J. Dairy Sci. 86:78-85
© American Dairy Science Association, 2003.
The Use of Lithium Chloride to Study Human Milk Micelles
S. M. Sood* and
C. W. Slattery*,
* Department of Biochemistry and Microbiology and
Department of Pediatrics, School of Medicine, Loma Linda University, Loma Linda, CA 92350
Corresponding author:
S. M. Sood; e-mail:
ssood{at}som.llu.edu.
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ABSTRACT
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Various methods have been used to study the dissociation of milk micelles in attempts to determine their structure and the interactions that stabilize them. These include the addition of urea, cooling to alter hydrophobic bonding, the addition of EDTA to sequester calcium, and changes in pH to alter molecular charge. For this study, the mild chaotropic agent LiCl was added to human milk micelles, and measurements were made on the relative percentages of the six different phosphorylation levels of ß-casein (CN) at various LiCl concentrations for different lengths of time and at different temperatures. Added LiCl had little effect at 37°C but caused maximal dissociation, mainly of the ß-CN species with higher phosphorylation levels, at 23°C and 4°C between 1 and 2 M concentration. Comparison was made with 2-M additions of NaCl, MgCl2, and KCl at 4°C, with LiCl showing the only appreciable change. The results suggest that Li+ may displace Ca+2 in protein-Ca+2-protein or protein-colloidal calcium phosphate-protein salt bridges and that the nonphosphorylated form of human ß-CN may change its conformation and mode of interaction upon phosphorylation. Lithium chloride may be useful to study the dissociation of the different CN in bovine milk micelles.
Key Words: milk micelles • chaotropic agents • lithium chloride • human ß-casein • micelle stabilization
Abbreviation key: ß-CN-0P to ß-CN-5P = phosphorylation level of human ß-casein ranging from zero to five as indicated by number preceding P, CCP = colloidal calcium phosphate
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INTRODUCTION
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The micelles of milk are composed of CN proteins that contain phosphorylated serine or threonine residues. These phosphate esters bind Ca+2, and this would lead to precipitation of the 
s1-CN, s2-CN and/or ß-CN were it not for micelle formation due to the stabilizing effect of the carbohydrate-containing 
-CN. These micelles are maintained under various environmental conditions by different types of intermolecular interactions. These interactions have been studied by several different means. Cooling at 4°C results in a large amount of the ß-CN dissociating from the micelles, leaving almost all of the s1-CN and s2-CN and most of the -CN intact (Morr, 1975; Sood et al., 1977; Ali et al., 1980; Ono et al., 1981; Davies and Law, 1983; Aoki et al., 1990). These results point toward hydrophobic bonding as a major stabilizing factor since it is weaker at lower temperatures (Mahler and Cordes, 1967). This dissociation was dependent upon the time that the milk was cooled and was also influenced by the size of the micelle, with the large micelles releasing most of the ß-CN and the small micelles that are rich in -CN supplying most of the -CN that was released (Ono et al., 1981). Hydrophobic bonds are also broken by urea, as are hydrogen bonds, and this reagent was used in a number of early batch and chromatographic separation procedures for the individual CN, described by Thompson (1971) and Mackinlay and Wake (1971). McGann and Fox (1974) later studied the extensive dissociation of milk micelles by urea.
In addition to CN proteins that bind Ca+2 (Farrell et al., 2002), micelles also are associated with inorganic calcium phosphate, called colloidal calcium phosphate (CCP). Removal of Ca+2 through dialysis or with EDTA caused dissociation of bovine CN micelles (Lin et al., 1972; Ono et al., 1978; 1981; Sood et al., 1979; Griffin et al., 1988). Furthermore, the CCP associated with the micelles may also be removed by dialysis against phosphate-free buffer (Holt et al., 1986), or the CCP may be dissolved out by adding acid to the milk (Dalgleish and Law, 1989) with maintenance of micelle integrity because of titration of the charge on the phosphate esters. Restoration of the charge to its original value causes micelle dissociation (Lucey et al., 1997). Thus, there seems to be a balance between attractive hydrophobic interactions and the electrostatic repulsion of charged groups, mainly phosphorylated amino-acid residues. These interaction modes may be accommodated by different models for micelle structure (Horne, 1998; Walstra, 1999).
