Electrophoretic Behaviors of α-Lactalbumin and β-Lactoglobulin Mixtures Caused by Heat Treatment * *

In order to study the reaction behaviors of bovine α-lactalbumin (α-La), β-lactoglobulin (β-Lg), and their mixtures during heat treatment, samples were analyzed using native-polyacrylamide gel electrophoresis (Native-PAGE), sodium dodecylsulfate (SDS)-PAGE, and two-dimensional (2-D)-PAGE. The electrophoresis demonstrated that the loss of native-α-La increased as temperature increased, and that the loss of apo-α-La was slightly higher than that of holo-α-La. The tests also showed that during heat treatment, a mixture of α-La and β-Lg was less stable than α-La alone. As such, it was assumed that β-Lg induced holo-α-La to be less stable than apo-α-La during heat treatment. The reaction behavior of α-La (holo-, apo-form) during heat treatment showed similar patterns in the 2-D-PAGE electropherogram, but the mixture of α-La and β-Lg created new bands. In particular, the results showed a greater loss of native α-La in the holo-α-La and β-Lg mixture than in the apo-α-La and β-Lg mixture. Thus, it can be concluded that the holo-α-La and β-Lg mixture was more intensively affected by heat treatment than other samples, and that free sulphydryl groups took part in the heat-induced denaturation. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 7 : 1041-1045)


INTRODUCTION
Bovine α-lactalbumin (α-La), with about 14.2 kDa molecular weight, is present at concentrations ranging from 1 to 1.5 g L -1 , and it consists of 123 amino acid residues.α-La is nearly spherical and has a highly compact, globular structure with four disulfide bonds (Wong et al., 1996).α-La is also susceptible to heat denaturation (Morr, 1976;Permyakov and Berliner, 2000;Ha, 2001).Ruegg et al. (1977) showed that this occured at 65.2°C and a pH 6.7, but that 80 to 90% of the denaturation can be reversed with cooling.The calcium-bound native α-La is holo-α-La, while the calcium-free α-La is apo-α-La (Hiraoka et al., 1980;Bryant and Andrews, 1984).Few results about reaction behavior of holo-α-La and apo-α-La during heat treatment have been reported (Bernal and Jelen, 1984;Relkin, 1996).
Bovine β-lactoglobulin (β-Lg) is the most abundant in the whey proteins, comprising about 50% of the total whey proteins, and is present at concentrations of 2 to 4 g L -1 in bovine milk.β-Lg B, a polypeptide of 162 amino acid residues, has a molecular mass of 18.3 kDa, and has been sequenced (Eigel et al., 1984).This protein exists in solution as a 36.6 kDa dimer between pH 4 and 6.5 and has an isoelectric point of 5.2.Below pH 3.5 and above 7.5, the dimer dissociates into a slightly expanded monomer.Between pH 3.5 and 5.2, the dimer of β-Lg A polymerizes into a 146 kDa octamer at low temperatures (Swaisgood, 1996).
The heat denaturation and aggregation of α-La and β-Lg have been studied extensively in milk and whey systems (Hillier and Lyster, 1979;Parris et al., 1991;Relkin, 1996;Havea et al., 1998;Oldfield et al., 2000), where almost all of the β-Lg was incorporated into the aggregates via disulfide bonds and, to a considerable extent, via hydrophobic interactions (Havea et al., 1998;Oldfield et al., 2000).
In this study, we investigated the electrophoretic reaction behavior of bovine α-La, β-Lg, and their mixtures during heat treatment to elucidate how the whey proteins affect each other and to provide for dairy industry with more information.

MATERIALS AND METHODS
Holo-α-La (L5385, Ca-bound), apo-α-La (L6010, Cafree), β-Lg, the molecular weight standards and the gel buffer salts were obtained from Sigma Chemical Co.(St.Louis, MO, USA).Most of the reagents used for the preparation of electrophoresis gels were obtained from BioRad Laboratories (Hercules, CA, USA).The water was purified using a MilliQ system (Millipore Corp., Bedford, MA, USA).
The protein concentration was adjusted to 2.0 mg mL -1 using a 15 mM phosphate buffer with a pH of 6.8.The samples were heated at 65 to 100°C in small plastic (Eppendorf) vials for 10 min and cooled immediately in iced water.In all cases, the mixtures of α-La and β-Lg were on a 1:1 volume ratio.
One-dimensional (1D) and two-dimensional (2D) PAGE was used to analyze the heated samples, as described by Havea et al. (1998).The 1D PAGE was either run on alkaline- (Andrews, 1983) or SDS-PAGE (Laemmli, 1970).After preparing the appropriate gel, 10 to 20 µl samples of 0.2 mg protein/ml solutions diluted with appropriate sample buffer were injected into sample wells and then electrophoresed to separate the proteins.For the alkaline 2D (second-dimensional)-PAGE, a strip from the alkaline gel containing the protein bands that had been separated in a first dimensional run, was cut and rinsed in SDS sample buffer.This strip was placed on the top of a SDS gel and run in a second dimension.For the SDS-reduced 2D-PAGE, a strip from the SDS gel containing the protein bands that had been separated in a first dimensional run, was cut and placed in hot (94°C) solution of SDS buffer containing 2mercaptoethanol (5 ml/L SDS sample buffer) for 30 s.The gel strip was then rinsed with water to remove the excessive 2-mercaptoethanol, then used for a second dimensional run on another SDS gel (Havea, 1998;Manderson et al., 1998).

