The Inhibitory Effect of EDTA and Mg2+ on the Activity of NADH Dehydrogenase in Lysozyme Lysis

Abstract

NADH dehydrogenase of Escherichia coli, a component of the electron transport chain, + oxidizes NADH to NAD. Previously, it was demonstrated that when E. coli K12 cells were lysed by the lysozyme lysis method, NADH dehydrogenase activity was inhibited; however, when cells were lysed by French press, NADH dehydrogenase activity was observed. In this study, components commonly used in lysozyme lysis were examined for their effect on 2+ NADH dehydrogenase activity. It was found that the combination of EDTA and Mg inhibit the enzyme’s activity. Hence, we have developed alternative protocols of lysing cells with lysozyme that do not require EDTA. One method involves the use of polymyxin B, while the other utilizes the mechanical freeze-thaw process. Our alternative methods eliminate the need for EDTA by utilizing different mechanisms of disrupting the outer membrane. Though these alternative methods did not give rise to high NADH dehydrogenase activity, it may be possible to further optimize their conditions to yield lysate containing functional NADH dehydrogenase.