Glycosylated kappa-caseins and the sizes of bovine casein micelles. Analysis of the different forms of kappa-casein.

Fast protein liquid chromatography (FPLC) of the kappa-casein from bovine casein micelles of different sizes is used to demonstrate that the proportions of glycosylated and non-glycosylated forms of kappa-casein do not vary with micellar size. The results suggest that glycosylated kappa-casein is distributed similarly to unglycosylated kappa-casein within the micellar structure.

A kinetic analysis of the dephosphorylation, by bovine spleen phosphoprotein phosphatase (EC 3.1.3.16) of a phosphopeptide derived from beta-casein.

A peptide containing the four closely grouped phosphoseryl residues present in beta-casein has been enzymatically dephosphorylated with bovine spleen phosphoprotein phosphatase (EC 3.1.3.16). The course of the dephosphorylation reaction has been followed by cellulose acetate electrophoresis and the amount of partially phosphorylated peptides present at each stage quantified by the same method. The phosphate groups are shown to be removed in a sequential manner and the rate constants for each stage of the dephosphorylation have been computed from the data obtained. The rate constants indicate that interaction in the intact peptide results in an enhancement of the activity of the phosphoseryl cluster.

The topography of bovine beta-casein at an oil/water interface as determined from the kinetics of trypsin-catalysed hydrolysis.

The topography of bovine beta-casein at a soya oil/water interface was studied by following the kinetics of the trypsin-catalysed hydrolysis. Tryptic peptides were identified from their amino acid compositions and the kinetics were compared with those obtained from beta-casein in solution. Whereas soluble beta-casein was initially hydrolysed at a number of trypsin-sensitive bonds, the hydrolysis of the protein at the interface was a more ordered event. The crucial initiating step was the cleavage of the N-terminal peptides 1-25 and 1-28 from the molecule. Hydrolysis at other trypsin-sensitive sites could then occur. This suggests that with the exception of the large hydrophilic moiety in the N-terminal region, most of the beta-casein molecule is inaccessible to the proteinase, and lies fairly flat on the oil/water interface. After removal of the N-terminal peptide, the remaining macropeptide can reorientate and other hydrophilic regions become accessible to the proteinase.

The thermochemistry of reactions between alpha-s1-casein and calcium chloride.

The enthalpies of reactions between alpha-s1-casein and Ca2+ in solution were measured using a gradient layer calorimeter. The reactions are exothermic between 0 and 4.3 mM CaCl2. In the region of 4.3 mM CaCl2 there is a change to an endothermic reaction corresponding to micellisation. A calcium-binding curve has been obtained under the same conditions as the calorimetry experiments and this shows two sigmoidal binding phases. Turbidity measurements show that there is an association process corresponding to the second sigmoidal phase. A tentative interpretation of the heat curve in the region before micellisation is given in terms of the site binding of Ca2+, conformational changes in the protein and association. The main thermal processes are taken to be exothermic intramolecular hydrogen bonding induced by calcium binding and endothermic hydrophobic bonding.

Measurement of particle sizes by elastic and quasi-elastic light scattering.

A method of measuring an average particle radius in a highly polydisperse dispersion using the wavelength dependence of turbidity is described. For particles which are no larger than 0.3 of the wavelength of light used, a polynomial representation of the scattering cross-section can be used. For larger particles, more extensive numerical calculations are required. The use of the method is illustrated by determining the average particle radius of casein micelles by both elastic and quasi-elastic light scattering techniques. A polydisperse homogeneous sphere model is found to be a reasonably accurate representation of casein micelles. Several modifications of the model which would improve the agreement between the two techniques are mentioned briefly.

A mechanism for the chymosin-induced flocculation of casein micelles.

