Successive analysis of antigen trapping and enzymatic digestion on membrane-immobilized avidin

Shimazaki, Youji; Kohno, Yusuke

doi:10.1016/j.ab.2011.12.023

Cited by 7 publications

(1 citation statement)

References 12 publications

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“…Ovomucoid, from egg white, is a serine protease inhibitor [4][5][6] that reversibly inhibits the activity of the serine protease trypsin [7]. The reversible inhibition by ovomucoid can be examined in the presence of the immobilized serine protease, because activity changes of serine protease, such as trypsin and carboxypeptidase Y (CPY), were analyzed by the combined methods of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the analysis of the enzyme immobilized on supports [8,9]. Based on the association and dissociation between ovomucoid and trypsin, ovomucoid is purified using trypsin-sepharose affinity chromatography [10].…”

Section: Introductionmentioning

confidence: 99%

Enzyme Inhibitory Activity of Ovomucoid Extracted Using a Carboxypeptidase Y-Immobilized Membrane

2018

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Reversible inhibition of enzymes is caused by association and dissociation between enzymes and inhibitors. Therefore, reversible inhibitors can be trapped and extracted using enzyme-inhibitor interaction. The purpose of this study was to establish a method in which the reversible inhibitors retaining the original inhibitory activities are extracted from a single drop of biological sample using the enzyme-inhibitor interaction on the surface of a membrane. A membrane-immobilized carboxypeptidase Y (CPY) was produced after the biotinylated CPY was bound to the avidin separated by nondenaturing electrophoresis, transferred to a polyvinylidene fluoride and stained by Ponceau S. Ovomucoid, possessing reversible CPY inhibitory activity, was trapped and extracted from a single drop of egg white and isolated using the membrane-immobilized CPY. The isolated ovomucoid using this membrane-immobilized CPY possessed a feature that more than 85 % of the relative carboxylesterase activity was suppressed. The results indicate that ovomucoid retaining enzyme inhibitory activities can be isolated from a single drop of egg white sample using enzyme-immobilized membrane.

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Section: Introductionmentioning

confidence: 99%

Enzyme Inhibitory Activity of Ovomucoid Extracted Using a Carboxypeptidase Y-Immobilized Membrane

2018

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Combined Method of Immunoaffinity Membrane Within Tubes and MALDI-TOF MS for Capturing and Analyzing Amyloid Beta

Shimazaki

Takatsu

2015

Appl Biochem Biotechnol

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Amyloid beta 1-40 peptide was specifically isolated and analyzed from human plasma spiked with amyloid beta using a combined method of biotinylated anti-amyloid beta antibody binding to membrane-immobilized avidin (immunoaffinity membrane) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (MALDI-TOF MS). A solution of 10 μL containing 13.6 ng to 2.9 μg of amyloid beta peptide was examined in this method. After the isolated amyloid beta peptide from the spiked human plasma containing 2.9 μg of amyloid beta peptide was incubated in the presence of trifluoroacetic acid, fibrillization of the peptides was observed using a thioflavin T assay. Furthermore, an immunoaffinity membrane present on the inner wall of a tube (diameter 2 mm) captured the amyloid beta peptide from the spiked human plasma. Our results indicate that the combination of the immunoaffinity membrane procedure and MALDI-TOF MS can be used to capture and analyze the target antigens such as amyloid beta in micro-spaces.

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Analysis of trypsin inhibition activity in human plasma proteins after separation by non-denaturing two-dimensional electrophoresis

Shimazaki

Michhiro

2013

Clinica Chimica Acta

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Successive analysis of antigen trapping and enzymatic digestion on membrane-immobilized avidin

Cited by 7 publications

References 12 publications

Enzyme Inhibitory Activity of Ovomucoid Extracted Using a Carboxypeptidase Y-Immobilized Membrane

Enzyme Inhibitory Activity of Ovomucoid Extracted Using a Carboxypeptidase Y-Immobilized Membrane

Combined Method of Immunoaffinity Membrane Within Tubes and MALDI-TOF MS for Capturing and Analyzing Amyloid Beta

Analysis of trypsin inhibition activity in human plasma proteins after separation by non-denaturing two-dimensional electrophoresis

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