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PubMed1977

Amino acid analysis by gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters. Complete resolution using a support-coated open-tubular capillary column.

Amino acids have been separated by gas-liquid chromatography as their N-heptafluorobutyryl isobutyl esters. Complete resolution of derivatives of all the common amino acids has been achieved using a high-performance support-coated open-tubular capillary column. The analysis time was 30 min. Modifications to the derivatization procedure of MacKenzie and Tenaschuk have been introduced. Acylation by heating at 150 degrees was shown to be destructive; 110 degrees has been selected for routine preparation. To obtain a volatile histidine derivative it has been found necessary to add an antioxidant and to heat samples with ethoxyformic anhydride prior to injection. Amino acid analysis of beta-lactoglobulin after 6 N HCl digestion yielded results in good agreement with those obtained by the conventional ion-exchange method. The method has also been successfully applied to estimation of the different caseins in whole casein and in purified fractions by amino acid analysis of residues liberated by carboxy-peptidase digestion.

Amino Acids↗
PubMed1983

Determination of native and denatured milk proteins by high-performance size exclusion chromatography.

High-performance size exclusion chromatography on a TSK 3000 SW column was used to separate and quantify the individual milk proteins in their native and/or denatured state. Quantitation of the individual native whey proteins within +/- 10% was achieved. The denatured proteins were dissociated into their monomer units by using sodium dodecyl sulphate and beta-mercaptoethanol. A good separation of the whey protein monomers was achieved and the quantitation of the individual whey proteins within +/- 10% was possible. Under these conditions, alpha s1- and beta-casein were eluted as one peak, whereas kappa-casein and gamma-casein were eluted in the same position as beta-lactoglobulin and alpha-lactalbumin respectively. A method for calculating the areas and hence the concentration of the individual proteins in a mixture of casein and whey proteins is described.

Animals↗
PubMed2001

Microcalorimetric studies of the interaction mechanisms between proteins and Q-sepharose at pH near the isoelectric point (pI) effects of NaCl concentration, pH value, and temperature.

This study examined the interaction mechanisms of beta-lactoglobulins A and B (Lg A, Lg B) with an anion exchanger, Q-Sepharose at pH near the isoelectric point at which the proteins are expected to be electrically neutralized under various NaCl concentrations and temperatures by the equilibrium binding analysis and the adsorption enthalpy directly measured by isothermal titration calorimetry. The data evaluated from isotherms fitted by the Langmuirean model reveal that the addition of NaCl considerably reduced the binding affinities and capacities of both the proteins with Q-Sepharose at pH 5.2, indicating that electrostatic forces are dominant during the adsorption. However, the hydrophobic interaction seems to be involved in adsorption as well at a higher NaCl concentration, and the adsorption enthalpies confirm this suggestion. In addition, the effects of temperature on the equilibrium binding behaviors for Lg A or Lg B with Q-Sepharose were found to be salt concentration-dependent, probably due to their different binding mechanisms at 0.03 M and 0.3 M NaCl. Where, at 0.3 M NaCl, the hydrophobic interaction plays a more pronounced role. This implication was again supported by the adsorption enthalpies. The presented data provide further insight to the interaction mechanisms between proteins and ion exchangers, facilitating the optimization of protein separations.

Calorimetry↗
PubMed1991

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. II. Effect of the stationary phase.

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. greater than 6000 dalton. No such effect was seen for polypeptides with mol. wt. less than 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the beta-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.

Acetates↗
PubMed1991

Column-friendly reversed-phase high-performance liquid chromatography of peptides and proteins using formic acid with sodium chloride and dynamic column coating with crown ethers.

Reversed-phase high-performance liquid chromatography of proteins, traditionally carried out with strong acids like trifluoroacetic acid or phosphoric acid, which can damage reversed-phase columns, can be performed with excellent results using the far milder formic acid in the presence of salt. For certain separations, dynamic coating of the column with crown ethers can bring added resolution. Examples given are for peptides from a digest of methionine growth hormone, protein separations from whey proteins containing alpha-lactalbumin and the beta-lactoglobulins A and B, and bovine and porcine insulins. The separation of methionine-growth hormone from growth hormone is also described.

