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PubMed2006

FTIR and nDSC as analytical tools for high-concentration protein formulations.

PURPOSE: The aim of the study is to evaluate Fourier-transform infrared spectroscopy (FTIR) as an analytical tool for high-concentrated protein formulations. METHODS: FTIR is used to determine the melting temperature (T(m (FTIR))) of various proteins, such as bovine serum albumin (BSA), immunoglobulin (IgG1), beta-lactoglobulin (beta-LG), and lysozyme (HEWL), at different protein concentrations (5-100 mg/mL), where four data interpretation methods are discussed. The obtained T(m (FTIR)) values are further compared to the T(m) measured by the nanodifferential scanning calorimetry (nDSC) technique. RESULTS: The T(m (FTIR)) values of IgG1 and beta-LG showed strong consistency and corresponded to the nDSC results irrespective of the method of data interpretation and the protein concentration applied. In contrast, the T(m (FTIR)) of BSA and HEWL is characterized by significant deviations. Only the midpoint of the second-derivative intensity-temperature curve of the intermolecular beta-sheet mode measured at a concentration of 100 mg/mL is consistent with the nDSC results. CONCLUSIONS: Determination of a T(m (FTIR)) is feasible by the midpoint of the intensity-temperature plot of the arising intermolecular beta-sheet band. More significant results are obtained for proteins, which are predominantly composed of intramolecular beta-sheet elements as well as at higher protein concentrations. A further study was started to assess the predictability of long-term protein stability by T(m (FTIR)).

Calorimetry, Differential Scanning↗
PubMed1997

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells.

A flow cytometric technique was developed to measure the relative concentration of whey protein and beta-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (alpha-lactalbumin + beta-lactoglobulin) or beta-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more beta-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.

Animals↗
PubMed1983

Ion-exchange displacement chromatography of proteins, using narrow-range carboxymethyldextrans and a new index of affinity.

A new method of characterizing carboxymethyldextrans with respect to their relative affinities for an anion exchanger can be applied not only to pure preparations but also to fractions of a displacement chromatogram in which these polyanions have been used as spacers. This, along with the development of a means of preparing carboxymethyldextrans with narrow ranges of affinity, as contrasted with the very heterogeneous preparations previously used, has greatly facilitated the focusing of resolving power on critical regions of the chromatogram. The effectiveness of this approach is illustrated by the separation of proteins that differ only slightly in affinity, using the genetic variants of beta-lactoglobulin as one model and the numerous components of purified ovalbumin as another.

Chemical Phenomena↗
PubMed1983

Separation of proteins by high-performance anion-exchange chromatography.

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.

Chromatography, High Pressure Liquid↗
PubMed1987

Polyacrylamide gel electrophoresis of several proteins in the presence of sodium oligooxyethylene dodecyl ether sulfates or a commercially available analog.

The behavior of water-soluble proteins and a typical membrane protein in polyacrylamide gel electrophoresis was studied in the presence of sodium oligooxyethylene dodecyl ether sulfates with a defined number of oxyethylene units or a commercially available analog with distribution and heterogeneity for the oxyethylene chain length and alkyl group, respectively. It was concluded that most water-soluble proteins do not interact with the anionic surfactants as long as their oxyethylene chain lengths are sufficiently long; the commercially available surfactant binds exceptionally well to beta-lactoglobulin without causing denaturation and subsequent dissociation; such surfactants are expected to solubilize membrane proteins without causing denaturation as judged from the result with Na+,K+-ATPase and are promising as new solubilizing agents for membrane proteins which enable efficient electrophoretic analysis or separation after the solubilization.

Animals↗
PubMed1988

Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry.

Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signal-to-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein beta-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.

Amino Acid Sequence↗
PubMed1987

Specificity of cellular retinol-binding protein in the transfer of retinol to nuclei and chromatin.

