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

Ovomucoid (OVM) is a protein found in chicken egg white. When it is hydrolyzed by a protease, subtilisin A from Bacillus licheniformis, it possesses Cu-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 Cu 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 Cu in the droplets by binding to the C4 and C18 chromatography media, respectively. Conversely, 0.6 and 1.0 nmol of Cu were removed by the non-digested OVM bound to the C4 and C18 media, respectively. The removal ratio of Cu increased as more OVM was digested by subtilisin A. The digested OVM polypeptides were stained with Cu 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 Cu within microdroplets of biological samples.