weight as bovine βLG A/B, according to their mobility in SDS-PAGE. The two fractions show only one single band by electrophoresis.

Identification of the combined fractions was confirmed by western blot with polyclonal antibodies to bovine βLG. Immunoblotting shows that βLG A/B and both eluted fractions are recognized by antiserum to βLG (Figure 2B).

Since IEF is the most sensitive method for charge distinction within different phenotypes, the isoelectric points of the two eluted fractions, separated by continuous native PAGE, were estimated also by isoelectric focusing at pH range 58. Commercial bovine βLG phenotype standards A, B, and A/B were analyzed for comparison. Previous data (Tulp et al.,³³ Conti et al.³⁴) shows that the difference between the isoelectric points (pI) between the phenotypes βLG A and βLG B is only 0.3 pH unit. Figure 3 shows that bovine βLG A/B focused into two bands with pIs of 5.1 and 5.3. Peak I (fractions 3543) has the same pI as phenotype βLG A (5.3), and peak II (fractions 4755) has the same pI as phenotype βLG B (5.1).

Conclusions
We have developed a novel method for purifying and separating bovine βLG A and B variants by preparative electrophoresis. The molecular weight and the isoelectric points of the eluted peak 1 (fractions 3543) and the eluted peak 2 (fractions 4755) separated by continuous electrophoresis, were analyzed by analytical SDS-PAGE and IEF. Since the molecular weight and the isoelectric points of peak 1 were the same as those of βLG A, and the molecular weight and the isoelectric point of fraction II the same as those of bovine βLG B, we suggest that the eluted fraction I and the eluted fraction II are bovine βLG A and B variants, respectively. Both fractions were recognized with antiserum to bovine βLG A/B. The molecular weight and isoelectric points of other milk proteins, used as controls, were different from those of the eluted fractions and were not recognized by antiserum to bovine βLG (data not shown). The results confirm that the two eluted fractions are specific βLG phenotypes and do not contain any impurities. The method could be used both in analytical and preparative scale in milk industry and when studying bovine milk allergy.

References
1. Conti, A., Godovac-Zimmerman, J., Pirchner, F., Liberatori, L., Braunitzer, G., Pig β-lactoglobulin I (Sus scrofa domestica, Artiodatyla), The Journal of Biological Chemistry, Hoppe-Seyler, 367, 871 (1986).

2. Godovac-Zimmerman, J., Conti, A., Liberatori, L., Braunitzer, G., The amino acid sequence of β-lactoglobulin II for horse colostrum (Equus caballus, Perisodactyla): β-lactoglobulins are retinol-binding protein, The Journal of Biological Chemistry, Hoppe-Seyler, 366, 601 (1986).

3. Pervaiz, S., Brew, K., Purification and characterization of the major whey proteins from the milk of the Bottlenose dolphin (Tursiops truncatus), the Florida manatee (Trichechus manatus latirostris) and the beagle (Canis familiaris), Archives of Biochemistry and Biophysics, 246, 846 (1986).

4. Halliday, J. A., Bell, K., Shaw, D. C., The complete amino acid sequence of feline β-lactoglobulin II and a partial revision of the equine β-lactoglobulin II sequence, Biochimica et Biophysica Acta., 1077, 25 (1991).

5. Hambraeus, L., Proprietary milk versus human breast milk, Milk in infant feeding, Pediatr. Clin. North Am., 24, 1236 (1977).

6. Brignon, G., Chtorou, A., Ribadeau-Dumas, B., Does β-lactoglobulin occur in human milk?, Journal of Dairy Research, 2, 249 (1985).

7. Monti, J. C., Mermoud, A. F., Jolles, P., Antibovine β-lactoglobulin antibodies react with a human lactoferrin fragment and bovine β-lactoglobulin present in human milk, Experientia, 45, 178 (1989).

8. Maeda, S., Morikawa, A., Tokuyama, K., Kuroume, T., The concentration of bovine IgG in human breast milk using different methods, Acta. Pdiatrica, 82 (12), 10121016 (1993).

9. Fox, P. J., Advanced Dairy Chemistry Volume 1: Proteins, Elsevier Applied Science, London, 767 (ISBN 1-85166-761-X) (1993).

10. Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Fondlay, J. B. C., Sivaprasadarno, R., Jones, T. A., Newcomer, M. E., Kraulis, P. J., The structure of -lactoglobulin and its similarity to plasma retinol-binding protein, Nature, 383385 (1986).

11. Godovac-Zimmerman, J., The structural motif of -lactoglobulin and retinol-binding protein: a basic framework for binding and transport of small hydrophobic molecules? Trends Biochem. Sci., 13, 6466 (1988).

12. Sawyer, L., Papiz, M. Z., North, A.C.T., Eliopoulos, E. E., Structure and function of -lactoglobulin, Biochem. Soc. Trans., 13, 265266 (1985).

