Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3

Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.     

Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin

The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.     

Structure and function of proteins involved in milk allergies

Allergy to milk proteins has been defined as any adverse reaction mediated by immunological mechanisms to one or several of proteins found in milk. The milk allergy has been classified according to the onset of symptoms as immediate or delayed type. The milk allergy seems to be manifested by three major proteins found in milk: α-lactalbumin, β-lactoglobulin and caseins. The structural comparison of allergenic sites in α-lactalbumin and β-lactoglobulin with the structure of lactoferrin has clearly shown that yet another major milk protein lactoferrin also possesses allergenic sites and thus may qualify to be an allergen. The heat treatment of milk proteins considerably reduces their allergenicity.     

Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

Solution structure and dynamics of bovine beta-lactoglobulin A

Using heteronuclear NMR spectroscopy, we studied the solution structure and dynamics of bovine beta-lactoglobulin A at pH 2.0 and 45 degrees C, where the protein exists as a monomeric native state. The monomeric NMR structure, comprising an eight-stranded continuous antiparallel beta-barrel and one major alpha-helix, is similar to the X-ray dimeric structure obtained at pH 6.2, including betaI-strand that forms the dimer interface and loop EF that serves as a lid of the interior hydrophobic hole. [1H]-15N NOE revealed that betaF, betaG, and betaH strands buried under the major alpha-helix are rigid on a pico- to nanosecond time scale and also emphasized rapid fluctuations of loops and the N- and C-terminal regions.     

Isolation and purification of serum and interfacial peptides of a trypsinolyzed β-lactoglobulin oil-in-water emulsion

Information on the conformation of proteins adsorbed to an oil–water interface is usually determined by following the time course of enzymatic hydrolysis of the protein in an oil-in-water emulsion. Unlike previous works reported in the literature, the research presented in this paper provides information on which peptides are actually in contact with the lipid bilayer (interfacial peptides) and those segments that project into the aqueous phase (serum peptides). In order to achieve this classification of peptides, we present a method to separate serum peptides from interfacial peptides by initial centrifugation steps followed by reversed-phase high-performance liquid chromatography. The effectiveness of the method was ascertained by performing proteolysis on β-lactoglobulin adsorbed to an oil-water interface in a soybean oil-water emulsion. It was found that more peptides are qualitatively and quantitatively found adsorbed to the oil–water interface as compared to peptides released into the serum.     

Crystal Structure and Molecular Conformation of the Cyclic Hexapeptide cyclo-(Gly-Aib-Gly)2

We have synthesized and crystallized the cyclic peptide (Gly-Aib-Gly) ₂. Its structure has been determined by conventional X-ray diffracti on methods. In the crystal it adopts a conformation with one β-turn (type I) and its mirror image at the other side of the ring. All conformation al angles are similar to those reported for these amino acid residues. In particular the Aib residue has a conformation intermediate between α- and 3₁₀-helical conformations. The ring is an adequate model for the β-turn conformation. A molecule of formic acid is found in the crystal which shows a very short hydrogen bond with one of the glycine carbonyl groups.     

A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space

The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc.     

Structure,Function, and Regulation of the Three β-Adrenergic Receptors

Three β-adrenergic receptor subtypes are now known to be functionally expressed in mammals. All three belong to the R₇G family of receptors coupled to G-proteins, and characterized by an extracellular glycosylated N-terminal and an intracellular C-terminal region and seven transmembrane domains, linked by three exta- and three intracellular loops. The catecholamine ligand binding domain, studied using affinity-labeling and site-directed mutagenesis, is a pocket lined by residues belonging to the transmembrane domains. The region responsible for the interaction with the Gs protein which, when activated, stimulates adenylyl cyclase, is composed of residues belonging to the parts most proximal to the membrane of intracellular loop i₃ and the C-terminal region. The pharmacology of the three subtypes is quite distinct: in fact most of the potent β₁/β₂ antagonists (the well known β blockers) act as agonists on β₃. The subtype is resistant to short-term desensitization mediated by phosphorylation through PKA or βARK, in stark contrast to the β₁ or β₂ subtypes. Various compounds (dexamethasone, butyrate, insulin) up regulate β₁ or β₁ subtypes while down-regulating β₃ whose expression strictly correlates with differentiation of 3T3-F442A fibroblasts into adipocytes, thus confirming that the expression of the three subtypes may each be regulated independently to exert a specific physiologic role in different tissues or at different stages of development.     