The chymosin-induced flocculation of casein micelles of bovine milk can be explained and calculated in terms of three relationships, which are (i) the action of chymosin upon the kappa-casein of the micelles; (ii) the probability that a micelle, with a given proportion of its kappa-casein destroyed, will aggregate, and (iii) the aggregation of micelles by a Smoluchowski mechanism. Details of the calculations are given, and the theory is shown to be in good agreement with experimental observations of the dependence of the clotting time with variations in enzyme and substrate concentrations.

Kinetics of aggregation of alpha s1-casein/Ca2+ mixtures: charge and temperature effects.

Time-dependent light-scattering studies have been made on mixtures of alpha s1-casein and Ca2+, at fixed temperature over a range of [Ca2+] and [alpha s1-casein], and also as functions of temperature. Measurements were also made of the extent of precipitate formation in the casein/Ca2+ mixtures, using centrifugation. The results are analysed in terms of a monomer-octamer equilibrium between calcium caseinate particles followed by a Smoluchowski aggregation in which only the octamers can participate. The equilibrium constant is dependent upon the charge on the protein/Ca2+ particles, and hence can be related to the extent of binding of Ca2+ to the alpha s1-casein. The Smoluchowski constant is likewise shown to be charge-dependent. The variation of the reaction rate with temperature can be ascribed solely to the changing charge of the alpha s1-casein/Ca2+ complex caused by changed binding of Ca2+ at different temperatures.

Orthokinetic flocculation of caseinate-stabilized emulsions: influence of calcium concentration, shear rate, and protein content.

Calcium-induced flocculation of caseinate-stabilized soybean oil-in-water emulsions in conditions of Couette flow was studied. A concentrated emulsion (20% oil, 0.5-2.0% sodium caseinate in 20 mM imidazole, pH 7) was diluted 20 times in buffer containing concentrations of CaCl(2) between 9 and 17 mM and sheared at rates between 335 and 1340 s(-)(1). The average particle size (d(43)), measured by integrated light scattering, increased in a sigmoidal manner with shearing time. An increased shear rate resulted in an increased flocculation rate, because of the increased number of collisions between particles, but a decreased value of the maximum d(43), because higher shear rates increasingly disrupted the flocs. The flocculation rate was increased by increasing the calcium concentration, indicating an increased collision efficiency. The orthokinetic stability of the emulsions was increased with increased protein content, and it is postulated that the increased surface coverage and hydrodynamic thickness of the adsorbed protein layer increased steric repulsion between droplets, so that higher calcium concentrations were necessary to induce sufficient conformational change of the proteins to allow flocculation. At high caseinate concentrations, calcium may also induce precipitation of unadsorbed caseins from the serum to the oil-water interface, thereby increasing steric repulsion and hence increasing orthokinetic stability.

Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins.

Quantitative analysis of competitive milk protein adsorption to air/water interfaces in aqueous foam was performed by capillary electrophoresis (CE). Foams were made by whipping protein solutions, in which skim milk powder (SMP) and whey protein isolate (WPI) were mixed at 0.5% protein in different proportions at different pH values and NaCl concentrations. Preferential adsorption of beta-casein into foam phases occurred under most solution conditions, if partial dissociation of the casein micelles had occurred. Preferential adsorption of beta-casein was not observed with added Ca2+, due to the re-association of casein micelles. Enrichment of caseins into the foam phase was more apparent than that of whey proteins. The foamability of SMP demonstrated a continuous improvement due to the gradually increasing dissociation of casein micelles when the concentration of NaCl increased from 0 to 0.8 M. The foamability of WPI increased when NaCl concentration rose from 0 to 0.1 M, and decreased with further increase in NaCl concentration. NaCl at low concentration (I < or = 0.4) did not show a significant effect on the competitive adsorption among milk proteins, indicating that electrostatic interactions do not play a key role in competitive adsorption. NaCl at higher concentration, e.g., 0.6 M, caused less whey protein to be adsorbed to the air/water interfaces. The whippability of WPI was highest at pH 4.5 and lowest at pH 3, and that of SMP was the opposite. The proportions of beta-lactoglobulin and alpha-lactalbumin in the foam phase were lower at acidic pH and higher at basic pH, compared with that at natural pH of WPI.