We have reported previously that cellular retinol-binding protein (CRBP) is able to transfer retinol to specific binding sites in nuclei and chromatin. In this report, we have examined the specificity of the interaction of the protein moiety of retinol-CRBP (R-CRBP) with chromatin and nuclei in the transfer process. We first determined the ability of apo-CRBP, apo-serum retinol-binding protein (RBP), and apo beta-lactoglobulin (BLG), all capable of retinol binding, to compete with R-CRBP in the transfer of retinol to chromatin and nuclei. Apo-CRBP was an effective competitor but apo-RBP and apo-BLG showed no competitive ability. On the other hand, cellular retinol-binding protein type II (CRBP(II], whose amino acid sequence shows a considerable similarity to CRBP, did compete for the transfer of retinol from the R-CRBP complex, but less effectively than CRBP. These results demonstrate that the interaction of the protein moiety of the R-CRBP complex with nuclei and chromatin is quite specific.

Animals↗
PubMed1975

Approach for an equation of state for adsorbed protein surfaces.

A surface equation of state for polymer interfaces is obtained by equating the chemical potentials of the solvent in the bulk and surface phases. This equation of state contains the fraction of the surface covered with polymer and the surface activity coefficient as parameters. These parameters may be obtained by measuring the thermodynamic modulus of elasticity. Moreover, if the amount of protein in the interface is known the degree of folding in the surface may be evaluated. The present theory was applied to adsorbed beta-lactoglobulin in surfaces. The results are in agreement with qualitative equations and results from other sources.

Animals↗
PubMed1993

A novel member of the lipocalin superfamily: tammar wallaby late-lactation protein.

The finding that tammar wallaby late-lactation protein is linked to beta-lactoglobulin prompted a search of current GenPeptide and NBRF-PIR protein databases for sequence similarities to late-lactation protein. Similarities were found to von Ebner's gland protein and other members of the lipocalin superfamily of proteins. A conservative replacement of Trp with Tyr suggests that late-lactation protein may represent an unusual member of this protein superfamily.

Amino Acid Sequence↗
PubMed1976

The effect of trypsin on bovine transferrin and lactoferrin.

The iron-saturated and iron-free (apo) forms of bovine transferrin and lactoferrin were digested with trypsin and the digests analysed by column chromatography and electrophoresis. Both of the iron-saturated proteins were more resistant to proteolysis than the corresponding apoproteins, and iron-transferrin was more resistant than iron-lactoferrin. Digestion of iron-transferrin yielded two iron-binding fragments with molecular weights of 32 000 and 38 500 whereas apotransferrin yielded only the larger fragment. In digests of lactoferrin, up to five different fragments with molecular weights ranging from 25 000 to 52 700 were detected, there being no obvious qualitative difference between digests of iron-lactoferrin and apolactoferrin. The susceptibility of apolactoferrin to tryptic digestion was only slightly reduced when apolactoferrin was complexed with beta-lactoglobulin, suggesting that complex-formation is not a mechanism for protecting lactoferrin against intestinal degradation. There was no immunological cross reaction between bovine transferrin or its digestion products against anti-lactoferrin antiserum, or vice-versa.

Animals↗
PubMed1981

Evidence for protein self-association induced by excluded volume. Myoglobin in the presence of globular proteins.

The fluorescence polarization of fluorescent derivatives of hemoglobin and myoglobin was measured as a function of the concentration of added polymers (PEG-6 000, PEG-20 000) and globular proteins (lysozyme, ribonuclease A, beta-lactoglobulin). The results indicated that the effective size and shape of 1-anilino-9-naphthalene sulfonate myoglobin are unaltered in the presence of up to 25 g/dl poly(ethylene glycol), whereas they are significantly altered in the presence of comparable concentrations of other proteins. The results are consistent with the hypothesis that in the presence of high concentrations of added protein, 1-anilino-9-naphthalene sulfonate myoglobin self-associates to form a dimer similar in size and shape to 1-anilino-9-naphthalene sulfonate hemoglobin.

Animals↗
PubMed1983

Protein structure by Fourier transform infrared spectroscopy: second derivative spectra.