Effect of heat on mixture of holo-α-La and β-Lg
When mixture (1:1, v/v) of holo-α-La and β-Lg was heated together and the resultant samples were analyzed by alkaline-and SDS-PAGE, the patterns shown in Figure 1 were obtained.Bands of native α-La and β-Lg began to lose their intensity at the temperature around 80°C, forming dimers, trimers, oligomers, and large aggregates that could not get through the stacking gel (Figure 1A).The  denaturation rate of native α-La was faster than that of native β-Lg, which is consistent with other reports (Havea et al., 2001;Hong and Creamer, 2002).A Comparison of the patterns shown in Figure 1 with those of earlier results (Lee and Hong, 2002) indicates a much greater rate of loss of the native holo-α-La when β-Lg is present.This is due to the substantial overlap of the α-La and β-Lg aggregate bands in the 1D PAGE patterns, as shown by the 2D patterns.The reactions between holo-α-La and β-Lg involve interprotein hydrophobic associations at a molecular level that allow segments of each molecule to interact with the other so that disulfide bond interchange can occur.

Effect of heat on mixture of apo-α-La and β-Lg
The differences between the patterns created by mixtures that contained the apo-α-La and those that contained the holo-α-La were slight but appeared to be moving in the same direction as the patterns created by α-La by itself; that is, apo-α-La reacted faster than holo-α-La (Figures 1 B, 2 B).It is known that α-La reversibly transforms into molten globule conformation at high temperature.The results shown in these Figures, which demonstrate that apo-α-La is more easily converted into non-native forms than holo-α-La, confirm the molten globule intermediate hypothesis (Hirose, 1993;Relkin, 1996;Hong and Creamer, 2002).
It was clear that the loss of native-like protein was greater for the apo-α-La than for the holo-α-La (Figure 1 A and Figure 2 A).In addition, more SDS dimers and higher aggregates were formed when the apo-α-La was given heat treatment (Hong and Creamer, 2002;Lee and Hong, 2002).
Figures 3 A and B (native→non-reduced SDS-PAGE) show 2-D electropherograms of holo-α-La, apo-α-La and β-Lg heated at 85°C, and suggest that the apo-α-La and β-Lg mixture produced less dimers, trimers and large aggregates than the holo-α-La and β-Lg.This implies that the β-Lg induces a greater loss of the holo-α-La than apo- α-La and is stimulated to form aggregates through denaturation of native α-La.The apparently lower rate of loss of SDS monomeric α-La (Figure 1 B), in comparison with the apparent loss of native-like α-La (Figure 1 A), was caused by the non-native α-Las, which were separate bands in native (alkaline)-PAGE but ran as a single band in the SDS-PAGE system (Figure 3 A).
Figure 4 A and B (native→ reduced SDS-PAGE) show 2-D electropherograms of holo-α-La, apo-α-La and β-Lg heated at 85°C and are not very different from the 2-D electropherograms of the holo-and apo-α-La.The trace spots of oligomers of apo-α-La and β-Lg mixture were slightly clearer than those found in the holo-α-La and β-Lg mixture.Similar results were recently reported by Hong and Creamer (2002).
The holo-α-La showed more stability than the apo-α-La during heat treatment, while the mixtures of holo-α-La, apo-α-La and β-Lg were less stable than α-La itself.These results are consistent with previous report that the holo-α-La itself was more heat stable than apo-α-La (Lee and Hong, 2002).Heated mixtures of α-La and β-Lg contained large aggregates that were held together by disulfide bonds, as well as intermediate-sized aggregates that were held together primarily by disulfide bonds and, to a lesser extent, by covalent bonding (Matsudomi et al., 1992;Dalgleish et al., 1997;Havea et al., 1998).
In the mixtures, β-Lg seems to catalyze more extensively during the heat denaturation of native-like α-La of holo-α-La than that of apo-α-La.The formation of nonnative β-Lg monomers, dimers, etc. was not influenced intensively.It is assumed that the free sulphydryl groups of β-Lg take part in the heat-induced aggregation reaction.
In conclusion, the loss of native-like protein was greater for apo-α-La than for holo-α-La.β-Lg induces a greater loss of holo-α-La than apo-α-La, and stimulates native α-La to form aggregates through denaturation.The free sulphydryl groups of β-Lg may take part in the heatinduced denaturation reaction.