Biochimiea et Biophysica Acta, 491 (1977) 76-81

© Elsevier/North-Holland Biomedical Press BBA 37596 H E T E R O G E N E I T Y IN a-LACTALBUMINS I. H U M A N a - L A C T A L B U M I N

JEAN-PAUL PR1EELS and JEAN SCHLUSSELBERG Laboratoire de Chimie Gdndrale I, Facultd des Sciences, Universitd Libre de Bruxelles, Av. F. D. Roosevelt, 50, B-1050, Bruxelles (Belgium)

(Received July 27th, 1976)

SUMMARY a-Lactalbumin from human milk shows an heterogeneous behaviour when subjected to ion exchange chromatography with DEAE-Sephadex. Two components have been separated, showing identical patterns in the following studies: amino acid compositions, fluorescence and circular dichroism spectra, transition temperature of denaturation, antigenicity, lactose synthase specifying activity and hydrodynamic properties. After rechromatography of either peak, these two components appeared to be in equilibrium. This equilibrium varies with the temperature and the pH of chromatography. Moreover, an increase of n-alcohol concentration in the eluting buffer also induces an increase of the second protein peak eluting at higher ionic strength. These two peaks seem to be the result of some conformational change induced upon the binding of the protein to the solid anionic matrix.

INTRODUCTION a-Lactalbumins from bovine, goat and sheep milks are known to be heterogeneous [1, 2, 3]. These heterogeneities were found to be due at least to four different causes. Minor glycosylated components of bovine a-lactalbumin [4, 5, 6] have been isolated. The presence of two genetically different a-lactalbumins [7] and of a non glycosylated minor component [8] have been described for bovine species. Apparent heterogeneity has also been reported for bovine and human alactalbumins [9]. Different interpretations have been given in order to explain this bimodality. They were dealing with buffer interaction [10] or as a result of ammonium sulfate precipitation [8]. In the present paper we want to relate the observed bimodality with a conformational change induced by the anionic matrix during the chromatographic process.

77 EXPERIMENTAL

Chromatographic procedures Human a-lactalbumin has been purified according to a previously described method I11 ]. Samples of a-lactalbumin (I 5 to 20 mg) were submitted to ion exchange chromatography with DEAE-Sephadex A-25. The column (Sephadex K 16/40) was maintained at constant temperature and packed with a constant volume (20 =k 0.5 ml) of resin equilibrated with 50 mM NaH2PO4-NaOH, pH 7.5. Elution was performed with NaC1 (0 to 0.3 M) by means of a linear gradient established with an LKB Ultrograd 11300 gradient mixer. Elution rate was constantly held to 0.5 ml/min. Transmittance at 280 nm of the effluent was recorded with a LKB Uvicord II detector. The value of absorbance of a 10% protein solution at 280 nm for a 1 cm path length cell was 1.6 [11].

Enzymatic activity assays Lactose synthase activity was measured following the rate of transfer of 14C from UDP[14C]galactose to lactose [12]. The A-protein of the lactose synthase system was obtained from human milk using the method described by Khatra et al. [12].

Heterogeneity tests Amino acid analyses were performed with a Bechman Unichrom II amino acid analyzer following acid hydrolysis in vacuo at 110 °C with 3 M methane sulfonic acid [13] containing 0.2 % of 3-(2-aminoethyl)-indole for 24, 48, 72 and 96 h.

Circular dichroism measurements Circular dichroism spectra were recorded between 195 and 300 nm using a Cary 61 spectropolarimeter. Protein concentration range was from 0.1 to 2 mg/ml in NaHzPO4.NaOH, 50 mM, pH 7.0, 7.5 and 8.0 using 1, 2 and 10-mm cells at 25 °C.

Polyacrylamide disc gel electrophoresis Disc gel electrophoresis were performed as described by Davis [14] using a Shandon analytical polyacrylamide electrophoresis apparatus. 7.5, 10.0 and 12.5% acrylamide gels were prerun with the various buffers used for gel filtration experiments. Samples of a-lactalbumin, 5/A (5, 10, 20 mg/ml), were added together with 50/zl of large pore gel solution (2.5% polyacrylamide) and polymerization was allowed for 20 rain before running the experiments. RESULTS

(1) Chromatography Human a-lactalbumin showed a single symmetrical peak when submitted to gel filtration with Sephadex G-75 under all experimental conditions which were tried. However if a single peak was obtained upon chromatography with DEAE Sephadex A-25, at pH 7.5 and 24.2 °C (Fig. la), when temperature was raised (28 °C) a second peak appeared eluting at higher ionic strength (Fig. lb). This second component also became more important when the pH of chromatography was raised while temperature was maintained constant (Fig. lc, ld).

