Monitoring of a conjugation reaction between fluorescein isothiocyanate and myoglobin by capillary zone electrophoresis

Banks, Peter; Paquette, Donald M.

doi:10.1016/0021-9673(94)01110-z

Cited by 31 publications

(23 citation statements)

References 11 publications

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“…'-with different reaction constants when using high molecular weight proteins exhibiting multiple reaction sides (a-amino acid and e-amino groups) of lysine [43]. The heterogeneity of the resulting protein-dye conjugates has already been reported [9,19,27,44] and analyzed by a wide variety of analytical techniques such as capillary electrophoresis [38,39,42,[45][46][47][48], isoelectric focusing, SDS-PAGE [47,49,50], HPLC [27,41,44,51] and mass spectroscopy [7,41,[52][53][54].…”

Section: Protein-dye Conjugatesmentioning

confidence: 97%

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Hubbuch

Kula

2008

Bioprocess Biosyst Eng

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In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.

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Section: Protein-dye Conjugatesmentioning

confidence: 97%

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Hubbuch

Kula

2008

Bioprocess Biosyst Eng

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“…An electrostatic component is then mostly recognized as having a role in various types of aggregate formation. Wild-type apomyoglobin could rapidly form amorphous aggregates upon heating at a high temperature and a pH close to neutral where it is uncharged (Banks and Paquette, 1995), while amyloid structures are generated when its net negative charge is increased. Adding NaCl to the protein environment may result in decreasing amounts of amorphous aggregates, and the effect is concentration-dependent (Fig.…”

Section: Amorphous Aggregationmentioning

confidence: 99%

“…In order to investigate amorphous aggregation of horse muscle apomyoglobin, acidic and alkaline pH values (4.2, 6.5 and 9) close to the protein pI of 7.3 (Banks and Paquette, 1995), were used in the 60-70 • C temperature range [(T m is 61 ± 2 • C (Fandrich et al, 2003)]. The process was assessed by the increase in the optical density of insoluble particles (Guagliardi et al, 1995;Paulikova et al, 1998;Li et al, 2001;Rafikova et al, 2003;Rezaei-Ghaleh et al, 2007b;Qiu and Macrae, 2008).…”

Section: Amorphous Aggregationmentioning

confidence: 99%

Heme binding site in apomyoglobin may be effectively targeted with small molecules to control aggregation

Azami-Movahed

Shariatizi

Sabbaghian

et al. 2013

The International Journal of Biochemistry & Cell Biology

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“…LIF detection of fluorescently labeled receptors can determine subnanomolar concentrations [31,32]. CE resolution of the free and complexed species and accuracy of quantitation are greatly enhanced by electrophoretically homogeneous reagents [21,33]. HCAII has a single cysteine residue in its sequence, which is remote from the active site.…”

Section: Fluorescent Labeling and Purification Of Hcaiimentioning

confidence: 99%

Ultratrace analysis of drugs in biological fluids using affinity probe capillary electrophoresis: Analysis of dorzolamide with fluorescently labeled carbonic anhydrase

2000

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This work demonstrates the use of affinity probe capillary electrophoresis (APCE) in the quantitative analysis of drugs in biological fluids at the low pM level. The interaction of human carbonic anhydrase II (HCAII) with the glaucoma drug dorzolamide (Dz) was chosen as a model system. HCAII was labeled at its single cysteine residue using a thiol‐specific fluorescein reagent. The peak area of HCAII complexed with the tight‐binding drug Dz provided a direct assay of the drug concentration in solution. A charged competitive ligand added to the running buffer was employed in APCE to distinguish Dz‐bound from free forms of the HCAII. Using laser‐induced fluorescence (LIF), the Dz detection limit was 16.5 pM in aqueous solution and 62.5 pM in both urine and plasma. Normalized peak area reproducibility of the drug was within 3.4% RSD. Each analysis was completed within 10 min, including incubation, and consumed only 0.3 pmol of labeled protein. The APCE approach provides an effective method for trace level detection of drugs in biological matrices.

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Monitoring of a conjugation reaction between fluorescein isothiocyanate and myoglobin by capillary zone electrophoresis

Cited by 31 publications

References 11 publications

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Heme binding site in apomyoglobin may be effectively targeted with small molecules to control aggregation

Ultratrace analysis of drugs in biological fluids using affinity probe capillary electrophoresis: Analysis of dorzolamide with fluorescently labeled carbonic anhydrase

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