Mercedes de Frutos

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78-4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.

Capillary electrophoresis characterization of the casein fraction of cheeses made from cows', ewes' and goats' milks.

Casein fractions and their breakdown products in Iberico-type cheeses made from the milk of cows, ewes or goats were analysed by capillary electrophoresis in order to characterize them. The actions of plasmin and chymosin on caseins were evaluated by comparing the electropherograms of caseins from milk and from cheese, both with and without treatment with plasmin. Characteristic capillary electrophoresis patterns were obtained for cheeses made from the milk of each of the three species, and the main components were identified. Caprine para-kappa-casein and bovine beta-caseins, eluting at the first and at the last part of the electropherogram respectively, were found to be indicative of the presence of the milks of these species.

First molecular characterization of a uridine diphosphate galacturonate 4‐epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1

Uridine diphosphate galacturonate 4‐epimerases (UDPGLEs) are enzymes that convert UDP‐glucuronate into UDP‐galacturonate. Although the presence of UDPGLEs has been reported in prokaryotic and eukaryotic organisms, the genes coding for these enzymes are completely unknown. The galacturonic acid‐containing capsular polysaccharide of Streptococcus pneumoniae type 1 is synthesized through the action of a specific UDPGLE. We have constructed a defined deletion mutant in the cap1J gene (one of the 15 cap1 genes responsible for the synthesis of the type 1 capsule) that exhibited an unencapsulated phenotype. This mutant was unable to synthesize UDPGLE, suggesting that Cap1J was the type 1‐specific UDPGLE of S. pneumoniae. Escherichia coli cells harbouring the recombinant plasmid pRMM38 (cap1J ) overproduced a 40 kDa protein, characterized as Cap1J on the basis of the N‐terminal amino acid sequence analysis, and expressed high levels of enzymatically active Cap1J epimerase. Cap1J was partially purified, although purification to electrophoretic homogeneity inactivated the enzyme irreversibly. The enzyme has the following characteristics: Kmfor UDP‐glucuronate, 0.24 mM; pH optimum, 7.5; equilibrium constant (in the direction of UDP‐galacturonate formation), 1.3; and an approximate Mr of 80 000 for the active form. The Cap1J protein exhibited a fluorescence emission spectrum similar to that of NADH. Upon inactivation with p‐hydroxymercuribenzoate, the addition of NAD+ and 2‐mercaptoethanol were sufficient to reactivate the enzyme. Among several compounds tested, UDP‐galactose and UDP‐xylose exhibited the highest inhibition of the UDPGLE activity. Inactivation of UDPGLE activity was also observed in the presence of UMP and several reducing sugars. To our knowledge, this is the first example of a thoroughly molecular characterization of a UDPGLE.

Comparison of different capillary electrophoresis methods for analysis of recombinant erythropoietin glycoforms

The authors are grateful to European Pharmacopoeia for providing rEPO BRP used as sample in this work and to M.T. Veledo for carrying out the CE-LIF experiments. This work was supported by an International Olympic Commit- tee project (1767/2000/HMS/sls) and by a CICYT project (AGL2000-1480).

Statistical evaluation of CZE-UV and CZE-ESI-MS data of intact α-1-acid glycoprotein isoforms for their use as potential biomarkers in bladder cancer

α‐1‐acid glycoprotein (AGP) is a highly heterogeneous protein that presents a vast number of isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). In the last years, several authors have studied the potential use of AGP as a cancer biomarker. These studies focus on the correlation of different features of AGP structure (i.e. fucosylation, antennarity) with cancer or on the total protein blood concentration. In this study, the potential of CZE‐UV and CZE‐ESI‐MS analysis of intact AGP isoforms to study the correlation of this protein with bladder cancer is shown. Samples from 16 individuals (eight healthy, eight bladder cancer) were analyzed and characterized in great detail including data on intact protein isoforms and on released glycans. The analytical data were evaluated employing different statistical techniques (ANOVA; principal component analysis, PCA; linear discriminant analysis; and partial least squares‐discriminant analysis). Statistical differences between the two groups of study were observed. The best results were obtained by linear discriminant analysis of the CZE‐ESI‐MS data for intact AGP isoforms (93.75% of correct classification). Due to MS characterization, it can be observed that differences between the samples are mainly due to higher abundance of AGP isoforms containing tri‐ and tetra‐antennary fucosylated oligosaccharides in cancer patients. The results show the great potential of CE‐MS in combination with advanced data processing for the use of intact protein isoforms as disease biomarkers.