Animals↗
PubMed2003

High-performance cation-exchange chromatofocusing of proteins.

Chromatofocusing using high-performance cation-exchange column packings, as opposed to the more commonly used anion-exchange column packings, is investigated with regard to the performance achieved and the range of applications possible. Linear or convex gradients in the range from pH 2.6 to 9 were formed using a variety of commercially available column packings that provide a buffering capacity in different pH ranges, and either polyampholytes or simple mixtures having a small number (three or fewer) of buffering species as the elution buffer. The resolutions achieved using cation-exchange or anion-exchange chromatofocusing were in general comparable, although for certain pairs of proteins better resolution could be achieved using one type of packing as compared to the other, evidently due to the way electrostatic charges are distributed on the protein surface. Several chromatofocusing methods were investigated that take advantage of the acid-base properties of commercially available cation-exchange column packings. These include the use of gradients with a composite shape, the use of very low pH ranges, and the use of elution buffers containing a single buffering species. The advantages of chromatofocusing over ion-exchange chromatography using a salt gradient at constant pH were illustrated by employing the former method and a cation-exchange column packing to separate beta-lactoglobulins A and B, which is a separation reported to be impossible using the latter method and a cation-exchange column packing. Trends in the apparent isoelectric points determined using cation- and anion-exchange chromatofocusing were interpreted using applicable theories. Results of this study indicate that cation-exchange chromatofocusing is a useful technique which is complementary to anion-exchange chromatofocusing and isoelectric focusing for separating proteins at both the analytical and preparative scales.

Albumins↗
PubMed2003

On-chip protein sample desalting and preparation for direct coupling with electrospray ionization mass spectrometry.

A membrane-based desalting step integrated in a MS microchip is presented: drugs, peptides and proteins are adsorbed on a hydrophobic poly(vinylidene difluoride) membrane, which allows the washing out of salts. The integration with microfluidics permits a controlled elution of analytes from the membrane and their direct mass spectrometric analysis by electrospray ionisation MS. The desalting process is demonstrated with picomole amounts of propanolol, insulin and cytochrome c. Moreover, this stop-and-go desalting process is tolerant to high concentrations of urea, and to the presence of reductants such as dithiothreitol. This particular feature allowed the chemical tagging of cysteines in beta-lactoglobulin A with iodoacetamide. Finally, the integration of chemical tagging, on-chip desalting and MS microchip paves the way for the development of high-throughput analytical procedure for structural proteomics.

Adsorption↗
PubMed1998

Protein binding on polyelectrolyte-treated glass. Effect of structure of adsorbed polyelectrolyte.

Polyelectrolyte adsorption can be used to modify the surface of chromatographic packings in order to make them more suitable for protein separations. We studied the binding of proteins to controlled pore glass (CPG) on which the polycation poly(diallyldimethylammonium chloride) (PDADMAC) was noncovalently immobilized through electrostatic interaction. We found that the selectivity of PDADMAC for bovine serum albumin vs. beta-lactoglobulin, identified in previous selective coacervation studies, is conserved after its immobilization on the CPG surface. Protein binding results showed that the pH, ionic strength, and mixing time for polyelectrolyte adsorption all affect subsequent protein binding, presumably via the molecular properties of the adsorbed polyelectrolyte layer. The polyelectrolyte adsorption layer thickness, for polyelectrolyte adsorbed at pH 9.0, ionic strength I = 0.001, was measured with size-exclusion chromatography as delta H = 2.5 +/- 0.5 nm. Quasielastic light scattering measurement of the polyelectrolyte hydrodynamic layer thickness (HLT) with a model system of PDADMAC and silica, supported a correlation between the structure of the adsorbed polyelectrolyte layer (e.g., loops and tails) and subsequent protein binding, although differences in magnitude between delta H and HLT suggest that adsorption onto silica may not mimic adsorption on CPG.