The probable importance of ß-CN to milk micelle structure is emphasized by the fact that it is present in appreciable quantities in the milk of all species, whereas the other major Ca+2-binding CN are not. For example, human milk has very little s1-CN (Rasmussen et al., 1995), nor has a human s2-CN been found, but it is estimated to contain 70% ß-CN and 27% -CN (Carroll et al., 1985). Additionally, the ß-CN of human milk is phosphorylated at different levels from zero to five (Groves and Gordon, 1970). This makes it a very good model system to study the effects of different micelle-dissociating agents and correlate them with possible micelle structure. Some of these agents may affect hydrophobic interactions only, and some may effect only charge-charge interactions that will vary with phosphorylation level.
In self-association studies in this laboratory on the six different phosphorylated forms of ß-CN purified from human milk (Sood et al., 1985, 1988, 1990, 1992; Javor et al., 1991; Sood and Slattery, 1994 Sood and Slattery, 1997, 2000), the predominant role of hydrophobic interactions was stressed (Slattery et al., 1989), along with electrostatic effects caused by pH changes (Sood and Slattery, 1995). Also, the intact native human milk micelle system has been examined to determine the contributions of hydrophobic interactions and electrostatic effects through cooling and addition of EDTA (Sood et al., 1997) and pH adjustments (Sood et al., 1998).
In addition to these types of interactions, hydrogen bonds (H-bonds) may play an important role in the stabilization of proteins and their secondary structure (Schulz and Schirmer, 1979). Intermolecular H-bonds may also be of importance in protein-protein interactions and are reported to be involved in the complex formation between bovine s1 -CN and -CN (Chiba et al., 1970; Sedmerova and Sicho, 1972). Furthermore, the strength of H-bonds is generally enhanced by hydrophobic interactions (Nemethy et al., 1963), which, as mentioned above, play a major role in the formation and structure of the CN micelles. Most chaotropic agents such as detergents, urea, or guanidine-hydrochloride tend to disrupt various of these interactions, and urea particularly is used in the preparation of purified human CN (Sood et al., 1985; Slattery et al., 1989), as well as bovine and others. These harsh treatments tend to denature the proteins and tell us little about protein-protein interactions. A mild chaotropic agent such as lithium chloride (LiCl) may be needed. Greater than 4 M LiCl is said to disrupt -helical and tertiary structure in proteins (Maruyama et al., 1977) and, therefore, must disrupt the H-bonds. Probably due to its smaller ionic radius (6.8 nm) it can penetrate deeper into the core of the molecule and can affect even buried residues. In addition, molar concentrations of salt may exert profound influences on protein structure through preferential interactions (Arakawa and Timasheff, 1984). These interactions generally result in preferential hydration, that is, increased water in the protein domain, but in certain cases salt binding can occur. The preferential interactions may either enhance or limit protein association reactions depending upon the types of specific forces involved in the protein-protein contacts. Concentrations from 1 to 4 M LiCl are apparently nondenaturing but have been used for the extraction of proteins from large complexes, such as the ribosomes of Escherichia coli (Dijk and Littlechild, 1979) and bovine mitochondria (Schieber and O’Brien, 1982) and from the aminoacyl-tRNA synthetase complex (Norcum, 1991). The effects of LiCl addition on human micelle ß-CN composition may be quantitated in human micelle pellets from skimmed milk (Dev et al., 1994).
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MATERIALS AND METHODS
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Mature, whole human milk samples were noted to be close to pH 7.2. The milk was skimmed according to the method of Sood et al. (1985) and divided into three aliquots; one was maintained at 37°C, one was equilibrated at room temperature (23°C), and one was kept at 4°C.
