FIGURE 11.7 Summary of heme binding and peroxidase activity exhibited by de novo four-helix bundle proteins. (A) Heme. (B) Heme binding assay. Each column shows a different sample performed in triplicate. Column 1 is heme alone in buffer. Column 2 is heme incubated with lysates from cells containing empty vector, which represents the activity of background E. coli proteins. Both columns 1 and 2 are negative controls and show a brown color. Columns 3 and 4 are cell lysates from cells expressing two different four-helix bundle (4HB) proteins incubated with heme showing various intensities of red. (C) The peroxidase reaction catalyzed by de novo four-helix bundle proteins using heme as cofactor and 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) as the reducing agent. (D) Peroxidase assay. Each column is a different sample performed in triplicate. Column 1 shows the background activity of buffer alone and Column 2 shows the background activity of endogenous E. coli proteins. Both controls show a light teal color. Columns 3 and 4 are two different four-helix bundle (4HB) proteins exhibiting peroxidase activity giving rise to a dark teal color.
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