Adsorption↗
PubMed1998

Displacement chromatography of proteins using a self-sharpening pH front formed by adsorbed buffering species as the displacer.

The preparative-scale separation of two proteins into adjoined, pure bands was accomplished using a novel, hybrid chromatography method which employs chromatofocusing using a self-sharpening pH front and displacement development. The method eliminates the use of a traditional displacer for accomplishing displacement chromatography, and was used to separate the A and B forms of beta-lactoglobulin using a strong-base anion-exchange column packing and a buffer system composed of acetic acid and either 3-(N-morpholino)propane-sulfonic acid (MOPS) or 2-(N-morpholino)ethanesulfonic acid (MES). Sample loads up to 150 mg of protein were applied to a 75 x 7.5 mm column to produce a displacement train composed of highly pure protein bands with greater than 90% recovery of protein. A discussion is given of the chromatographic behavior of proteins under concentration overloaded conditions for the case where a self-sharpening pH front formed using adsorbed buffering species is used to desorb proteins from an anion-exchange column packing.

Adsorption↗
PubMed1999

Measuring conformational stability of proteins using an optimized temperature-controlled capillary electrophoresis approach.

The thermal denaturation process of a model protein, bovine beta-lactoglobulin, was analyzed using capillary zone electrophoresis (CZE). For this purpose, a commercial CE apparatus was improved, allowing efficient control and accurate measurement of the temperature up to 95 degrees C. Under various pH conditions, transition temperature (Tm), enthalpy change (delta H) and entropy change (delta S) associated with the thermal denaturation were determined. Moreover, the technique is unique in its ability to estimate the heat capacity change (delta Cp). This work shows that CZE, performed even when electroosmotic flow occurs, is an innovative approach for determining the stability curves of proteins. Accordingly, CZE is a powerful tool to study protein unfolding/folding quickly and with minimal sample requirements.

Animals↗
PubMed1996

Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays.

Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.

Amino Acid Sequence↗
PubMed1995

Efficacy and safety of hydrolyzed cow milk and amino acid-derived formulas in infants with cow milk allergy.

OBJECTIVE: To determine the antigenicity, nutritional adequacy, and growth-promoting efficacy of protein hydrolysate or amino acid-derived formulas in infants with cow milk allergy. STUDY DESIGN: Several protein hydrolysate or amino acid-derived formulas were graded for beta-lactoglobulin content and skin reactivity in 74 atopic children with cow milk allergy proved by a double-blind, placebo-controlled challenge. A randomized, prospective follow-up study of 9 months included 22 infants with a mean age of 6 months (95% confidence interval, 4 to 7), who were fed an extensively hydrolyzed whey formula (group We), and 23 infants with a mean age of 17 (95% confidence interval, 4 to 7) months, who were given an amino acid-derived formula (group AA). RESULTS: Both formulas were clinically and biochemically tolerated. The mean concentration of essential amino acids in plasma was lower in group We but higher in group AA compared with values for breast-fed control infants (p = 0.001). There was a different trend between the groups in weight (p = 0.09) and length (p = 0.006). Growth was promoted in group AA during the follow-up; it was constant during the first months, followed by a gradual decline in rate in group We. In both groups, atopic eczema improved significantly and progressively, and a downward trend was found in serum total and milk-specific IgE concentrations, proving the efficacy of both formulas. CONCLUSIONS: Extensively hydrolyzed formulas are safe and effective for most infants; an amino acid-derived formula may be preferable for infants with multiple food allergies, especially for the maintenance of normal growth.

Amino Acids↗
PubMed1995

Chronic constipation as a symptom of cow milk allergy.