For analysis, 1 ml of LiCl at the proper concentration was added to 4 ml samples of skimmed milk at the designated temperature to give the desired final concentration. These were equilibrated at temperature for 2 h for regular measurements or for longer periods when determining the effect of time. Samples were then centrifuged at 110,000 x g for 90 min to produce pellets of CN micelles that were each dissolved in Buffer A (4.5 M urea, 10 mM Tris, 0.46 mM DTT, pH 8.0) and assayed for total protein by measuring the ultraviolet absorbance at 280 nm, A280 (corrected for light scattering at A320). To separate and quantitate the different levels of human ß-CN, the samples were analyzed on a Mono Q HR5 (LKB Pharmacia, Piscataway, NJ) fast-protein liquid chromatography column using a linear gradient between Buffer A and Buffer B (1.0 M NaCl in Buffer A) as described by Sood et al. (1997). The output from the fast-protein liquid chromatography detector was fed into a computer and analyzed by means of the 3000 Series Chromatography Data System Software (Nelson Analytical, Cupertino, CA) for determination of peak areas. Purified human (ß-CN-0P), -1P, -2P, -3P, and -4P samples were used as standards. Finally, in order to determine whether the changes were the results of simple ionic effects or due to disruption of bonds by LiCl, solutions of NaCl, MgCl2, and KCl at pH 7.2 were added to portions of the sample maintained at 4°C to get a final concentration of 2 M of each. They were equilibrated for 2 h, and pellets of micelles were obtained and analyzed as above.
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RESULTS AND DISCUSSION
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Dev et al. (1994) described the separation of the micellar proteins on the Mono Q column and identified the various peaks by polyacrylamide gel electrophoresis. Sood et al. (1997) reported the completeness of the separation of the different forms of ß-CN, showing the Mono Q profile of a standard mixture of the highly purified forms of human ß-CN phosphorylated at six different levels. The positions of these six discrete peaks were used to identify the peaks found upon analysis of the micelle pellets obtained after adding LiCl. Integration of the six peak areas with the Nelson software, after assignment of a baseline to eliminate background and overlapping peaks, was translated into relative proportions for the different ß-CN in the micelles and expressed as the percentage of the total ß-CN composition in the pellet (Sood et al., 1997).
The effects of the addition of different concentrations of LiCl (from 0.1 M to 4.0 M) at 37°C and compared to the control (0.0 M LiCl) are presented in Figure 1
. No appreciable change in the relative percentages of different forms of human ß-CN was observed upon addition of LiCl to this milk sample at 37°C. This implies that there are no intermolecular bonds that may be broken by LiCl that are necessary for micelle stability at this temperature. These would include intermolecular H-bonds and salt bridges and hydrophobic bonds that could presumably be affected by a disruption of secondary structure. This stability was maintained for up to 6 h with 2 M LiCl, as indicated for the sample in Figure 2, although a small effect may be occurring progressively with time in which there is perhaps a decrease in the percentage of the 3P, 4P, and 5P forms relative to the 0P, 1P, and 2P forms. Although some experiments were carried out to 16 h at lower temperatures, the milk was not generally stable for long periods of time at this high temperature due to proteolytic action.
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Figure 1. Effect of LiCl concentration at 37°C. Bar graph showing the percentage of the different forms of ß-CN in human milk micelles at LiCl concentrations from 0 to 4 M, incubated for 2 h at 37°C before analysis.
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Figure 2. Effect of incubation time at 37°C with 2 M LiCl. Bar graph showing the percentage of the different forms of ß-CN in human milk micelles incubated at 37°C for the time indicated.
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Figure 3 shows the effect of different LiCl concentrations at 23°C. There is a change in composition at this lower temperature that appears to be a maximum at 1 to 2 M LiCl. Incubating a sample with 2 M LiCl for increasing lengths of time (Figure 4) indicates an appreciable change in micelle composition that is apparently almost complete by 2 h. Compared with the standard at 37° (0.0 M LiCl in Figure 1
) or even for 2 M LiCl at 23°C and zero time, an appreciable increase in the relative percentage of the 0P and 1P forms and a decrease of higher phosphorylation levels was observed. Lowering the temperature further to 4°C again shows that 1 to 2 M LiCl had an effect on micelle composition (Figure 5) that appeared to be largely complete by 1 h (Figure 6).