78

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i

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200

Fig. 1. Effect of temperature and pH on ion exchange chromatography of human a-lactalbumin with DEAE Sephadex A-25 (7 x 1.6 cm) equilibrated with 0.05 M sodium phosphate and eluted with a linear gradient of NaCl varying from 0 to 0.3 M; elution was read at 280 nm. Temperatures of chromatography are 24.2 °C, pH 7.5 (Fig. la), 28.0 °C, pH 7.5 (Fig. lb), 28.6 °C, pH 7.0 (Fig. lc) and 28.6 °C, pH 7.5 (Fig. ld).

The same effect can be observed when n-alcohols in different concentrations are added in the eluting buffer (Fig. 2a, b, c and d). At high temperature, alcohols may induce a denaturation of the protein. This was observed by the appearance of a third peak which has no more lactose synthase activity but presents optical rotatory dispersion characteristics of thermal denatured a-lactalbumin. Reversibility of the equilibrium is shown in Fig. 3 where a first chromatography at 28.6 °C was made followed by a desalting with Sephadex G-25 equilibrated with 50 m M phosphate buffer, p H 7.5. The two eluted peaks were submitted separately to a rechromatography in the same conditions. The same bimodality was found for the two species.

(2) Properties of a-lactalbumins of the two peaks Protein material from either peak had amino acid composition identical to the known sequence as well as identical mobilities on disc electrophoresis in the buffers used for chromatographies.

79

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Fig. 2. Effect of n-alcohols concentration on ion exchange chromatography of human ct-lactalbumin with DEAE-Sephadex A-25. Experimental conditions are the same as Fig. 1 except for T ° (24.2 °C) and pH (7.5); n-alcohol concentration: 10% w/w methanol (Fig. 2a), 2% w/w ethanol (Fig. 2b), 1% w/w propanol (Fig. 2c), 0.8 % w/w butanol (Fig. 2d).

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Fig. 3. Ion exchange chromatography of human a-lactalbumin with DEAE-Sephadex A-25. Conditions are the same as Fig. 1 (T ° 28.6 °C and pH 7.5). i , first chromatography; A, rechromatography of 1st peak; t , rechromatography of 2nd peak. The area of the two rechromatographies were normalized to the area of the original chromatography.

There was negligible difference in circular dichroism spectra and samples from either peak gave the same kinetic parameters for lactose synthase activity. Antigenic behaviour as determined by double immunodiffusion against antibodies prepared as previously described [15] showed no spurs between the two precipitin lines showing a complete identity of antigenicity between the two components. DISCUSSION

The identity of the amino acid compositions of the two chromatographic peaks, together with their same electrophoretic mobilities on polyacrylamide gels, indicates that they are obviously not is,proteins (e.g. no differences in amino acids sequence).

80 The argument is reinforced by the fact that the bimodality is reproduced upon rechromatography indicating a real equilibrium between two forms. Separations of two forms in equilibrium by use of methods like chromatography or moving boundary electrophoresis have largely been reviewed [16]. Rapid association dissociation reactions have already been observed, either for protein polymerization [17] or for protein interaction with small uncharged constituents of buffer [18]. Moreover, rapid macromolecular isomerizations were also observed during electrophoresis and chromatography showing multiple zone depending on the rate of interconversion [11, 10, 21]. In the present case only the last possibility would apply to what was observed. Polymerization of a-lactalbumin in the eluting buffer can be excluded by the result of gel filtration and boundary analysis where a-lactalbumin was found to behave as a single and homogeneous species. Specific interactions of a-lactalbumin with phosphate buffer can be excluded on the basis of several observations: nonheterogeneity was observed on polyacrylamide disc gel electrophoresis as compared with bovine a-lactalbumin in Tris. HCI buffer, bimodality was always observed when 50 mM morpholinopropane sulfonic buffer was used in place of phosphate. Moreover, for a given temperature, the relative amount of the two peaks did not depend on concentration of phosphate in the eluting buffer. From these data, it appears that human a-lactalbumin may undergo some isomerization. But as no difference in any of the following properties could be observed between the two eluting species: circular dichroism, optical rotatory dispersion, ultraviolet and fluorescence spectra, sedimentation velocity and partial specific volumes, lactose synthase activity and Nacetyllactosamine inhibition experiments and antigenic properties; we rather suspect that the bimodality is induced by the anionic matrix itself during chromatographic process which can be schematized as follows. c~LAs

