Edward D. Wickham

Secondary structural studies of bovine caseins: temperature dependence of β-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization

Abstract To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between β-casein and the other caseins, three-dimensional models have been presented. Secondary structural prediction algorithms and molecular modeling techniques were used to predict β-casein structure. The secondary structure of bovine β-casein was re-examined using Fourier transform infrared and circular dichroism spectroscopies to test these predictions. Both methods predict a range of secondary structures for β-casein (28–32% turns, 32–34% extended) at 25°C. These elements were highly stable from 5 to 70°C as viewed by circular dichroism. More flexible conformational elements, tentatively identified as loops, helix and short segments of polyproline II, were influenced by temperature, increasing with elevated temperatures. Another view is that as temperature decreases, these elements are lost (cold denaturation). Several distinct transitions were observed by circular dichroism at 10, 33 and 41°C, and another transition, extrapolated to occur at 78°C. Calculations from analytical ultracentrifugation indicate that the 10, 33 and 41°C transitions occur primarily in the monomeric form of the protein. As β-casein polymers are formed, and increase in size, the transitions at higher temperature may reflect changes in the more flexible conformational elements as they adjust to changes in surface charge during polymer formation. The transition at 10°C may represent an actual general conformational change or cold denaturation. Over the range of temperatures studied, the sheet and turn areas remain relatively constant, perhaps forming a supporting hydrophobic core for the monomers within the micelle-like polymer. This interpretation is in accord with the known properties of β-casein, and those predicted from molecular modeling.

Environmental Influences on Bovine κ-Casein: Reduction and Conversion to Fibrillar (Amyloid) Structures

The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of κ-casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the κ-casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem.17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-κ). Surprisingly, at only 37°C, the RCM-κ-casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM-κ-caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and κ turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in κ-casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM-κ-casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.

Effect of self-association of αs1-casein and its cleavage fractions αs1-casein(136–196) and αs1-casein(1–197), on aromatic circular dichroic spectra: comparison with predicted models

Abstract The self-association of native αs1-casein is driven by a sum of interactions which are both electrostatic and hydrophobic in nature. The dichroism of aromatic side chains was used to derive regio-specific evidence in relation to potential sites of αs1-casein polymerization. Near-ultraviolet circular dichroism (CD) revealed that both tyrosine and tryptophan side chains play a role in αs1-casein associations. Spectral evidence shows these side chains to be in an increasingly nonaqueous environment as both ionic strength and protein concentration lead to increases in the degree of self-association of the protein from dimer to higher oligomers. Near-UV CD investigation of the carboxypeptidase A treated peptide, αs1-casein(1–197), indicated that the C-terminal residue (Trp199) may be superficial to these interactions, and that the region surrounding Trp164 is more directly involved in an aggregation site. Similar results for the cyanogen bromide cleavage peptide αs1-casein(136–196) indicated the presence of strongly hydrophobic interactions. Association constants for the peptides of interest were determined by analytical ultracentrifugation, and also were approximated from changes in the near-UV CD curves with protein concentration. Sedimentation equilibrium experiments suggest the peptide to be dimeric at low ionic strength; like the parent protein, the peptide further polymerizes at elevated (0.224 M) ionic strength. The initial site of dimerization is suggested to be the tyrosine-rich area near Pro147, while the hydrophobic region around Pro168, containing Trp164, may be more significant in the formation of higher-order aggregates.

Environmental effects on disulfide bonding patterns of bovine kappa-casein.

Bovine κ-casein, the stabilizing protein of the colloidal milk protein complex, has a unique disulfide bonding pattern. The protein exhibits varying molecular sizes on SDS-PAGE ranging from monomer to octamer and above in the absence of reducing agents. Heating the samples with SDS prior to electrophoresis caused an apparent decrease in polymeric distribution: up to 60% monomer after 30min at 90°C as estimated by densitometry of SDS-PAGE. In contrast, heating the samples without detergent at 90 or 37°C caused a significant increase in high-molecular-weight polymers as judged by electrophoresis and analytical ultracentrifugation. In 6 M urea, the protein could be completely reduced, but upon dialysis, varying degrees of polymer reformation occurred depending on the dialysis conditions. Spontaneous reoxidation to polymeric forms is favored at low pH (<5.15) and low ionic strength. The results are discussed with respect to the influence of the method of preparation on the polymer size of κ-caseins and on their resultant physical chemical properties.

Investigation of molecular interactions between β-lactoglobulin and sugar beet pectin by multi-detection HPSEC.

Molecular interactions between β-lactoglobulin (β-LG) and sugar beet pectin (SBP) were studied using online multi-detection high performance size exclusion chromatography (HPSEC) at neutral pH and 50mM ionic strength. The hydrodynamic properties of various interacting polymer fractions were characterized in detail and compared with those of β-LG and SBP. Results showed that ∼6.5% (w/w) of native dimeric β-LG molecules formed complexes with over 35% SBP molecules of varying sizes, 800, 110 and 75 kDa. Although the β-LG molecules bind to SBP molecules of all sizes and shapes, they tend to favor the intermediate (110 kDa) and small sized (75 kDa) SBP molecules. All resulting complexes possess altered shapes and hydrodynamic properties when compared to unbound SBP and β-LG. About half of the interacting β-LG (∼3.5%) molecules were thought to bind to a small amount of non-covalently bound feruloyl groups, possibly present in SBP. When pre-heat treated β-LG and SBP were combined, more than 16% of β-LG formed complexes with at least 45% of SBP molecules of varying sizes, Mw∼750-800, 110, and 55-80 kDa. The complexes formed between β-LG aggregates and/or oligomers and the large SBP molecules (750-800 kDa) adopt the shape of β-LG aggregates, random coil. Both groups of complexes formed between β-LG intermediate (110 kDa) and small sized (55-80 kDa) SBP take on the shape of rigid rod. It was speculated that half of the interacting heat-treated β-LG molecules (∼8%) are complexed with non-covalently bound feruloyl groups in SBP.

