Use of acidic and basic pH and calcium ion addition in the capillary zone electrophoretic characterization of recombinant human deoxyribonuclease, a complex phosphoglycoprotein (original) (raw)
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Journal of Chromatography A, 1997
The use of phytic acid to improve protein analysis by capillary electrophoresis (CE) is becoming more and more popular, Due to its size and number of negative charges (up to 12) it provides a high ionic strength combined with a low conductance resulting in an efficient decrease of wall adsorption for proteins. Because of its twelve acidic groups, phytic acid can be used as a buffer over a wide pH range (pH 2-11). The limited wall adsorption of proteins using phytic acid-containing buffers is observed for buffers with a pH of 5.5 and higher. With a monoprotic buffer, most of the investigated proteins show wall adsorption at the pH values studied. In case of a phytic acid buffer, wall adsorption is reduced by a factor of 2-4. The use of phytic acid both as a modifier and as a pH buffer results in more pronounced differences between the various protein mobilities compared with the use of monoprotic buffers. As a result this feature can be used to improve resolution in protein separations.
Characterization of the aggregated states of glycogen phosphorylases by gel electrophoresis
Biochemistry, 1969
The disc gel electrophoretic method developed previously for the characterization of size and/or charge differences in proteins has been applied t o glycogen phosphorylase. We have concluded that phosphorylases a and b have the same molecular size but different net charges under the conditions of electrophoresis employed. The enzymes are both dimers with molecular weights of 170,00~180,000 g/mole at p H 8.5, 35", and a protein concentration of 0.5 mg/ml. Phosphorylase dimer can be dissociated to monomers using sodium dodecyl sulfate or 2 M urea a t 4". The binding of various ligands (adenosine monophosphate, glucose 6-phosphate, and dextrins) changes the relative mobility of phosphorylase. Plots of the logarithm of the relative mobility L'S. gel concentration in the presence of ligands yields lines parallel to the line obtained with phosphorylase alone. This
Journal of Chromatography A, 1993
Successful separations of alcohol dehydrogenase (pZ= 5.4), /l-amylase (pZ= 5.2) and albumin (pZ= 4.7) by capillary electrophoresis in uncoated fused-silica capillaries are reported. Different electrophoretic conditions, consisting in variation of temperature, applied voltage and ionic strength of the buffer used as electrolyte, were tested in order to compare the separation efficiency, resolution and selectivity of the acidic proteins. The results were compared with those obtained by size-exclusion chromatography. Rinsing of the capillary between runs, in order to eliminate adsorbed proteins, can shorten its useful lifetime.
Analytical Chemistry, 2002
To improve the separation efficiency while achieving high sensitivity for the analysis of proteins' microheterogeneity, a segmental-filling technique has been developed and tested in capillary electrophoresis with laser-induced native fluorescence using a pulsed Nd:YAG laser. Using a short plug of SDS applied to the capillary and the anticonvectant poly(ethylene oxide) (PEO), the microheterogeneities of a number of proteins with pI values ranging from 4.5 to 11.1 could be detected. This high resolving power is due to reduced adsorption on the capillary wall, sieving, and the interaction with SDS. Consequently, the length and the concentration of the SDS plug play a significant role in determining the resolution and sensitivity. The method has been applied to the analysis of salivary and cerebrospinal fluid (CSF) samples. Without any sample pretreatment, using a 10-s 1× SDS plug, six r-amylase isoforms in a salivary sample were resolved in 17 min and three more peaks were detected in a CSF sample. With simplicity, high resolving power, and rapidity, the method has shown great potential for proteomics.
Journal of Chromatography A, 1997
We report the use of mixtures of cationic and anionic fluorosurfactants as additives for free-flow capillary electrophoresis of proteins. Effective deactivation of the capillary wall is obtained, which allows the use of raw fused-silica capillaries. The magnitude and direction of the electroosmotic flow is strongly affected by the composition of the surfactant mixture, and a suggested model for this behaviour in terms of micellation and formation of admicellar surfactant layers is described. By utilizing mixtures of the oppositely charged surfactants, it is possible to separate both positively and negatively charged proteins in the same run. Due to charge interactions of the fluorosurfactants with the proteins, it is possible to tune the separation selectivity, without having to change the buffer strength or pH. This was demonstrated in particular for the model substances myoglobin and ribonuclease, where the order of elution could be reversed, compared to their elution order in a normal buffer system. Another advantage of the fluorosurfactant additives is their effectiveness in low concentrations (< 100 Ixg/ml) even when buffers of low ion strength are employed. Thus, rapid separations at a high field strength can be accomplished, without suffering from excessive Joule heating. This is demonstrated with an example, where a mixture of positively and negatively charged proteins is separated in less then 2 min in a 10 mM phosphate buffer at pH 7.
Journal of Chromatography A, 1996
A capillary electrophoresis (CE) method using acidic buffers and capillaries coated with Polybrene, a cationic polymer has been developed for the separation of glycoproteins and glycopeptides. Electrophoretic conditions have been optimized to provide resolution of individual glycoforms observed for different glycoprotein preparations. These conditions were found to be entirely compatible with the operation of electrospray mass spectrometry (ESMS), which facilitated the assignments of possible carbohydrate compositions of glycopeptides arising from digests of glycoproteins. By using operating conditions enhancing the formation of oxonium fragment ions prior to mass spectral analysis, selective identification of glycopeptides was achieved for complex samples such as those from proteolytic digests or chemical cleavages. Examples of applications are presented for ribonuclease B, ovalbumin, horseradish peroxidase, and a lectin from Erithrina corallodendron using both CE-ESMS and CE with ultraviolet detection (CE-UV).
Glycoconjugate Journal, 1992
The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomannose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B with A. saitoi ~(1-2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.
Determination of protein charge by capillary zone electrophoresis
Analytical Biochemistry, 2004
The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a ''charge ladder'' approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein.