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. Author manuscript; available in PMC: 2014 Dec 2.
Published in final edited form as: Curr Protoc Chem Biol. 2011 Jun 1;3(2):ch100155. doi: 10.1002/9780470559277.ch100155

Figure 4.

Figure 4

Km determinations for TDP-KDX (left panel) and NADPH (right panel). The protocol was based on that described under Steps 12-24, Km determinations. Data points represent the mean of triplicate determinations; error bars depict standard deviation. Left-Hand Panel. The concentration of TDP-KDX was varied from 0 to 1000 μM in the presence of a fixed concentration of NADPH (25 μM). The fluorescence change in the presence of a mixture of 2.63 × 10−4 μg/μl Rml C, 6.53 × 10−4 μg/μl Rml D in 1× assay buffer was monitored over 90 min at 25°C. Data were fit to a sigmoidal plot using GraphPad Prism, and the Km of TDP-KDX was calculated to be 200 μM. Right-Hand Panel. The concentration of NADPH was varied from 0 to 36 μM in the presence of a fixed concentration of TDP-KDX (200 μM). The fluorescence change in the presence of a mixture of 2.63 × 10−4 μg/μl Rml C, 6.53 × 10−4 μg/μl Rml D in 1× assay buffer was monitored over 90 min at 25°C. Data were fit to a sigmoidal plot using GraphPad Prism, and the Km of NADPH was calculated to be 12.5 μM.