/\ \Z

aLAs is the unique species of a-lactalbumin in solution.

aLA'b and aLA"b are the two forms of a-lactalbumin bound to the solid matrix.

etLAs

According to the chromatographic conditions, a linear increase of salt, an apparent heterogeneity will be observed if one of the two forms, for example aLAg is more tightly bound to the anionic matrix than aLA~,', aLA~, will be eluted at a higher ionic strength and will be responsible for the apparition of a second peak during the chromatography. Moreover if the induction by the anionic matrix of this conformational change is dependent on the temperature, pH or n-alcohol concentration, any change in one of these parameters will modify the ratio of aLA~,/aLA;,' and produce different chromatographic patterns. The induction of such a conformational change by an anionic matrix can be related to the reversible conformational change observed in solution at pH above 9 [22]. It is also tempting to relate this conformational change to the previous hypothesis of Singer [23] who suggested that a-

81 lactalbumin, as a peripheral membrane protein, would have two conformational states from which only one would be able to interact with the galactosyltransferase. The conversion from one conformational state to another being induced either by a ligand either by the galactosyltransferase itself. Chemical studies on human a-lactalbumin [24] are n o w in progress in order to establish if such a hypothesis can find any experimental support. REFERENCES 1 Schmidt, D. V. and Ebner, K. E. (1972) Biochim. Biophys. Acta 263, 714-720 2 Pr6aux, G., De Weer, P., Peirsman, E., Tielemans, K. and Lontie, R. (1964) Arch. Int. Physiol. Biochem. 73, 1 3 Prieels, J. P., Cludts, M., Dolmans, M. and L6onis, J. (1974) Arch. Int. Physiol. Biochem. 82, 194 4 Barman, T. E. (1970) Biochim. Biophys. Acta 214, 242-244 5 Hopper, K. E. and McKenzie, H. A. (1973) Biochim. Biophys. Acta 295, 352-363 6 Hindle, E. J. and Wheelock, J. V. (1971) Chimia 25, 188-190 7 Lyster, R. L. T. (1972) J. Dairy Res. 39, 279-318 8 Hopper, K. E. (1973) Biochim. Biophys. Acta 293, 364-370 9 Hopper, K. E. and MacKenzie, H. A. (1974) Mol. Cell. Biochem. 3, 93-107 10 Rawitch, A. B. and Gleason, M. (1971) Biochem. Biophys. Res. Commun. 45, 590-597 11 Barel, A. O., Prieels, J. P., Maes, E., Looze, Y. and L6onis, J. (1972) Biochim. Biophys. Acta 257, 288-296 12 Khatra, B. S., Herries, D. G. and Brew, K. (1974) Eur. J. Biochem. 44, 537-560 13 Lin, T. Y. (1972) Methods in Enzymology, Vol. XXV, Part B 44-45 14 Davis, B. S. (1965) Ann. N.Y. Acad. Sci. 121,404-427 15 Prieels, J. P., Poortmans, J., Dolmans, M. and L6onis, J. (1975) Eur. J. Biochem. 50, 523-527 16 Cann, J. R. (1972) Methods in Enzymology, Vol. XXV, Part B, 157-178 17 Johnson, P., Shooter, E. M. and Rideal, E. K. (1950) Biochim. Biophys. Acta 5, 376-396 18 Cann, J. R. (1970) Interacting Macromolecules, Acad. Proc. N.Y. 58-83 19 Scholten, P. C. (1961) Arch. Biochem. Biophys. 93, 568-575 20 Cann, J. R. and Bailey, H. R. (1961) Arch. Biochem. Biophys. 93, 576-579 21 Keller, R. A. and Gidings, J. C. (1960) J. Chromatogr. 3, 205-220 22 Barel, A. O. and Prieels, J. P. (1975) Eur. J. Biochem. 50, 463-473 23 Singer, J. T. (1974) Annu. Rev. Biochem. 43, 805-834 24 Schindler, M., Sharon, N. and Prieels, J. P. (1976) Biophys. Biochem. Res. Commun. 69, 167-173