Determination of the Affinity Constants of Concanavalin A for Monosaccharides by Fluorescence Affinity Probe Capillary Electrophoresis

Shimura, Kiyohito; Kasai, Ken Ichi

doi:10.1006/abio.1995.1269

Cited by 70 publications

(49 citation statements)

References 2 publications

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“…Data Handling in Migration Shift Affinity CE-Peak appearance times (t) were equalized with respect to variations in electro-osmotic flow and shifts in buffer dielectric constant from run to run by subtracting the inverse appearance time of the added marker peptide from the inverse ␤ 2 m peak appearance times. This figure is proportional to the electrophoretic mobility (31). Triplicate sets of control runs consistently had a relative S.D.…”

Section: Methodsmentioning

confidence: 94%

“…Mobility shifts were then expressed as the difference between the normalized 1/t values at various concentrations (c) of additive. Linearization of the plot for binding constant estimation took place according to the equation: (31,32). Binding constants were also derived from direct nonlinear curve fits in plots of ⌬(1/t) as a function of c (GraphPadPRISM, v. 2.01; GraphPad Software, Inc., San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

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Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Heegaard

Roepstorff

Melberg

et al. 2002

Journal of Biological Chemistry

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␤ 2 -Microglobulin, a small protein localized in serum and on cell surfaces, can adopt specific aggregating conformations that generate amyloid in tissues and joints as a complication to long-term hemodialysis. We characterize a proteolytic variant of ␤ 2 -microglobulin (cleaved after Lys 58 ) that as a trimmed form (Lys 58 is removed) can be demonstrated in the circulation in patients with chronic disease. An unexpected electrophoretic heterogeneity of these two cleaved variants was demonstrated by capillary electrophoresis under physiological conditions. Each separated into a fast and a slow component while appearing homogeneous, except for a fraction of oxidized species detected by other techniques. The two components had different binding affinities for heparin and for the amyloid-specific dye Congo red, and the equilibrium between the two forms was dependent on solvent conditions. Together with analysis of the differences in circular dichroism, the results suggest that ␤ 2 -microglobulin cleaved after Lys 58 readily adopts two equilibrium conformations under native conditions. In the cleaved and trimmed ␤ 2 -microglobulin that appears in vivo, the less populated conformation is characterized by an increased affinity for Congo red. These observations may help elucidate why ␤ 2 -microglobulin polymerizes as amyloid in chronic hemodialysis and facilitate the search for means to inhibit this process.1 is a small protein (M r 11,729) localized in the circulation and on cell surfaces, where it constitutes the nonpolymorphic light chain of the class I major histocompatibility complex (1). ␤ 2 m can be cleaved C-terminally to Lys 58 (Lys 58 -␤ 2 m) by activated complement component C1s. In serum, the exposed Lys 58 residue of this cleaved molecule is subsequently removed by a carboxypeptidase B-like activity. This generates a cleaved and trimmed ␤ 2 m variant called desLys 58 -␤ 2 m (␤ 2 -microglobulin cleaved C-terminally to Ser 57 and lacking Lys 58 ) (2). Wild-type ␤ 2 m is a one-chain protein belonging to the immunoglobulin superfamily characterized by two antiparallel ␤-sheets connected by an internal disulfide bridge in a ␤-sandwich topology without helical structures (3, 4). The Lys 58 -␤ 2 m and des-Lys 58 -␤ 2 m variants are disulfide-linked two-chain heterodimers (Fig. 1). des-Lys 58 -␤ 2 m has been demonstrated in sera from patients in hemodialysis treatment (5) and in patients with malignancies and autoimmune and immunodeficiency diseases (2, 6, 7), but, to our knowledge, analyses of the conformational structure of the cleaved variants have not previously been published. In chronic renal failure, plasma concentrations of ␤ 2 m may increase 10 -70 times despite dialysis, and within 2 years after the onset of dialysis, about 20% of patients suffer from complications due to the formation of insoluble deposits of ␤ 2 m (␤ 2 m amyloid) in tissues and joints (8). The events leading to the formation of amyloid in this and other chronic diseases such as Alzheimer's disease are still largely unknown (9). There a...

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Section: Methodsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Heegaard

Roepstorff

Melberg

et al. 2002

Journal of Biological Chemistry

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“…The inner wall of a fused silica capillary (25 pm ID, 375 pm OD) was coated with succinylpolylysine as described previously [5], and then placed in a cartridge with a separation distance of 50 cm. The capillary was filled with an electrophoresis buffer (0.1 M Tris-acetic acid buffer, pH 7.9, containing 0.02% NaN, as a preservative) with or without the affinophore in the high-pressure-rinse mode for 3 min.…”

Section: Capillary Affnophoresismentioning

confidence: 99%

Capillary affinophoresis of pea lectin with polyliganded affinophores: A model study of divalent‐polyvalent interactions

Shimura

Kasai

1998

Electrophoresis

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Affinophoresis is a type of affinity electrophoresis using an affinophore, a soluble ionic carrier bearing affinity ligand(s). It was reported previously that an affinophore, prepared by coupling multiple p-aminophenyl alpha-D-mannoside ligands to a part of the carboxyl groups of succinylated polylysine, specifically changed the mobility of pea lectin in agarose gel. The affinophoresis of this divalent lectin with the polyliganded affinophore was investigated by using capillary electrophoresis. Analysis of the mobility change of the lectin in the presence of differently modified affinophores showed that the affinity was larger for affinophores having higher ligand density. Analysis of the inhibition of the mobility change by a neutral ligand, with a known affinity constant for the lectin, allowed estimation of the contributions of monovalent and divalent interactions to the binding in the lectin-affinophore complex. The proportion of divalent complexes was greater for affinophores having higher ligand density. This approach to estimate the contribution of divalency in complex formation should be generally applicable to the analysis of divalent interactions with different techniques other than electrophoresis.

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“…The other choice was using dynamic complexation affinity electrophoresis, where complex formation occurs during the run with the buffer containing one of the interacting components [38]. This approach requires that the value of 1/k d be much shorter than the migration time of labeled C4 [39].…”

mentioning

confidence: 99%

Characterization of the interaction between human complement protein C4 and a single‐chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance

Seifar¹,

Cool²,

Quax³

et al. 2004

Electrophoresis

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Immunoaffinity capillary electrophoresis and surface plasmon resonance have been used for the characterization of the interaction between two large-sized proteins, the human complement protein C4 and the single-chain variable fragment C43. The rather high kinetic rate constants as determined by surface plasmon resonance pointed out that a capillary electrophoresis method had to be applied, in which the labeled C4 is preincubated with C43 before injection and the same concentration of C43 is included in the running buffer. Analysis of the concentration dependence of the small mobility shift of the fluorescent C4 signal upon binding of C43 resulted in a dissociation constant that was comparable to the one obtained with surface plasmon resonance. This study is one of the few examples where capillary electrophoresis is successfully used to characterize the interaction between large proteins.

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Determination of the Affinity Constants of Concanavalin A for Monosaccharides by Fluorescence Affinity Probe Capillary Electrophoresis

Cited by 70 publications

References 2 publications

Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Capillary affinophoresis of pea lectin with polyliganded affinophores: A model study of divalent‐polyvalent interactions

Characterization of the interaction between human complement protein C4 and a single‐chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance

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