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

  • Youji ShimazakiEmail author
  • Suzuka Inoue


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.


Copper removal Electrophoresis Reverse-phase chromatography media Subtilisin A ZipTip 



Polyvinylidene difluoride






Coomassie Brilliant Blue


Bovine serum albumin


Compliance with Ethical Standards

Conflict of Interest

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


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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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