Enrichment and Purification of Casein Glycomacropeptide from Whey Protein Isolate Using Supercritical Carbon Dioxide Processing and Membrane Ultrafiltration

Whey protein concentrates (WPC) and isolates (WPI), comprised mainly of β-lactoglobulin (β-LG), α-lactalbumin (α-LA) and casein glycomacropeptide (GMP), are added to foods to boost nutritional and functional properties. Supercritical carbon dioxide (SCO2) has been shown to effectively fractionate WPC and WPI to obtain enriched fractions of α-LA and β-LG, thus creating new whey ingredients that exploit the properties of the individual component proteins. In this study, we used SCO2 to further fractionate WPI via acid precipitation of α-LA, β-LG and the minor whey proteins to obtain GMP-enriched solutions. The process was optimized and α-LA precipitation maximized at low pH and a temperature (T) ≥65 °C, where β-LG with 84% purity and GMP with 58% purity were obtained, after ultrafiltration and diafiltration to separate β-LG from the GMP solution. At 70 °C, β-LG also precipitated with α-LA, leaving a GMP-rich solution with up to 94% purity after ultrafiltration. The different protein fractions produced with the SCO2 process will permit the design of new foods and beverages to target specific nutritional needs.

Structural and thermal stability of β-lactoglobulin as a result of interacting with sugar beet pectin.

Changes in the structural and thermal stability of β-lactoglobulin (β-LG) induced by interacting with sugar beet pectin (SBP) have been studied by circular dichroism (CD), Fourier transform infrared, and steady-state as well as time-resolved fluorescence spectroscopic techniques. It has been demonstrated that SBP not only is capable of binding to native β-LG but also causes a significant loss in antiparallel β-sheet, ∼10%, accompanied by an increase in either random coil or turn structures. In addition, the interaction also disrupted the environments of all aromatic residues including Trp, Phe, and Tyr of β-LG as evidenced by near-UV CD and fluorescence. When preheated β-LG was combined with SBP, the secondary structure of β-LG was partially recovered, ∼4% gain in antiparallel β-sheet, and Trp19 fluorescence was recovered slightly. Although forming complexes with SBP did not significantly impact the thermal stability of individual secondary structural elements of β-LG, the environment of Trp19 was protected considerably.

Reactions between β-lactoglobulin and genipin: kinetics and characterization of the products.

In this paper, we present the first detailed study of the reaction kinetics and the characterization of the products from the endothermic reactions between β-lactoglobulin and genipin. The effects of the concentration, temperature, and pH were investigated. In the temperature range studied, the reaction was approximately a pseudo-first-order with respect to genipin and 0.22-order and -0.24-order with respect to β-lactoglobulin for pH 6.75 and 10.5 with corresponding activation energy (E(a)) estimated to be 66.2 ± 3.8 and 9.40 ± 0.36 kJ/mol, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies, validated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, showed the presence of oligomeric, i.e., di-, tri-, quadri-, and pentameric, forms of cross-linked β-lactoglobulin by genipin at neutral but not alkaline pH; however, an extensive cross-linked network was not observed, consistent with the atomic force microscopy images. It was demonstrated that the reaction temperature and the concentration of genipin but not that of β-lactoglobulin positively affected the extent of the cross-linking reactions.

Distribution of ADPase activity in the lactating rat mammary gland and its possible role in an ATP cycle in the Golgi apparatus

A Ca2+/Mg(2+)-stimulated ADPase has been found to occur in the lactating rat mammary gland. The enzyme is membrane associated and occurs in mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 6.3 and one at pH 7.4. Studies with inhibitors and activators indicate that the enzyme is similar to ADPases found in other tissues and is distinct from the uridine nucleoside diphosphatase previously reported in the mammary Golgi apparatus. The occurrence of ADPase in the Golgi apparatus indicates a possible role for this enzyme in the milk secretory process, while the microsomal enzyme could be involved in extracellular activities.

Localization of acid phosphatase in lactating rat mammary glands

SummaryLocalization of acid phosphatase in mammary glands of lactating rats was studied by both biochemical and cytochemical methods. Cytochemically, acid phosphatase activity was detected by using lead citrate as the capture agent for the inorganic phosphate released from p-nitrophenyl phosphate. The activity was predominantly localized in the lumina of the endomembrane system and in the milk that had been secreted into the alveolar lumen. Biochemically, acid phosphatase was present in all the subcellular fractions with higher activities in the membrane-associated fractions. The localization of tartrate-resistant acid phosphatases within the endomembrane system of fully lactating rat mammary tissue suggests a possible role for these enzymes in milk secretory processes.