The structure of a doripenem‐bound OXA‐51 class D β‐lactamase variant with enhanced carbapenemase activity

OXA‐51 is a class D β‐lactamase that is thought to be the native carbapenemase of Acinetobacter baumannii. Many variants of OXA‐51 containing active site substitutions have been identified from A. baumannii isolates, and some of these substitutions increase hydrolytic activity toward carbapenem antibiotics. We have determined the high‐resolution structures of apo OXA‐51 and OXA‐51 with one such substitution (I129L) with the carbapenem doripenem trapped in the active site as an acyl‐intermediate. The structure shows that acyl‐doripenem adopts an orientation very similar to carbapenem ligands observed in the active site of OXA‐24/40 (doripenem) and OXA‐23 (meropenem). In the OXA‐51 variant/doripenem complex, the indole ring of W222 is oriented away from the doripenem binding site, thereby eliminating a clash that is predicted to occur in wildtype OXA‐51. Similarly, in the OXA‐51 variant complex, L129 adopts a different rotamer compared to I129 in wildtype OXA‐51. This alternative position moves its side chain away from the hydroxyethyl moiety of doripenem and relieves another potential clash between the enzyme and carbapenem substrates. Molecular dynamics simulations of OXA‐51 and OXA‐51 I129L demonstrate that compared to isoleucine, a leucine at this position greatly favors a rotamer that accommodates the ligand. These results provide a molecular justification for how this substitution generates enhanced binding affinity for carbapenems, and therefore helps explain the prevalence of this substitution in clinical OXA‐51 variants.     

Association of bovine β‐casein protein variant I with milk production and milk protein composition

The aim of this study was to detect new polymorphisms in the bovine β‐casein (β‐CN) gene and to evaluate association of (new) β‐CN protein variants with milk production traits and milk protein composition. Screening of the β‐CN gene in genomic DNA from 72 Holstein Friesian (HF) bulls resulted in detection of 19 polymorphisms and revealed the presence of β‐CN protein variant I in the Dutch HF population. Studies of association of β‐CN protein variants with milk composition usually do not discriminate protein variant I from variant A2. Association of β‐CN protein variants with milk composition was studied in 1857 first‐lactation HF cows and showed that associations of protein variants A2 and I were quite different for several traits. β‐CN protein variant I was significantly associated with protein percentage and protein yield, and with α_s1‐casein (α_s1‐CN), α_s2‐casein (α_s2‐CN), κ‐casein (κ‐CN), α‐lactalbumin (α‐LA), β‐lactoglobulin (β‐LG), casein index and casein yield. Inferring β‐κ‐CN haplotypes showed that β‐CN protein variant I occurred only with κ‐CN variant B. Consequently, associations of β‐κ‐CN haplotype IB with protein percentage, κ‐CN, α‐LA, β‐LG and casein index are likely resulting from associations of κ‐CN protein variant B, while associations of β‐κ‐CN haplotype IB with α_s1‐CN and α_s2‐CN seem to be resulting from associations of β‐CN variant I.     

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log₁₀ reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65–3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.     

Packing constraints of hydrophobic side chains in (α/β)8 barrels

An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between C_α atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.     