Second derivative Fourier transform infrared spectra of the proteins ribonuclease A, hemoglobin, and beta-lactoglobulin A (native and denatured) have been obtained in deuterium oxide solution from 1350 to 1800 cm-1. The relationship of the original spectra to their second derivatives is briefly discussed. In the second derivative spectra, clearly resolved peaks are observed which can be associated with the alpha-helix, beta-strands, and turns. No protein spectra with such resolution have heretofore been reported. Tentative assignments are proposed, and the observed peaks are related to the secondary structure of the proteins studied. The data appear to present the first direct spectroscopic evidence of turns in a native protein.

Animals↗
PubMed1983

Studies of oleate binding to rat liver plasma membranes.

The binding of [14C]oleate to rat liver plasma membranes was examined under various conditions in vitro. In protein free incubation mixtures binding was saturable, inhibited by excess cold oleate, virtually abolished by heat denaturation of the membranes, reversible by post-incubation with cold oleate or albumin, and pH and temperature dependent. When [14C]oleate was incubated in the presence of albumin or beta-lactoglobulin, the amount of membrane bound oleate was a function of the calculated free oleate concentration in the incubation mixture. Trypsin significantly inhibited binding. Further analysis suggests that membranes contain 10(15) high affinity (Kav = 2 x 10(8)M-1) oleate binding sites/mg protein.

Albumins↗
PubMed1995

Specific IgA antibody increases in schizophrenia.

IgA antibody levels in serum were examined in two groups of schizophrenic patients. All were diagnosed according to DSM-III-R criteria. One group of 36 males and 12 females were compared to historical controls. The other group consisted of 13 males off drugs for at least 3 months; these were compared with age- and sex-matched controls. An increase in specific IgA antibodies was found. More schizophrenics than controls showed IgA antibody levels above the upper normal limit to gliadin, beta-lactoglobulin, and casein.

Adolescent↗
PubMed1996

Incorporation of proteins in sphingomyelin-water gel phases.

Sphingomyelin from bovine milk and water form lipid gel phases at room temperature. A sample was used which incorporated of about 55% water, and X-ray diffraction data indicate an aqueous layer thickness of about 28 Angstrom. In order to accommodate proteins in the gel phase, the aqueous layer thickness was increased by solubilizing sodium palmitate into the sphingomyelin bilayer. In this way the gel phase could take up about 80% water. The incorporation of lysozyme, beta-lactoglobulin, and alpha-lactalbumin, was followed and the protein concentration for phase separation to occur was determined. It was found that the degree of incorporation was dependent on the salt concentration, thus the protein used was extensively dialysed. The amount of protein which can be dissolved in the thin aqueous layer of the gel phase was suggested to be limited by the dimensions of the layer. These are likely to be reduced as a consequence of the osmotic stress exerted by the 'outside' solution phase at high enough protein concentration.

Animals↗
PubMed1987

Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism.

Kinetic refolding reactions of ferricytochrome c and beta-lactoglobulin have been studied by stopped-flow circular dichroism by monitoring rapid ellipticity changes of peptide backbone and side-chain chromophores. In both proteins, a transient intermediate accumulates within the dead time of stopped-flow mixing (18 ms), and the intermediate has an appreciable amount of secondary structure but possesses an unfolded tertiary structure. It is suggested that the rapid formation of a secondary structure framework in protein folding is a common property observed in a variety of globular proteins.

Circular Dichroism↗
PubMed1993

Human gamma-interferon expression in the mammary gland of transgenic mice.

Transgenic mice carrying a hybrid gene consisting of ovine beta-lactoglobulin gene sequences and human gamma-interferon (hIFN-g) cDNA were produced. hIFN-g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN-g in the milk was estimated as being ca. 1800 IU/ml. The hIFN-g ability to express in the mammary gland was found in the progeny of transgenic founder male.

Animals↗
PubMed1994

The mammary factor MPBF is a prolactin-induced transcriptional regulator which binds to STAT factor recognition sites.

Site-directed mutagenesis of the three binding sites for the mammary factor MPBF in the beta-lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon gamma-activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor-induced transcription factors.

Animals↗