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Removal of Copper in Microdroplets by Ovomucoid Hydrolysates Bound to Reverse-Phase Chromatography Media Within Pipette Tips

  • Youji ShimazakiEmail author
  • Suzuka Inoue
Article
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Abstract

Ovomucoid (OVM) is a protein found in chicken egg white. When it is hydrolyzed by a protease, subtilisin A from Bacillus licheniformis, it possesses Cu2+-chelating activity. In the present work, we demonstrate that the resulting OVM hydrolysates bind to reverse-phase chromatography media in pipette tips and can be applied to remove Cu2+ within microdroplets. 1.4 nmol of purified OVM was digested in the presence of 17 pmol of subtilisin A at 55 °C for 3 h. The OVM hydrolysates efficiently removed 2.1 and 2.4 nmol of Cu2+ in the droplets by binding to the C4 and C18 chromatography media, respectively. Conversely, 0.6 and 1.0 nmol of Cu2+ were removed by the non-digested OVM bound to the C4 and C18 media, respectively. The removal ratio of Cu2+ increased as more OVM was digested by subtilisin A. The digested OVM polypeptides were stained with Cu2+ after they were separated by non-denaturing electrophoresis. These results indicate that OVM hydrolysates bound to chromatography media in a pipette tip can be applied to remove Cu2+ within microdroplets of biological samples.

Keywords

Copper removal Electrophoresis Reverse-phase chromatography media Subtilisin A ZipTip 

Abbreviations

PVDF

Polyvinylidene difluoride

TEMED N, N, N′,

N′-tetramethylenediamine

Tris

2-Amino-2-hydroxymethyl-1,3-propanediol

CBB

Coomassie Brilliant Blue

BSA

Bovine serum albumin

Notes

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest..

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry and Biology, Graduate School of Science and Engineering (Science Section)Ehime UniversityMatsuyama CityJapan
  2. 2.Faculty of ScienceEhime UniversityMatsuyamaJapan

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