Observing and measuring charge regulation between two redox states of myoglobin using protein charge ladders and capillary electrophoresis

Abstract

Charge regulation occurs when a protein modulates its net charge in a way that minimizes some perturbance, e.g. the binding of a metal cofactor. Net charge is a fundamental biophysical property that is nonetheless poorly characterized for many proteins. Although charge regulation has been observed in a handful of cases, the extent to which this phenomenon occurs, its mechanisms, and its function are not well understood (1,3). This study examined charge regulation in myoglobin in the transition between two redox states of the heme iron, oxy-Mb (Fe2+) and met-Mb (Fe3+). Protein charge ladders and capillary electrophoresis were used to measure the net charge of each myoglobin species (Zoxy-Mb and Zmet-Mb). It was determined that Zoxy-Mb = -1.67 ± 0.09, while Zmet-Mb = -1.02 ± 0.06. The difference between the net charge of these two states, 0.6 ± 0.1, was smaller than the expected 1.0 unit. The net charge of myoglobin was regulated by 0.4 ± 0.1 units in the transition between oxy-Mb and met-Mb. This study demonstrated the existence of charge regulation in myoglobin, suggesting that this phenomenon may be more widespread than previously considered.