Quantitative analysis in capillary zone electrophoresis with conductivity and indirect UV detection

Ackermans, MT Mariëtte; Everaerts, FM Frans; Beckers, JL Jozef

doi:10.1016/s0021-9673(00)91445-6

Cited by 100 publications

(36 citation statements)

References 4 publications

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“…These were injected into the capillary after different time intervals had elapsed from dilution. Peak areas obtained for all experiments have been divided by their corresponding retention time in order to express them as normalized areas (14). Normalized areas are further divided by the total peak areas to express every conformational population as a percentage of total protein.…”

Section: Methodsmentioning

confidence: 99%

A Partially Structured Species of β2-Microglobulin Is Significantly Populated under Physiological Conditions and Involved in Fibrillogenesis

Chiti

Lorenzi

Grossi

et al. 2001

Journal of Biological Chemistry

144

188

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The folding of ␤ 2 -microglobulin (␤ 2 -m), the protein forming amyloid deposits in dialysis-related amyloidosis, involves formation of a partially folded conformation named I 2 , which slowly converts into the native fold, N. Here we show that the partially folded species I 2 can be separated from N by capillary electrophoresis. Data obtained with this technique and analysis of kinetic data obtained with intrinsic fluorescence indicate that the I 2 conformation is populated to ϳ14 ؎ 8% at equilibrium under conditions of pH and temperature close to physiological. In the presence of fibrils extracted from patients, the I 2 conformer has a 5-fold higher propensity to aggregate than N, as indicated by the thioflavine T test and light scattering measurements. A mechanism of aggregation of ␤ 2 -m in vivo involving the association of the preformed fibrils with the fraction of I 2 existing at equilibrium is proposed from these results. The possibility of isolating and quantifying a partially folded conformer of ␤ 2 -m involved in the amyloidogenesis process provides new opportunities to monitor hemodialytic procedures aimed at the reduction of such species from the pool of circulating ␤ 2 -m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.Dialysis-related amyloidosis represents an inevitable and severe complication of long term hemodialysis (1-4). Under this pathological condition, protein aggregates known as amyloid fibrils, accumulate in essential tissues, such as the skeletal muscle, interfering with their normal functions. A major constituent of the amyloid fibrils related to this pathological condition is ␤ 2 -microglobulin (␤ 2 -m).1 In its native form, this 99-residue protein is constituted by two ␤-sheets packed against each other to form a fold typical of the immunoglobulin superfamily (5). The two ␤-sheets, constituted by three and four strands, respectively, interact by means of hydrophobic interactions and a disulfide bridge that stabilizes further the ␤-sandwich structure.␤ 2 -m constitutes the light chain of the major histocompatibility complex class I (MHCI). A significant pool of ␤ 2 -m is also normally present in the plasma as a consequence of the constant release from the MHCI to allow the process of catabolic degradation in the kidney (1, 2). In chronic dialysis patients, the artificial membrane induces an inflammatory reaction, which causes the production and release of ␤ 2 -m to increase significantly (6). In addition, ␤ 2 -m cannot be filtered efficiently through the artificial membrane, resulting in an increase of the levels of soluble ␤ 2 -m from 0.3 to 30 g/ml, the range of concentrations observed within healthy individuals, to ϳ40 g/ml (7). The increase of free circulating ␤ 2 -m, the preferential substrate for amyloid deposition by this protein, is responsible, at least in part, for this form of amyloidosis in these patients (1, 2). Big efforts have been expended to improve the biocompatibility and performance of dialysis approaches. ...

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Section: Methodsmentioning

confidence: 99%

A Partially Structured Species of β2-Microglobulin Is Significantly Populated under Physiological Conditions and Involved in Fibrillogenesis

Chiti

Lorenzi

Grossi

et al. 2001

Journal of Biological Chemistry

144

188

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“…Indirect photometric detection (IPD) is a well-established detection mode for capillary electrophoresis (CE) to detect nonabsorbing analytes [1][2][3][4][5][6]. IPD is performed by the addition to the background electrolyte of an absorbing ion of the same charge as analytes, typically referred to as a probe, and monitoring the absorbance at a wavelength at which the probe absorbs strongly.…”

Section: Introductionmentioning

confidence: 99%

Design and performance of a light‐emitting diode detector compatible with a commercial capillary electrophoresis instrument

2004

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Indirect photometric detection in capillary electrophoresis (CE) has been predominantly performed in the UV region, in part due to a lack of suitable high-intensity and low-noise light sources in the visible spectral region. A new photometric detector based on light-emitting diodes (LEDs) as light sources and compatible with a commercially available CE instrument has been designed and constructed and its performance evaluated. The utility of this detector was successfully demonstrated by the indirect photometric detection of anions using a dye as probe and absorbance measured in the visible region. The detector exhibited very low baseline noise (around 0.03 mAU), stable output, and improved upper limit of detection linearity (502 mAU) compared with previously used LED detectors. The detector was tested for indirect detection of anions separated with an electrolyte containing 4 mM Orange G as the indirect detection probe, 10 mM histidine as an isoelectric buffer, and 0.05% hydroxypropylmethylcellulose to suppress the electroosmotic flow. Extremely low detection limits were obtained ranging from 0.16-0.36 microM (excluding chloride 0.56 microM), with separation efficiencies in the range of 154,000-274,000 theoretical plates.

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“…If the ionic mobility of the sample ions is higher than that of the coions, the transfer ratio is smaller than unity, implicating that the decrease in concentration of the coions is smaller than 5 10 ±4 M, and an increase in ionic strength and an increase in concentration of the counterElectrophoresis 1999, 20, 518±524 Peaks in CZE: Fact or fiction 519 ions are the result. Application of UV-absorbing counterions leads to peaks for UV-transparent sample components in the latter case [15].…”

Section: Panels B D F and Hmentioning

confidence: 99%

Peaks in capillary zone electrophoresis: Fact or fiction

Beckers

Boček

1999

Electrophoresis

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Quantitative analysis in capillary zone electrophoresis with conductivity and indirect UV detection

Cited by 100 publications

References 4 publications

A Partially Structured Species of β2-Microglobulin Is Significantly Populated under Physiological Conditions and Involved in Fibrillogenesis

A Partially Structured Species of β2-Microglobulin Is Significantly Populated under Physiological Conditions and Involved in Fibrillogenesis

Design and performance of a light‐emitting diode detector compatible with a commercial capillary electrophoresis instrument

Peaks in capillary zone electrophoresis: Fact or fiction

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