Twenty-seven consecutive infants (mean age, 20.6 months) with chronic "idiopathic" constipation were studied to investigate the possible relation between constipation and cow milk protein allergy (CMPA). The infants were initially observed on an unrestricted diet, and the number of stools per day was recorded. Subsequently the infants were put on a diet free of cow milk protein (CMP) for two periods of 1 month each, separated by two challenges with CMP. During the CMP-free diet, there was a resolution of symptoms in 21 patients; during the two consecutive challenges, constipation reappeared within 48 to 72 hours. In another six patients the CMP-free diet did not lead to improvement of constipation. Only four of the patients who improved on the CMP-free diet had concomitant symptoms of suspected CMPA, but a medical history of CMPA was found in 15 of the 21 patients cured and in only one of the six patients whose condition had not improved (p < 0.05); in addition, in 15 of the 21 cured patients, results of one or more laboratory tests (specific IgE, IgG, anti-beta-lactoglobulin, circulating eosinophils) were positive at the time of diagnosis, indicating hypersensitivity, compared with one of the six patients whose condition did not improve (p < 0.05). The endoscopic and histologic findings at the time of diagnosis showed proctitis with monocytic infiltration in two patients cured with the CMP-free diet; after 1 month on this diet, they were completely normal. We conclude that constipation in infants may have an allergic pathogenesis.

Animals↗
PubMed1999

Cow's milk challenge through human milk evokes immune responses in infants with cow's milk allergy.

OBJECTIVES: In order to measure the immune response evoked in breast-fed infants with cow's milk allergy (CMA) by cow's milk challenge through human milk, mothers were given increasing doses of cow's milk after they had been on a cow's milk elimination diet. Another objective was to study the secretion of beta-lactoglobulin (BLG) into human milk before and during milk challenge in relation to the appearance of symptoms in infants. STUDY DESIGN: Seventeen asymptomatic mothers who had infants with challenge-proven CMA and 10 asymptomatic mothers who had healthy infants were recruited. Infants ranged in age from 1.8 to 9.4 months. A solid-phase enzyme-linked immunoassay (ELISPOT) was used to assess the total number of immunoglobulin-secreting and specific antibody-secreting cells. Flow cytometry was used to enumerate different lymphocyte subpopulations among peripheral blood lymphocytes primed during provocation by cow's milk antigens. BLG levels were assessed in human milk before the challenge and 1, 2, 3, and 4 hours after the commencement of the challenge. RESULTS: All but one of the infants with CMA showed symptoms of CMA during cow's milk challenge through human milk. There was a significant rise in the total number of immunoglobulin-secreting cells in the IgA and IgG classes associated with a positive cow's milk challenge response, but the proportions of peripheral blood B cells bearing CD19, CD23, CD19 and 23, CD5, or CD19 and CD5 were comparable. BLG levels were comparable in both study groups. CONCLUSIONS: Most of the infants with CMA reacted to cow's milk challenge through human milk. Hypersensitivity reactions to food antigens through human milk may be more common than previously thought.

Animals↗
PubMed1998

Proteic composition of corpora amylacea in the bovine mammary gland.

Histochemical studies of mammary gland sections obtained from healthy lactating Prim' Holstein cows contained numerous corpora amylacea, mainly located in active alveoli. Observations by electron microscopy revealed a cauliflower shape, with a fibrillar or multilayered organization. Mineral studies confirmed the presence of high calcium levels (12.3% of dry matter) and phosphorus (7.4%) in the corpora amylacea composition. These bodies stained positive to Von Kossa silver nitrate and to Periodic acid-Schiff. However, depending on the gland of origin, corpora amylacea stained positive or negative to Congo red. Histochemical studies seemed, therefore, insufficient to determine the presence or absence of amyloid. The amount of total protein varied by approximately 25%. Immunoblotting and analysis of the amino acid sequence of a peptidic fragment obtained from corpora amylacea gave clear evidence of the occurrence of caseins, beta-lactoglobulin and alpha-lactalbumin. However, the comparison between the amino acid composition of corpora amylacea and those of the main milk proteins indicated the presence of other proteins. Electrophoretic analysis also gave evidence of the presence of several other proteins, i.e. glycoproteins. Therefore, it is probable that corpora amylacea composition is much more complex than previously reported.

Animals↗
PubMed2003

Effect of native phosphocaseinate on the in vitro preservation of fresh semen.

The fertilization capacity of goat sperm stored in milk extenders is approximately 12-24h. Long-term storage of goat sperm (up to 3 days) is desirable as it would confer greater flexibility to breeding farms. The aim of this study was to evaluate in vitro motility parameters of buck spermatozoa for up to 7 days of storage using skim milk or chemically defined extender supplemented with native phosphocaseinate (NPPC). Four experiments were conducted to determine optimum temperature (4 or 15 degrees C) and storage conditions (aerobic versus anaerobic), the effect of seminal plasma on sperm survival, the optimal concentration of NPPC and the effect of beta lactoglobulin (BL). Both skim milk and NPPC were found to be more efficient for preserving goat sperm at 4 degrees C than at 15 degrees C (P<0.01). Furthermore, when sperm was stored at 4 degrees C, no detrimental effects of seminal plasma were observed. Our results showed that motility parameters can be maintained with success until Day 4. However, NPPC-based extenders extend the in vitro survival to 7 days of storage. The optimal concentration of NPPC for the preservation of sperm cells for 4 days of storage was 81g/l and for 7 days of storage was 81 and 54g/l. No effect of the supplementation of the NPPC extender with BL was found.

Animals↗
PubMed2001

Esterified milk proteins inhibit DNA replication in vitro.

DNA replication was studied in vitro in the presence of native and esterified milk proteins [alpha-lactalbumin (ALA), beta-lactoglobulin (BLG) and beta-casein (BCN)]. Addition of unmodified proteins to the PCR medium did not change the result of the reaction seen by electrophoresis, even at excessive ratios of basic amino acids in proteins:phosphate groups in DNA as high as 100:1. Addition of esterified proteins greatly reduced the intensity of the bands corresponding to the newly synthesized DNA, at ratios as low as 1:1 and 5:1 in case of methylated-BLG and methylated-ALA, respectively. The inhibitory effect of esterified proteins was directly proportional to their extent of esterification and strongly related to their DNA-binding capacity. Generally, inhibition of PCR with esterified proteins was similar to what can be observed with histones. However, stronger inhibition was observed with highly esterified proteins when using a higher ratio of basic:acid residues (1:1) when compared with 0.5:1 ratio in case of histones. Highly esterified BCN did not exert any inhibitory effect because of its relatively lower pI when compared with that of other esterified milk proteins and due to its lower positive net charge at the pH used for PCR. During a second PCR run, only the addition of new DNA template was able to reinitiate the reaction, giving rise to new synthesized DNA. Addition of Taq DNA polymerase did not enhance DNA synthesis, showing that inhibition was performed only by binding of DNA template and not by the inhibition of the polymerase.

Animals↗
PubMed2003

Influence of a sugar moiety (rhamnosylglucoside) at 3-O position on the reactivity of quercetin with whey proteins.

The reaction of whey proteins (WP) with quercetin and rutin (quercetin-3-O-rhamnosylglucoside) is influenced by the glycosidic bound sugar moiety. The protein derivatives formed showed a blocking of tryptophan (max. 49%), free amino (max. 32%) and thiol groups (max. 24%). The amount of quercetin and rutin bound covalently (up to 94 and 31nmol mg(-1), respectively) was estimated by their characteristic absorbance between 300 and 340 nm. At least one molecule of the phenolic reactant was covalently bound to a beta-lactoglobulin molecule (beta-Lg). High molecular protein fractions were detected by SDS-PAGE (cross-linking with quercetin). All results confirm that quercetin is more reactive than rutin. The pH-dependent solubility of the derivatives decreased, although their hydrophilic character increased. The structural changes (circular dichroism (CD)) showed that especially rutin causes perturbation of the secondary (decrease of alpha-helix elements accompanied by an increase in random coil) and tertiary structure. The in vitro proteolytic digestibility, especially of the rutin derivatives was elevated, due to an increase in denaturation of the derivatives.

Chromatography, High Pressure Liquid↗