Surface shear viscometry as a probe of protein-protein interactions in mixed milk protein films adsorbed at the oil-water interface.

Time-dependent surface viscosities are reported for films adsorbed from binary mixtures of the proteins alpha-lactalbumin, beta-lactoglobulin and beta-casein. The measurements were made at a planar interface between n-tetradecane and various protein solutions (10(-3) wt% of each protein, pH 7, 25 degrees C) using a Couette-type torsion-wire surface viscometer operating at very low shear-rate. Differences in behaviour between simultaneous and sequential exposure of the pairs of proteins to the interface were investigated. Some experiments were performed with chemically modified beta-lactoglobulin samples whose disulphide bonds had been cleaved and blocked. Displacement of one protein by another (e.g. alpha-lactalbumin by beta-casein) is indicated by a sudden drop in surface viscosity immediately after addition of the second protein. In systems containing beta-lactoglobulin, the long-time surface viscosity is very sensitive to the adsorption time of beta-lactoglobulin prior to addition of the second protein. Blocking the disulphide bonds in beta-lactoglobulin leads to a much faster approach to a steady-state surface viscosity. This is interpreted in terms of a much more rapid unfolding of the disordered molecules of modified beta-lactoglobulin at the oil-water interface. We conclude that surface viscosity experiments give useful and sensitive information about competitive adsorption and cooperative interactions in mixed protein films.

Particle development in apple juice determined by light scattering and electron microscopy.

Light scattering was used to detect the kinetic development of particles in apple juices produced with and without oxidation and in procyanidin extracts prepared from these two juices. Particle size developed exponentially in both oxidized and unoxidized juices, suggesting an enzymatic origin for the particle forming reactions. The procyanidin extract from oxidized juices produced particles which grew linearly, suggesting diffusion-controlled aggregation or coalescence of particles. Procyanidins from unoxidized juice showed no particle development over at least 60 days of storage. Electron microscopy showed particles similar to those seen previously and some newly described morphologies. The mechanism of haze particle development is discussed in the light of the light scattering and electron microscopic results.

Analysis by fast protein liquid chromatography of variants of kappa-casein and their relevance to micellar structure and renneting.

Fast protein liquid chromatography was used to study the kappa-casein fraction of casein micelles from bulk milk and from milk from individual animals homozygous for kappa-caseins A and B. The extent of glycosylation of the kappa-casein appeared to have no effect on its distribution in casein micelles of different sizes, nor did it affect the rate at which kappa-casein was destroyed during renneting. The rate of breakdown of kappa-casein during renneting was also almost independent of micellar size. The results may indicate a difference between methods which analyse for intact kappa-casein or for the product macropeptide during renneting.

Quantitation of alpha S1-casein aggregation by the use of polyfunctional models.

A model for the coagulation of Ca2+ alpha S1- caseinate is described wherein the casein molecules are considered to aggregate as polyfunctional units. The functionalities on the protein molecules are produced as a consequence of the binding of Ca2+, and the multiple equilibria which are established during this binding produce a distribution in which not all casein molecules have the same functionality. The theory is compared with experimental data of the precipitation reaction, and is shown to provide an adequate quantitative description of the precipitation.

Circular-dichroism studies on two -lactamases from Bacillus cereus.

The circular-dichroism (CD) spectra of beta-lactamases I and II from Bacillus cereus 569/H are reported, along with that of the beta-lactamase II free from carbohydrate. The results show that carbohydrate makes an appreciable contribution to the optical activity of beta-lactamase II in the far-ultraviolet, and that removal of carbohydrate greatly affects the optical activity of several aromatic side chains of the protein moiety. Both tyrosyl and tryptophanyl residues are affected, showing that some of these residues must be near to the surface of the protein moiety, close to the site of attachment of the carbohydrate. Although the far-ultraviolet CD spectrum of beta-lactamase II resembles that of a protein containing some beta-structure, it can be shown that this is a consequence of the optical activity of carbohydrate in this region of the spectrum, and that the protein is likely to contain alpha-helix rather than beta-pleated sheet structure. The overall structures of the protein components of beta-lactamases I and II are similar, but not identical, as shown by the dissimilarity of the CD spectra when calculated on a mean residue basis.