13. Brownlow, S., Morais Cabral, J. H., Cooper, R., Flower, D. R., Yewdall, S. J., Polikarpov, I., North, A. C., Sawyer, L., Bovine -lactoglobulin at 1,8 resolutionstill an enigmatic lipocalin, Structure, 5 (4), 481495 (1997).

14. Prez, M. D., Calvo, M., Interaction of -lactoglobulin with Retinol and Fatty Acid and Its Role as a Possible Biological Function for This Protein; A Review Journal of Dairy Science, 78, 978988 (1995).

15. Narayan, M., Berliner, L. J., Fatty acids and retinoids bind independently and simultaneously to -lactoglobulin, Biochemistry, 36 (7), 19061911 (1997).

16. Piastra M., Stabile, A., Fioravanti, G., Castagnola, M., Pani, G., RIA, F., Cord blood mononuclear cell responsiveness to -lactoglobulin: T-cell activity in atopy-prone and non-atopy-prone newborns, International Archives of Allergy & Immunology, 104 (4), 358365 (1994).

17. Mkinen-Kiljunen, S., Detection and characterization of allergens derived from cows milk and natural rubber latex, Thesis, Faculty of Medicine of the University of Helsinki, 69 (1996).

18. Duchen, K., Einarsson, R., Grodzinsky, E., Hattevig, G., Bjorksten, B., Development of IgG1 and IgG4 antibodies against -lactoglobulin and ovalbumin in healthy and atopic children, Annals of Allergy, Asthma & Immunology, 78 (4), 363368 (1997).

19. Hst A., Samuelsson, E. G., Allergic reactions to raw, pasteurized, and homogenized/pasteurized cow milk: a comparison, A double-blind placebocontrolled study in milk allergic children, Allergy, 43 113118 (1988).

20. Dufour, E., Genot, C., Haertle, T., -lactoglobulin binding properties during its folding changes by fluorescence spectroscopy, Biochimica et Biophysica Acta., 1205 (1), 105112 (1994).

21. Qi X. L., Brownlow, S., Holt, C., Sellers, P., Thermal denaturation of -lactoglobulin: effect of protein concentration at pH 6.75 and 8.05, Biochimica et Biophysica Acta., 1248 (1), 4349 (1995).

22. Kalan, E. B., Greenberg, R. Walter, M., Studies of -lactoglobulin A, B, and C. 1. Comparison of chemical properties. Biochemistry., 4, 991997 (1965).

23. Otte, J. A. H. J., Kristiansen, K., Zakora, M., Qvist, K. B., Separation of individual whey proteins and measurement of a-lactalbumin and -lactoglobulin by capillary zone electrophoresis, Neth. Milk Dairy J., 48, 81 (1994).

24. Paterson, G. R., Otter, D. E., Hill, J. P., Application of Capillary Electrophoresis in the Identification of Phenotypes Containing the -lactoglobulin C Variant, Journal of Dairy Science, 78, 26372644 (1995).

25. De Frutos, M., Molina, E., Amigo, L., Applicability of Capillary Electrophoresis to the Study of Bovine -lactoglobulin Polymorphism, Milchwissenschaft, 51 (7), 374378 (1996)

26. Pearce, R. J., Analysis of whey proteins by high performance liquid chromatography, Aust. J. Dairy Technol., 38, 114 (1981).

27. Lowe, R., Anema, S. G., Paterson, G. R., Hill, J. P., Simultaneous Separation of the -lactoglobulin A, B, and C Variants Using Polyacrylamide Gel Electrophoresis, Milchwissenschaft, 50 (12), 663669 (1995).

28. MacKenzie, H. A., -lactoglobulins in milk proteins, Chemistry and Molecular Biology, MacKenzie H. A., Murphy, M. Eds., Academic Press, New York, Vol. II, 257330 (1971).

29. Bradford, M., Anal. Biochem., 72, 248 (1976).

30. Ornstein, L., Davis, B. J., Anal. NY Acad. Sci., 121, 321 (1964).

31. Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T, Nature, 680686 (1970).

32. Towbin, H., Staehelin, T., Gordon, J., Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76, 43504353 (1979).

33. Tulp, A., Verwoerd, D., Hard, A.A., Density gradient isoelectric focusing of protein in artificial pH gradients made up of binary mixtures of amphoteric buffers, Electrophoresis, 18 (5), 767773 (1997).

34. Conti A., Napolitano, L., Cantisani, A.M., Davoli, R., Dallolio, S., Bovine -lactoglobulin H: isolation by preparative isoelectric focusing in immobilized pH gradient and preliminary characterization, Journal of Biochemical & Biophysical Methods, 16 (23), 205214 (1988).

Coomassie is a trademark of ICI Organics, Inc.
Hybond and Amersham are trademarks of Amersham Pharmacia Biotech.

back to top
'"/>

Source: