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Micro-Scale Extraction and Analysis of Intact Carboxylesterase after Trapping on an Immunoaffinity Membrane Surface

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Abstract

Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm2) of this membrane and eluted by rinsing with 5 μL of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme’s activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.

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Abbreviations

PVDF:

polyvinylidene difluoride

TEMED:

N,N,N′, N′-tetramethylenediamine

MALDI-TOF MS:

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Tris:

2-amino-2-hydroxymethyl-1,3-propanediol

4-MUA:

4-methylumbelliferyl acetate

acrinol:

6,9-diamino-2-ethoxyacridine

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Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 25410145.

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Correspondence to Youji Shimazaki.

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Kimura, A., Shimazaki, Y. Micro-Scale Extraction and Analysis of Intact Carboxylesterase after Trapping on an Immunoaffinity Membrane Surface. Appl Biochem Biotechnol 172, 4053–4061 (2014). https://doi.org/10.1007/s12010-014-0807-4

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  • DOI: https://doi.org/10.1007/s12010-014-0807-4

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