Binding of calcium ions to bovine beta-casein.

The isotherms for Ca++ binding to bovine alpha-casein have been measured at 5 temperatures in the range 4-40 degrees C and at 4 different ionic strengths. The results are interpreted by an interactive-site binding model, and are compared with results previously obtained on alpha s1-casein. The affinity of beta-casein for the first Va2+ to bind is similar to the affinity of alpha s1-casein for the same binding event: however, binding of subsequent Ca2+ to beta-casein is weaker than the binding to alpha s1-casein. The results are discussed in terms of precipitability of the 2 caseins caused by the binding of Ca2+.

A study of beta-casein tertiary structure by intramolecular crosslinking and mass spectrometry.

The objective of this study was to obtain experimental evidence to extend the discussion on the 3-D structure of beta-casein (beta-CN). The approach involved the preparation of homobifunctional crosslinkers, bis(sulfosuccinimidyl) derivatives of dicarboxylic acids of several lengths, which specifically react with primary amines of lysinyl residues or the N-terminal in the protein. The intramolecular crosslinks formed were determined by enzymatic digestion and by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry combined with comparison against the theoretical digestion patterns. This procedure allowed the measurement of distances between the crosslinked residues. Ten different masses arising from 8 different specific intramolecular crosslinks were identified. Of these, 5 crosslinks were in good agreement with a published model (Kumosinski et al., 1993). Two other crosslinks each connected 2 residues that are much closer together, according to the model, than the maximum length of the crosslink. However, one of the crosslinks apparently connected 2 residues that are predicted by the model to be 16.7 A farther apart than the crosslink's stretched length. This disparity might be explained by structural flexibility. The structure expressed by the model is probably one of several energetically favorable conformations of the beta-CN molecule, whose structure is best described as rheomorphic rather than either a fixed structure or a random coil.

Production of a novel ingredient from buttermilk.

The presence of material derived from the milk fat globule membrane (MFGM) makes buttermilk (the byproduct of butter making) distinct from any other dairy product. Membrane filtration of commercial buttermilk was carried out to obtain isolates rich in MFGM material. The separation of MFGM from the skim milk proteins present in commercial buttermilk was carried out by the addition of sodium citrate followed by microfiltration through a membrane of 0.1-microm nominal pore size. The sodium citrate caused the dissociation of casein micelles and allowed permeation of a large proportion of the skim-milk derived proteins through the membrane. This process successfully concentrated MFGM material in the retentate, and demonstrated that membrane filtration can be employed to produce MFGM fractions from commercial buttermilk. The utilization of MFGM isolates from buttermilk is of increasing importance in light of recent studies suggesting the role of phospholipids in many health-related functions: buttermilk is an untapped resource of these functional components.

The coagulation of differently sized casein micelles by rennet.

Fractions of bovine casein micelles of different sizes were prepared by successive centrifugation steps, and dilute suspensions of the different fractions were reacted by rennet. The molecular weight increase with time after addition of rennet was measured by light scattering. After an initial lag stage, where the increase was zero, and a short intermediate stage, the molecular weight became linearly proportional to time, indicating complete conversion of the kappa-casein substrate by the enzyme. The slope of the final portion of the growth profile can be used to define values of the Smoluchowski rate constant governing the aggregation. No significant differences in this constant were found with micelles of different sizes, and the value of the rate constant suggested that the aggregation reaction of completely renneted micelles is only moderately retarded relative to the diffusion-controlled process, even at room temperature.