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Figure 3. Effect of LiCl concentration at 23°C. Bar graph showing the percentage of the different forms of ß-CN in human milk micelles at LiCl concentrations from 0 to 4 M, incubated for 2 h at 23°C before analysis.
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Figure 4. Effect of incubation time at 23°C with 2 M LiCl. Bar graph showing the percentage of the different forms of ß-CN in human milk micelles incubated at 23°C for the time indicated.
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Figure 5. Effect of LiCl concentration at 4°C. Bar graph showing the percentage of the different forms of ß-CN in human milk micelles at Li concentrations from 0 to 4 M, incubated for 2 h at 4°C before analysis.
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Figure 6. Effect of incubation time at 4°C with 2 M LiCl. Bar graph showing the percentage of the different forms of ß-CN in human milk micelles incubated at 4°C for the time indicated.
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A comparison of these results with the effect of cooling only (Sood et al., 1997) indicates that these changes are similar to those seen normally after almost 24 h at 4°C. In order to check further if these changes were merely the effect of diminished hydrophobic interactions at lower temperatures, studies were also carried out to determine the percentage of different forms of human ß-CN at 4°C upon incubation for 2 h with 2 M NaCl, MgCl2, and KCl, as well as LiCl. The results are shown in Figure 7. It is apparent that there is a major difference in the sample containing Li+ but relatively minor changes when the other cations are present. Apparently, any preferential interactions (Arakawa and Timasheff, 1984) normally induced at 2M concentrations of NaCl, MgCl2, or KCl have little influence on CN micelle stability. This suggests that the dissociation does not come simply from charge shielding by high ionic strength but from some specific effect of the Li+ ion.
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Figure 7. Effect of 2 M concentration of different cations at 4°C. Bar graph showing the percentage of the different forms of ß-CN in human milk micelles incubated at 4°C for 2 h with the addition of 2 M chloride salts of the cations listed.
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It seems likely that human milk micelles, which contain mostly ß-CN and -CN, are formed and maintained through a combination of hydrophobic interactions and salt bridges involving protein phosphate esters and CCP. It is important to note that when Ca+2 binds to the phosphate esters, the positive charge on the cation is not completely neutralized. The most plausible explanation for the results given here is that the Li+ ion replaces Ca+2 bound to the phosphate esters, the charge is mostly neutralized, and the reduction in charge disrupts the ester-ester or ester-CCP-ester salt bridges. However, at 37°C, the stability of the micelle is maintained by the hydrophobic interactions. This is in contrast to the effects of EDTA, where the micelle is disrupted even at 37°C, as the Ca+2 is removed (Sood et al., 1997) and the negative charge of the phosphates is restored. Because of the salt bridges, the micelles at 4°C are surprisingly stable and do not completely dissociate as might be expected. Rather long periods of cooling are required. However, LiCl allows them to dissociate much more readily. The maximum effect is from 1 to 2 M Li+, and there is less dissociation as the ion concentration increases. This implies that in addition to breaking the salt bridges, the high ionic strength of the LiCl, or perhaps preferential hydration, shields the residual charges on the proteins from each other and prevents the repulsion that tends to dissociate the micelles.
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CONCLUSIONS
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When the mildly chaotropic agent, LiCl, is added to human milk CN micelles, Li+ appears to replace Ca+2 and permit ready dissociation of the micelles upon reduction of the hydrophobic bonding by cooling. The fact that neither cooling alone nor cooling after LiCl addition causes a dissociation of the forms of ß-CN with lower levels of phosphorylation that is proportional to those with higher levels implies that the 0P form may participate in interactions that are not available to the phosphorylated forms. This implies that phosphorylation of the 0P form may cause a change in conformation of the molecule. It would also be of interest to see if LiCl would have a differential effect on the dissociation of s1-, s2-, and ß-CN in bovine milk micelles, perhaps giving some insights into their contributions to micelle structure.
Received for publication April 17, 2002.
Accepted for publication June 11, 2002.
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ABSTRACT
INTRODUCTION
