Biochemistry


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0 Q&A 9485 Views Oct 5, 2016
Copper is an essential micronutrient and functions as a cofactor in many enzymes such as heme-Cu oxygen reductases, Cu-Zn superoxide dismutases, multi-copper oxidases and tyrosinases. However, due to its chemical reactivity, free copper is highly toxic (Rae et al., 1999) and all organisms use sophisticated machineries for controlling uptake, storage and export of Cu. The strict control of the cellular Cu homeostasis prevents toxic effects but sustains synthesis of cuproproteins. Monitoring the copper levels within the cell and within different cellular compartments is an essential approach for identifying the contribution of different proteins in maintaining the cellular copper equilibrium. Therefore, whole cells and whole-cell lysates, which can be further fractionated into cytoplasm and periplasm, were digested and the protein concentration was determined by Lowry assay. Subsequently, the copper content was measured by atomic absorption spectroscopy (AAS) and the Cu content per mg of protein was calculated. This provides a simple and cost-effective method of producing quantifiable results about the cellular Cu content. To exemplify this method, we used the phototrophic α-proteobacterium Rhodobacter capsulatus, which is commonly used as a model organism for studying Cu-trafficking in bacterial cells (Ekici et al., 2012).



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