Crystal structure of (L-Arg)-BO bovine insulin at 0.21 nm resolution

The crystal structure of (L-Arg)-B0 bovine insulin has been determined, using data to 0.21 nm and atomic parameters of 2Zn porcine insulin as a starting model, by the difference Fourier method, the restrained least square method and X-PLOR package, interspersed with careful review of the electron density, to a final R-factor of 0.182 and r.m.s. deviation of 0.002 2nm for the bond lengths and 4.3° for the bond angles. The electron densities of additional (L-Arg)-B0 residues to B-chain N-terminus of two monomers in each asymmetric unit are very dear. The crystallographic micro-environment of the N-terminus of the B-chain is different from that of rhombohedral 2-zinc insulin.     

Examination of actin polymerization and viscosity induced by cations and ionic strength when cross-linked by alpha-actinin

Increasing potassium chloride concentration from 0 to 100 mM and magnesium chloride from 0 to 2 mM show a parallel rate increase in polymerizing actin, whereas increasing calcium chloride concentration from 0 to 0.2 mM decreases the rate of polymerizing actin. The presence of alpha-actinin has little influence on the polymerization kinetics of actin under these conditions. Viscometric measurements indicate that the presence of various mono- and divalent cations, ionic strength, and alpha-actinin in combination are responsible for changes in the mechanical properties of solutions containing actin. The actin filament dynamic behavior is drastically reduced under these conditions as confirmed by quasi-elastic light scattering.     

Transglutaminase-mediated modification of glutamine and lysine residues in native bovine beta-lactoglobulin

Bovine beta-lactoglobulin (BLG) is a major component in whey and its physical properties are important for the texture of many dairy-based foods. Modification of proteins with transglutaminase from Streptoverticillium mobaraense (MTGase) can be used to alter their physical properties. MTGase-mediated modification of native BLG was until now, however, not effective. Here we report a method that allows for the enzymatic modification of native BLG with MTGase. Lysines 8, 77, and 141 were modified with alpha-N-carbobenzyloxy-glutamine-glycine and glutamines 35, 59, 68,and 155 were modified with 6-aminohexanoic acid under nonreducing and nondenaturing conditions. MTGase-mediated BLG crosslinking is hampered by the low reactivity of the lysines and enzymatic deamidation of the glutamines prevails. Modification of BLG with poly-lysine yields a BLG derivative with increased affinity for the water-air interface and stronger surface tension lowering capacities than normal BLG. Hence, this modification method offers the opportunity to change the functional properties of BLG and to prepare novel protein foods.     

Antagonistic and synergistic interactions between aluminum and manganese on growth of Vigna unguiculata at low ionic strength

Concentrations of soluble aluminum (Al) and manganese (Mn) frequently reach phytotoxic levels in acid soils. While dose response relationships for these metals are well documented, the effects of combined exposure have received less attention. We have examined the effect of combinations of Al and Mn on growth and metal accumulation in Vigna unguiculata (L.) Walp. grown in solution culture under conditions of low ionic strength (conductivities typically < 100 µS cm⁻¹). The nature of interaction between these metals varied with the specific physiological response, the part of the plant investigated, and the relative amount of stress imposed. Analysis of growth data provided evidence for amelioration of metal toxicity (antagonistic effects), although this effect was dose dependent. Analysis of metal content data provided evidence for antagonistic and synergistic (exacerbation of toxicity) effects, again depending on dose. Analysis of foliar symptoms also provided evidence for antagonisms and synergisms, with the nature of the response dependent on the specific physiological response and specific plant part investigated. In contrast with previous reports, evidence for antagonistic, synergistic, and multiplicative effects on growth, metal uptake, and expression of foliar symptoms have been obtained under physiologically and environmentally relevant conditions. These results suggest a more detailed analysis of the potential for interactions between metals in the environment is required.     

Structure prediction and fold recognition for the ferrochelatase family of proteins

An α/β barrel is predicted for the three-dimensional (3D) structure of Bacillus subtilis ferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI. © 1997 Wiley-Liss Inc.     

Crystallographic and mutational analyses of tannase from Lactobacillus plantarum

Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of α/β‐hydrolase superfamily with an additional “lid” domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate. Proteins 2013; 81:2052–2058. © 2013 Wiley Periodicals, Inc.