Rapid analysis of whey proteins from different animal species by reversed-phase high-performance liquid chromatography

ZusammenfassungIn der vorliegenden Arbeit werden die Möglichkeiten der Anwendung von Umkehrphasen-Hochleistungsflüssigchromatographie (RP-HPLC) zur Analyse der Molkenproteine in Kuhmilch und Milch anderer Tiere geprüft. Es wird eine RP-HPLC-Methode zur Trennung und quantitativen Analyse der Rindermolkenproteine vorgeschlagen, hierbei werden Rindermolkenalbumine, α-Lactalbumin,β-Lactoglobulin A undβ-Lactoglobulin B in weniger als 7 min getrennt. Es wurde festgestellt, daß in unbenutzten Säulen (d. h. jene Säulen, die zuvor nicht zur Trennung des Proteins benutzt worden sind) eine irreversible Adsorption stattfindet. Aus diesem Grunde ist, um eine gültige Quantifizierung der Molkenproteine zu erhalten, eine vorherige Konditionierung der Säule erforderlich, und zwar durch Elution einer großen Probenmenge. Die Eichkurve (Peakfläche gegenüber Proteinkonzentration) der Hauptmolkenproteine war linear. Die Methode erlaubt auch eine gute Trennung der Ziegen- und Schafmolkenproteine sowie die Trennung anderer homologer Molkenproteine verschiedener tierischer Gattungen. Unter Anwendung dieser Methode konnten Mischungen von Milch verschiedener tierischer Gattungen nachgewiesen werden.SummaryThis paper explores the possibilities of reversed-phase high performance liquid chromatography (RP-HPLC) for analysing whey proteins from the milk of cows and other animal species. An RP-HPLC method is proposed to separate and quantify bovine whey proteins. Using this method, bovine serum albumin, α-lactalbumin,β-lactoglobulin A andβ-lactoglobulin B were separated in less than 7 min. It is demonstrated that irreversible adsorption of bovine whey proteins occurs on unused columns (i.e. those not previously used to separate proteins). Therefore, prior conditioning of the column with whey proteins is required for valid protein quantification. Conditioning can be achieved by eluting a large amount of at least one of the bovine whey proteins through the unused column. The calibration curve (peak area vs. protein concentration) for the main bovine whey proteins was linear. This method also allowed good separation of caprine and ovine whey proteins and separation of some homologous whey proteins of different animal species. Detection of milk mixtures from different animal species was carried out using this method.

Evaluation of the effect of the immunopurification-based procedures on the CZE-UV and CZE-ESI-TOF-MS determination of isoforms of intact α-1-acid glycoprotein from human serum

Differences in α‐1‐acid glycoprotein (AGP) peptidic and glycan moieties originate several isoforms, whose modifications have been related to different pathophysiological situations. Differences in the isoforms of AGP existing in serum of individuals suffering from different diseases compared to healthy ones could be potentially used as biomarkers. CZE has been proven to be a useful technique for the analysis of glycoprotein isoforms. However, direct CZE analysis of AGP isoforms in serum samples needs efficient purification methods that allow the protein analysis. In this work two new and fast methods to purify AGP from human serum are evaluated in regard to their effect on the determination of isoforms of the intact glycoprotein by CZE‐UV and by a developed CZE‐ESI‐TOF‐MS method. Both preparation methods, which differ in the pre‐treatment of the sample prior to an anti‐AGP immunochromatographic step are shown to be adequate to analyze isoforms of intact AGP. Comparison of both purification methods by CZE‐UV and CZE‐ESI‐TOF‐MS indicates that serum AGP purified without acidic precipitation as pre‐treatment is more adequate due to AGP higher yield, which leads to better CZE‐Mass spectra. Both CZE methods show no indication that acidic precipitation influences the glycosylation (including sialylation) of AGP.

Adsorption kinetics of β-lactoglobulin on a polyclonal immunochromatographic support

Beta-Lactoglobulin is one of the main components of whey proteins. Among other reasons, its allergenicity makes its determination in hypoallergenic foods and bio-pharmaceutical products necessary. Immunoaffinity chromatography is a widely accepted technique for purification and analysis of proteins. Knowledge of the apparent kinetics of the adsorption of beta-lactoglobulin onto the anti-beta-lactoglobulin immunochromatographic column is important to optimize the analytical process. High-performance frontal affinity chromatography was used to study the apparent kinetics of the adsorption process. Langmuir and bi-Langmuir kinetic models, assuming one and two kinds of binding sites, respectively, were used to characterize the adsorption kinetics of beta-lactoglobulin B on a polyclonal immunoadsorbent. Very good fits were obtained with the bi-Langmuir model for two different concentrations of beta-lactoglobulin and this allowed us to calculate the apparent adsorption rate constants and the column capacities for both kinds of sites. Experimental results indicate the possibility that the adsorption process is not irreversible. The values of the apparent dissociation rate constants leading to the best fit were estimated and the affinity constants were calculated.

A new sample preparation method compatible with capillary electrophoresis and laser-induced fluorescence for improving detection of low levels of β-lactoglobulin in infant foods

Beta-lactoglobulin (betaLG) is the main allergenic protein in cows milk and can cause allergy even when present at very low concentration. The aim of this work is to develop an innovative sample preparation method fully compatible with capillary electrophoresis and laser-induced fluorescence detection for improving the sensitivity when analyzing betaLG. Different types of baby food were on purpose contaminated with diverse dairy desserts and submitted to thermal treatment to simulate potential contamination at production. Sample preparation prior to CE analysis was performed by the classical extraction method and by the innovative one, and the results were compared. Analysis was performed by capillary electrophoresis with laser-induced fluorescence detection. The innovative method permitted to detect contaminations as low as 1 part of yoghurt in 10,000 parts of baby food.

Multiple Peaks in HPLC of Proteins: Bovine Serum Albumin Eluted in a Reversed-Phase System

The authors thank F. Soriano for his skillful technical assistance. This work was supported by CICYT (project ALI 97 – 0630).